Food Chemistry xxx (xxxx) xxx–xxx

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Biorefining of industrial hemp (Cannabis sativa L.) threshing residues into

cannabinoid and antioxidant fractions by supercritical carbon dioxide,
pressurized liquid and enzyme-assisted extractions
⁎
Vaida Kitrytė, Dovyda Bagdonaitė, Petras Rimantas Venskutonis
Department of Food Science and Technology, Kaunas University of Technology, Radvilėnų rd. 19, Kaunas LT-50254, Lithuania

A R T I C L E I N F O A B S T R A C T

Keywords: C. sativa threshing residues were biorefined by consecutive supercritical carbon dioxide (SFE-CO2) pressurised
Hemp threshing residues liquid (PLE) and enzyme-assisted extractions (EAE). SFE-CO2 at optimised parameters yielded 8.3 g/100 g of
High-pressure extraction lipophilic fraction containing 0.2 and 2.2 g of cannabidiol and cannabidiolic acid per 100 g of threshing residues,
Enzyme-assisted extraction respectively. The recovery of cannabinoids from plant material was > 93%. PLE gave 4.3 and 18.9 g/100 g of
Cannabinoids
flavonoid-containing polar extracts, while EAE added 20.2% (w/w) of water-soluble constituents and increased
Antioxidants
the release of mono- and disaccharides by up to 94%. Antioxidant capacity of non-polar and polar fractions was
in the range of 1.3–23.5 mg gallic acid equivalents/g DW and 0.6–205.2 mg Trolox equivalents/g DW, with the
highest activities of PLE-EtOH/H2O extract. The combined SFE-CO2, PLE and EAE reduced antioxidant capacity
of starting plant material by 90–99%, showing that suggested multistep fractionation procedure is efficient in the
recovery of a major part of the antioxidatively active constituents from hemp threshing residues.

1. Introduction medicinal properties of phytocannabinoids. Among them, Δ9-tetra-

Industrial hemp (Cannabis sativa) is one of the oldest annual crops
with multi-purpose cultivation for a wide variety of products such as
hemp stem cellulose and fibre for paper and textile, hemp seed oil for
food, cosmetics and pharmaceutical industries. Hemp seed oil being
rich in polyunsaturated fatty acids (up to 80%) with nutritionally pre-
ferable linoleic (ω-6) to linolenic (ω-3) acid ratio (∼3:1), tocopherols
and minor bioactive constituents in unsaponifiable fraction (sterols,
aliphatic and triterpene alcohols, squalene) is among the most valuable
oils (Oomah, Busson, Godfrey, & Drover, 2002; Montserrat-de la Paz,
Marín-Aguilar, García-Giménes, & Fernández-Arche, 2014). It is ob-
tained mainly via cold pressing or hydrocarbon solvent (Latif & Anwar,
2009) and, more recently, supercritical carbon dioxide (SFE-CO2) ex-
traction (Da Porto, Voinovich, Decorti, & Natolino, 2012; Da Porto,
Decorti, & Tubaro, 2012; Da Porto, Natolino, & Decorti, 2015; Tomita
et al., 2013; Aladić et al., 2015).
More recently the interest in hemp has remarkably increased due to
the presence of specific phytochemicals in its leafy anatomical
parts. > 70 biologically-active and unique to Cannabis terpenophenolic
compounds, phytocannabinoids, have been found in hemp (Flores-
Sanchez & Verpoorte, 2008a, 2008b; Andre, Hausman, & Guerriero,
2016). A large number of studies demonstrated health promoting and
hydrocannabinol (Δ9-THC) is a well-known natural psychotropic com-
pound; therefore, today only the approved cultivars of C. sativa accu-
mulating less than 0.2–0.3% of Δ9-THC, are officially allowed in
Canada, USA and many European countries.
Non-psychotropic cannabidiol (CBD) and its parent compound
cannabidiolic acid (CBDA) were reported in various C. sativa cultivars
as the major quantitatively cannabinoids (Welling, Liu, Shapter,
Raymond, & King, 2016). CBD and CBDA were shown to exert mod-
ulating effects of human endocannabinoid system, which have been
associated with various beneficial medicinal and therapeutic properties
such as analgesic, antibacterial, antidiabetic, antiemetic, antiepileptic,
antiinflammatory, antiproliferative, antipsychotic, antispasmodic, etc.
Therefore, cannabinoids are considered as promising natural com-
pounds in treating epilepsy, pain, depression, anorexia, cancer and
other diseases and disorders (Mechoulam, Parker, & Gallily, 2002;
Flores-Sanchez & Verpoorte, 2008a; Takeda et al., 2012; Andre et al.,
2016).
Harvesting and processing of hemp, either for oil or fibre generates
vast amounts of by-products containing substantial amounts of im-
portant nutrients, e.g. phytochemical antioxidants; it was recently de-
monstrated for different hemp seed meal fractions (Pojić et al., 2014),
inflorescences (Da Porto, Decorti, & Natolino, 2014), kernels and seed

⁎
Corresponding author.
E-mail address: rimas.venskutonis@ktu.lt (P. Rimantas Venskutonis).

http://dx.doi.org/10.1016/j.foodchem.2017.09.080
Received 1 March 2017; Received in revised form 5 September 2017; Accepted 14 September 2017
0308-8146/ © 2017 Elsevier Ltd. All rights reserved.

Please cite this article as: Kitryte, V., Food Chemistry (2017), http://dx.doi.org/10.1016/j.foodchem.2017.09.080
V. Kitrytė et al. Food Chemistry xxx (xxxx) xxx–xxx

Table 1
Central composite design matrix (levels of independent variables and variation levels in natural values) for SFE-CO2 optimisation and values of observed responses for the extraction of
non-polar constituents from C. sativa threshing residues.

No. SFE-CO2 parameters SFE-CO2 extract yield CBD yield CBDA yield

Pressure, MPa Temperature, °C Time, min g/100 g DW1 mg/g extract g/100 g DW1 mg/g extract g/100 g DW1

1 10 35 60 1.76 ± 0.03b 64.18 ± 2.98e 0.11 ± 0.01ab 157.6 ± 6.0a 0.28 ± 0.01a
2 30 70 90 7.93 ± 0.08cd 25.75 ± 1.31d 0.20 ± 0.00ef 185.6 ± 8.3ab 1.47 ± 0.06bc
3 30 52.5 90 7.62 ± 0.09cd 19.13 ± 0.73ab 0.15 ± 0.01bcd 232.5 ± 0.5bcd 1.77 ± 0.00de
4 10 52.5 90 0.63 ± 0.00a –nd –nd –nd –nd
5 30 52.5 60 7.65 ± 0.08cd 18.31 ± 0.72a 0.14 ± 0.01abc 236.2 ± 14.0bcd 1.81 ± 0.11de
6 10 70 120 0.27 ± 0.01a –nd –nd –nd –nd
7 30 52.5 90 7.90 ± 0.13cd 18.46 ± 3.61ab 0.15 ± 0.03bcd 231.6 ± 9.9bcd 1.83 ± 0.08de
8 30 52.5 90 7.61 ± 0.05cd 21.00 ± 0.72abcd 0.16 ± 0.01cd 233.3 ± 1.5bcd 1.78 ± 0.01de
9 10 70 60 < 0.00a –nd –nd –nd –nd
10 30 52.5 90 7.56 ± 0.00cd 20.89 ± 0.17abcd 0.16 ± 0.00c 237.8 ± 3.1cd 1.80 ± 0.02de
11 50 52.5 90 8.25 ± 0.19d 21.41 ± 0.21 abcd 0.18 ± 0.00de 231.5 ± 2.4bcd 1.91 ± 0.02ef
12 10 35 120 2.51 ± 0.02b 44.22 ± 1.94e 0.11 ± 0.01a 203.8 ± 7.9b 0.51 ± 0.02a
13 50 70 60 9.63 ± 0.26e 24.15 ± 0.89bcd 0.23 ± 0.01f 204.1 ± 7.3b 1.97 ± 0.07ef
14 30 52.5 120 7.80 ± 0.17cd 20.16 ± 0.96abcd 0.16 ± 0.01cd 221.1 ± 12.5bc 1.73 ± 0.10cde
15 30 35 90 7.30 ± 0.13c 23.80 ± 0.54bcd 0.17 ± 0.00de 236.2 ± 12.1bcd 1.72 ± 0.09cde
16 50 70 120 10.36 ± 0.31e 21.52 ± 0.84abcd 0.22 ± 0.01f 223.7 ± 14.1bc 2.32 ± 0.15f
17 30 52.5 90 7.50 ± 0.16cd 20.89 ± 0.17abcd 0.16 ± 0.00cd 239.3 ± 1.0cd 1.80 ± 0.01de
18 50 35 60 7.39 ± 0.21cd 19.42 ± 0.89abc 0.14 ± 0.01abcd 179.9 ± 9.2ab 1.33 ± 0.07b
19 30 52.5 90 7.63 ± 0.05cd 20.07 ± 0.60abc 0.15 ± 0.01cd 230.4 ± 9.9bc 1.76 ± 0.08de
20 50 35 120 7.51 ± 0.04cd 18.28 ± 0.68a 0.14 ± 0.01abc 209.1 ± 7.6b 1.57 ± 0.06bcd
Optimal conditions:
46.5 70 120 8.30 ± 0.01d 24.72 ± 0.31cd 0.21 ± 0.00ef 261.4 ± 2.2d 2.17 ± 0.02f

1
: SFE-CO2 extract, CBD and CBDA yields were expressed as g/100 g DW of sample prior SFE-CO2; –nd: not detected. CBD: cannabidiol; CBDA: cannabidiolic acid; SFE-CO2: supercritical
carbon dioxide extraction; Different superscript letters within the same column indicate significant differences (one way ANOVA and Tukey’s test, p < 0.05).

hulls (Chen et al., 2012). Therefore, there is an obvious scientific and
industrial interest in utilising such by-products or waste more effi-
ciently. Soluble bioactive substances are usually isolated by the con-
ventional solvent extraction, however such method, as a rule, may re-
cover target constituents only partially, depending on their solubility in
the selected solvent; therefore, development of multi-step fractionation
processes is considered as a more promising strategy for the effective
valorisation of various agro food by-products, which would enable to
convert them into the higher added value functional ingredients. From
this point of view, wider application of various innovative green tech-
nologies has become very attractive. For instance, SFE-CO2 has been
recognised as a good alternative to soli-liquid extraction with hydro-
carbon solvents (e.g. hexane, petrol ether) for the isolation of lipophilic
compounds. SFE-CO2 does not require the removal of toxic solvent re-
sidues from the oils and extracts obtained; it also enables to achieve
partial extraction selectivity by a proper selection of process pressure
and temperature. Higher polarity components such as polyphenolic
antioxidants and pigments may be extracted from the defatted residues
using combinations of fast and efficient separation techniques such as
pressurised liquid (PLE), ultrasound, microwave and/or enzyme-as-
sisted extractions (EAE). The advantages of a multistep application of
SFE-CO2, PLE and EAE for the isolation of valuable ingredients was
reported for amaranth seeds (Kraujalis & Venskutonis, 2013), brewers
spent grain (Kitrytė, Šaduikis, & Venskutonis, 2014), berry pomace
(Kryževičiūtė, Kraujalis, & Venskutonis, 2016; Grunovaitė, Pukalskienė,
Pukalskas, & Venskutonis, 2016; Oktay Basegmez et al., 2017; Kitrytė
et al., 2017), wheat and rye bran (Povilaitis, Šulniūtė,
Venskutonis, & Kraujalienė, 2015).
The aim of this study was to develop a multistep biorefining tech-
nology for the isolation of valuable phytocannabinoids and antioxidant
fractions from industrial hemp threshing residues via consecutive ap-
plication of SFE-CO2, PLE and EAE. It is expected that such systematic
approach may provide a promising platform in developing industrial
scale clean production processes for converting hemp processing by-
products into novel bioactive ingredients with functional food, nu-
traceutical and pharmaceutical applications.
2. Materials and methods
2.1. Materials
Dried threshing residues of C. sativa cultivar ‘Beniko’ remaining
after harvesting and cleaning of industrial fiber-type hemp seeds was
provided by the JSC ‘Agropro’ (Vilnius, Lithuania). It was a mixture of
leaves, floral bracts, flower fragments and immature seeds. Plant ma-
terial was ground by an ultra centrifugal mill ZM 200 (Retsch, Haan,
Germany) using 0.2 mm hole size sieve prior to the extraction. All other
chemicals and solvents were of analytical and HPLC-grade
(Supplementary Material).
2.2. Supercritical CO2 extraction (SFE-CO2)
SFE-CO2 was performed in a supercritical fluid extractor Helix
(Applied Separation, Allentown, PA) by the modified procedure of
Kraujalis and Venskutonis (2013) from 10 ± 0.1 g of hemp threshing
residue loaded into a 50 mL stainless steel extractor. Its temperature
was controlled by the surrounding heating cover. The volume of CO2
was measured by a ball float rotameter and digital mass flow meter in
standard litres per minute (SL/min) at standard state (PCO2 = 100 kPa,
TCO2 = 20 °C, ρCO2 = 0.0018 g/mL). The flow rate of CO2 was con-
trolled manually by the micro-metering valve and kept at 2–3 SL/min
during all experiments. Other important extraction parameters, pres-
sure (P), temperature (T) and time (τ) were optimized using response
surface methodology (RSM) and central composite design (CCD). The
range of variables, P, T and τ were 10–50 MPa, 35–70 °C and 60–120
min, respectively. As response factors (RF), the total yield of lipophilic
extract and the yields of extracted CBD and CBDA were selected (RFI,
RFII and RFIII, respectively). The model, consisting of 20 experimental
runs with 8 factorial points, 6 axial points and 6 centre points (Table 1)
was established using Design-Expert software trial version 8.0.7.1
(Stat–Ease Inc., Minneapolis, MN) as previously reported by Kraujalis
and Venskutonis (2013). A constant static time of 10 min was included
into the total extraction time of all extractions. The extracts were col-
lected in the glass bottles, weighed ( ± 0.001 g) and stored at −20 °C.

2
V. Kitrytė et al.
The solid residue after SFE-CO2 was collected and kept in a dry, well-
ventilated place prior to the analysis.
2.3. Pressurised liquid extraction (PLE)
PLE was performed from hemp material residue remaining after
SFE-CO2 as described by Povilaitis et al. (2015) in an accelerated sol-
vent extraction apparatus ASE350 (Dionex Sunnyvale, CA, USA) using
different polarity solvents, namely acetone (PLE-Ac) and mixtures of
ethanol/water (PLE-EtOH/H2O). The residue (5 ± 0.1 g) was mixed
with 5 g of diatomaceous earth (1:1) and placed in 34 mL Dionex
stainless-steel extraction cells equipped with stainless steel frits and
cellulose filters at the ends. PLE-Ac was tested at 30, 70, 100 and 130 °C
temperature and 15 min (3 cycles × 5 min), 45 min (3 cycles × 15
min), and 75 min (3 cycles × 25 min) extraction time. Finally, PLE-
EtOH/H2O was performed at 100 °C during 45 min (3 cycles × 15 min)
using the mixtures of EtOH/H2O (1:4, 1:1 and 4:1, v/v). The pressure
(10.3 MPa), pre-heating time (5 min), cell flush volume (100%) and
purge time (120 s) with nitrogen to collect the extract were constant in
all experiments. Organic solvents were evaporated in a Büchi V–850
Rotavapor R–210 (Flawil, Switzerland), while the residual water was
freeze-dried (−50 °C, 0.5 mbar). PLE-Ac and PLE-EtOH/H2O extracts
were kept under the nitrogen flow for 15 min to remove solvent re-
sidues, weighed ( ± 0.001 g) and stored at −20 °C. The solid residues
after PLE were collected and kept in a dry, well-ventilated place prior to
the analysis.
2.4. Enzyme-assisted extraction (EAE)
EAE was carried out as reported by Oktay Basegmez et al. (2017).
The residue of PLE-EtOH/H2O (4/1, v/v) was weighed (10 ± 0.1 g) in
a 250 mL polyethylene flat-bottom centrifugation bottle and suspended
in 100 mL of 50 mM sodium acetate buffer (pH 3.5). Afterwards cel-
lulolytic enzyme mixture Viscozyme® L was added to reach the en-
zyme/substrate (E/S) ratio of 6% v/w (corresponds to 72 FBGU/10 g
plant material) and incubated in thermostatically controlled shaker
(800 rpm) at 40 °C for 7 h. EAE was terminated by immersing cen-
trifugation bottle in a boiling water bath for 10 min, followed by the
rapid cooling and centrifugation (9000 rpm, 10 min). Appropriate
control (sample + buffer) and blank samples A (enzyme + buffer) and B
(buffer) were prepared simultaneously. The resulting supernatants
(water-soluble fractions) and solid residues (water-non soluble frac-
tions) were freeze-dried (−50 °C, 0.5 mbar), weighed ( ± 0.001 g) and
stored at −20 °C.
2.5. Cannabinoid analysis by HPLC-DAD
Quantitative determination of CBD and CBDA in SFE-CO2 extracts
(1 mg/mL) and hemp threshing residue (0.5 ± 0.01 g) before and after
SFE-CO2 was performed by the procedure of UN Office on Drugs and
Crime (UNODC, 2009) on a Simadzu HPLC system under isocratic
elution conditions, using CH3CN/ultra-pure H2O mobile phase (4:1)
with 0.1% formic acid (v/v). The external calibration curves (peak area
versus injected amount of CBD and CBDA reference compounds) were
used for quantification (Supplementary Material).
2.6. Sugar analysis by UPLC–MS
The content of monosaccharide glucose and disaccharide maltose in
EAE-derived supernatant and corresponding control sample (1 mg/mL)
was determined by the modified procedure of Grunovaitė et al. (2016)
on an Acquity UPLC Heclass system under isocratic conditions, using
CH3CN/ultra-pure H2O (3:1) mobile phase with 0.1% NH4OH (v/v).
The peaks were identified by comparing their retention times with
those of the corresponding standards. The external calibration curves
(peak area versus injected amount) of reference compounds (glucose for
3
Food Chemistry xxx (xxxx) xxx–xxx
hexoses and maltose for dihexoses) were used for quantification
(Supplementary Material).
2.7. In vitro antioxidant activity assessment
Total phenolic content (TPC), ferric reducing antioxidant power
(FRAP), ABTS%+/DPPH% scavenging capacities and oxygen radical ab-
sorbance capacity (ORAC) of extracts (dilutions of 30–4000 μg/mL)
were evaluated by the modified procedures of Singleton, Orthofer, and
Lamuela-Raventós (1999), Benzie and Strain (1996), Re et al. (1999),
Brand-Williams, Cuvelier, and Berset (1995), and Prior et al. (2003),
respectively. The absorbance and fluorescence were measured with
Spectronic Genesys 8 spectrophotometer (Thermo Spectronic, Roche-
ster, NY) and FLUOstar Omega reader (BMG Labtech, Offenburg, Ger-
many), respectively. Antioxidant capacity of solid substances was de-
termined by QUENCHER method (Gökmen, Serpen, & Fogliano, 2009)
using 10 mg of sample (solid dilutions in microcrystalline cellulose at
1–100 µg/mg) or cellulose (blank), as described elsewhere (Kitrytė
et al., 2014). The total phenolic content (TPC) and Trolox equivalent
antioxidant capacity (TEAC) were expressed as gallic acid (mg GAE/g
extract or DW) and Trolox (mg TE/g extract or DW) equivalents, re-
spectively by means of dose-response calibration curves
(Supplementary Material).
2.8. Phytochemical characterisation by UPLC/ESI–QTOF–MS
Phytochemical composition of extracts was screened on an Acquity
UPLC system (Waters, Milford, USA) by the modified procedure of
Grunovaitė et al. (2016). Peak identification was carried out by com-
paring the retention times with those of the standards, accurate masses,
using literature sources and free chemical databases (Supplementary
Material).
2.9. Statistical analysis
Extraction experiments and phytochemical composition analysis
were performed in duplicate; cannabinoid and sugar content – in tri-
plicate; antioxidant activity assessment – at least in quadruplicate.
Mean values and standard deviations were calculated using MS Excel
2003. One-way analysis of the variance (ANOVA), followed by the
Tukey’s posthoc test to compare the means that showed significant
variation (p < 0.05), also bivariate correlation analysis and Pearson
correlation coefficients between different antioxidant activity indices
were performed and calculated using GraphPad Prism 6.01 software
(2012).
3. Results and discussion
Multistep extraction scheme (Fig. S1, Supplementary Material) was
designed and tested for biorefining of harvesting by-products of in-
dustrial hemp, which consisted of a mixture of leaves, floral bracts,
flower fragments and immature seeds, in order to evaluate the possi-
bilities to valorise hemp processing waste for obtaining several higher
added value fractions. In terms of methodology, several consecutively
performed extraction processes, including high pressure (SFE-CO2 and
PLE) and enzyme-assisted (EAE) techniques were selected for separ-
ating soluble substances from the hemp threshing residues. SFE-CO2
parameters were optimized for the highest lipophilic extract yield and
the main quantitatively cannabinoids, namely CBD and CBDA by using
CCD and RSM, whereas PLE solvents and parameters were selected to
extract other fractions, which were analysed for total phenolics, in vitro
antioxidant potential and phytochemical composition. In addition, an-
tioxidant capacity indicators were measured in solid residues, which
remain after each extraction step, in order to evaluate the efficiency of
such steps in the overall biorefining scheme.
V. Kitrytė et al. Food Chemistry xxx (xxxx) xxx–xxx

Table 2 yield, g/100 g DW) and RFIII (CBDA yield, g/100 g DW) is summarised
Analysis of variance of the regression parameters for response surface quadratic model for by the analysis of variance (ANOVA) presented in Table 2. The ade-
SFE-CO2 extract, CBD and CBDA yields (g/100 g DW) from C. sativa threshing residues.
quacy of the model, as it may be judged from the total determination
Source SS df MS F-Value p-Value coefficient R2 (RFI: 0.9979; RFII: 0.9756; RFIII: 0.9829), indicates
reasonable fit of the models to the experimental data. Model analysis
RF I: SFE-CO2 extract yield (g/100 g DW): also showed good agreement between the adjusted and predicted
Model 195.26 9 21.70 525.26 < 0.0001*
coefficients of determination (R2): 0.9960 and 0.9763 (RFI); 0.9537 and
Pressure (P, MPa) 144.17 1 144.17 3490.40 < 0.0001*
Temperature (T, 0.30 1 0.30 7.16 0.0232* 0.8247 (RFII); 0.9675 and 0.8563 (RFIII), respectively. Calculated
°C) adequate precision (Press) values of 71.693 (RFI), 23.885 (RFII) and
Time (τ, min) 0.41 1 0.41 9.88 0.0105* 24.314 (RFIII), which compare the range of the predicted values to the
PT 10.33 1 10.33 250.05 < 0.0001* average prediction error at the experimental design points (desirable
Pτ 3.613 · 10−3 1 3.613 · 10−3 0.087 0.7735**
signal to noise ratio > 4), indicate that the signal is adequate and the
Tτ 2.113 · 10−3 1 2.113 · 10−3 0.051 0.8256**
P2 25.47 1 25.47 616.57 < 0.0001* model can be used to navigate the design space. Quadratic regression
T2 0.048 1 0.048 1.16 0.3074** model analysis for SFE-CO2 yield (Table 2) showed that the model was
τ2 0.16 1 0.16 3.89 0.0767** significant according to the Student test (p < 0.05) with a calculated F-
Residual 0.41 10 0.041
value of 525.26; consequently, ‘lack of fit’ was not significant relative to
Lack of fit 0.32 5 0.064 3.35 0.1053**
Pure error 0.095 5 0.019 the pure error (p = 0.1053). The results obtained showed that P, τ, T,
Corrected Total 195.68 19 PT interaction and second-order term of P2 were significant on the total
RF II: CBD yield (g/100 g DW):
SFE-CO2 extract yield (RFI) in the following order: P
Model 0.082 9 9.092 · 10−3 44.47 < 0.0001* (p < 0.0001) > P2 (p < 0.0001) > PT (p < 0.0001) > τ
Pressure (P, MPa) 0.048 1 0.048 232.86 < 0.0001* (p = 0.0105) > T (p = 0.0232). For RFII and RFIII the model itself
Temperature (T, 3.610 · 10−5 1 3.610 · 10−5 0.18 0.6832** with F-values of 44.47 and 63.82 for CBD and CBDA yield, respectively,
°C)
and the factors P, PT and P2 were significant, as for SFE-CO2 extract
Time (τ, min) 4.000 · 10−7 1 4.000 · 10−7 1.956 · 10−3 0.9656**
PT 0.020 1 0.020 97.33 < 0.0001* yield (RFI). However, in this case significant ‘lack of fit’ values were
Pτ 2.813 · 10−5 1 2.813 · 10−5 0.14 0.7185** obtained for RFII (CBD yield; p < 0.0125) and RFIII (CBDA yield;
Tτ 1.250 · 10−7 1 1.250 · 10−7 6.114 · 10−4 0.9808** p < 0.0002). It may be assumed that the changes of CO2 properties at
P2 0.011 1 0.011 56.03 < 0.0001* different P-T levels have more pronounced effect on the cannabinoid
T2 3.555 · 10−3 1 3.555 · 10−3 17.39 0.0019*
τ2 5.682 · 10−5 1 5.682 · 10−5 0.28 0.6096**
solubility and extraction efficiency, as compared to the total SFE-CO2
Residual 2.045 · 10−3 10 2.045 · 10−4 yield; this may partially explain the significant ‘lack of fit’ of the model
Lack of fit 1.857 · 10−3 5 3.715 · 10−4 9.91 0.0125* in the case of RFII and RFIII. For example, remarkably lower yields of
Pure error 1.873 · 10−4 5 3.747 · 10−5 the total extract, as well as the yields of CBD and CBDA were obtained
Corrected Total 0.084 19
at the lowest applied P = 10 MPa and T = 35 °C; after increasing
RF III: CBDA yield (g/100 g DW): T > 50 °C phytocannabinoids were not found in the extracts at all
Model 10.47 9 1.16 63.82 < 0.0001*
(Table 1). It may be explained by the remarkable decrease of CO2
Pressure (P, MPa) 6.90 1 6.90 378.38 < 0.0001*
Temperature (T, 0.011 1 0.011 0.63 0.4456**
density at lower pressure levels by increasing temperature, resulting in
°C) a weaker diffusivity and solvating power (Kryževičiūtė et al., 2016). At
Time (τ, min) 0.056 1 0.056 3.05 0.1111** P > 30 MPa, the increase of T had positive effect both on the total SFE-
PT 0.59 1 0.59 32.27 0.0002* CO2 yield and the yields of phytocannabinoids, reaching maximum
Pτ 0.016 1 0.016 0.88 0.3693**
values at 40–50 MPa and 70 °C, most likely due to the so-called “en-
Tτ 1.953 · 10−3 1 1.953 · 10−3 0.11 0.7501**
P2 1.51 1 1.51 82.92 < 0.0001* hanced solubility effect”, which occurs when the increasing vapour
T2 0.026 1 0.026 1.44 0.2574** pressure of solute outweighs the decreasing solvating power of CO2
τ2 0.014 1 0.014 0.75 0.4053** (Kraujalis & Venskutonis, 2013; Da Porto et al., 2014). Similar ob-
Residual 0.18 10 0.018 servations regarding the varying molar solubility of non-psychotropic
Lack of fit 0.18 5 0.036 56.38 0.0002*
Pure error 3.176 · 10−3 5 6.352 · 10−4
CBD and cannabigerol as well as psychoactive THC and cannabinol in
Corrected Total 10.65 19 supercritical CO2 at different temperature (313–334 K) and pressure
(11.3–21.1 MPa) ranges were previously reported by Perrotin-Brunel
*
: significant; **: not significant; df: degree of freedom; F: Fisher value.; MS: mean square; et al., 2010.
RF: response factor; SS: sum of square. Second order polynomial regression model, describing relationship
between dependent and independent variables (P, T, τ), is given in the
3.1. Optimization of SFE-CO2 parameters for hemp lipophilic fraction and equations (1−3). Predicted values were calculated using a second order
its main phytocannabinoids polynomial equation and compared with experimental values in Fig. S2
(Supplementary Material).
SFE-CO2 has been proved as an effective green technology for the
isolation of high added value products from botanicals. Therefore, it YieldSFE − CO2 (RFI ) = 7.58 + 3.80 × P + 0.17 × T + 0.20 × τ
was selected as a 1st step for valorising hemp threshing residues. So far + 1.14 × (PT )−0.021 × (Pτ )
as the effectiveness of SFE-CO2 depends on several process parameters, + 0.016 × (Tτ )−3.04 × (P 2) + 0.13 × (T 2) + 0.24 × (τ 2) (1)
particularly P, T and τ, CCD and RSM were used to optimize the effect of
those independent variables on the total SFE-CO2 yield and the yields of YieldCBD (RFII ) = 0.15 + 0.069 × P−0.0019 × T −0.0002 × τ
the two most important lipophilic bioactive constituents, CBD and
+ 0.05 × (PT )−0.001875 × (Pτ )
CBDA. The model selected 20 experimental sets, their characteristics
and results are listed in Table 1. It may be observed that process − 0.0000125 × (Tτ )−0.065 × (P 2) + 0.036 × (T 2) + 0.004545 × (τ 2)
parameters had remarkable effects on the selected responses: the total (2)
yield from hemp threshing residue was from 0.3 to 10.4 g/100 g DW,
containing 18.3–64.2 mg/g of CBD (0.1–0.2 mg/g DW) and YieldCBDA (RFIII ) = 1.75 + 0.38 × P + 0.034 × T + 0.075 × τ
157.6–239.3 mg/g of CBDA (0.3–2.3 g/100 g DW). + 0.27 × (PT ) + 0.045 × (Pτ )
Model evaluation for RFI (extract yield, g/100 g DW), RFII (CBD − 0.016 × (Tτ )−0.74 × (P 2)−0.098 × (T 2) + 0.071 × (τ 2) (3)

4
V. Kitrytė et al. Food Chemistry xxx (xxxx) xxx–xxx

Fig. 1. Response surface 3D and 2D plots showing the effects of independent variables on SFE-CO2 extract, CBD and CBDA yields (g/100 g DW) from C. sativa threshing residues: A – effect
of temperature and pressure at constant time of 90 min; B – effect of time and pressure at constant temperature of 52.5 °C; C – effect of time and temperature at constant pressure of
30 MPa.

Response surface plots showing the effect of independent variables
and their interactions on RFI, RFII and RFIII are presented in Fig. 1. It
may be observed that at τ = 90 min (Fig. 1A), the yields increased 5–8-
fold when P increased from 10 to 50 MPa; moreover, strong effect of P2
may be noticed at P > 40 MPa. At the latter pressure levels the change
of T from 35 to 70 °C did not have significant effect on the total SFE-CO2
yield, whereas some significant positive effect on CBD extraction was
observed.
At T = 52.5 °C (Fig. 1B), the effect of P was more important than
that of τ; the yield of SFE-CO2 fraction and the recovery of CBD and
CBDA increased on average 4-fold when P increased from 10 to 30 MPa.
It is in agreement with Perrotin-Brunel et al. (2010) who reported that
at constant T (53 °C) the increase of P from 11.3 to 20.6 MPa favoured
up to 3-fold higher CBD solubility in supercritical CO2. 3-D diagram in
Fig. 1C shows that the major parts of extracts and recovered phyto-
cannabinoids are obtained during 60 min; however, further increase of
SFE-CO2 extract, CBD and CBDA yields by 8%, 14% and 9%, respec-
tively, was recorded after prolonging extraction time to 120 min at

Fig. 1. (continued)

5
V. Kitrytė et al.
Fig. 1. (continued)
P = 30 MPa within T interval of 52.5 and 70 °C.
Considering all responses, the following optimal conditions for ob-
taining the highest yields of SFE-CO2 extract, CBD and CBDA from
hemp threshing residues were as follows: P = 46.5 MPa, T = 70 °C,
τ = 120 min. Under these conditions, hemp by-product yielded 8.3 g/
100 g DW of lipophilic fraction with 24.7 mg/g of CBD and 261.4 mg/g
of CBDA (0.2 and 2.2 g/100 g DW of starting material, respectively).
However, it should be noted, that the highest yields were obtained at
maximal τ value; therefore, optimisation in this case means determi-
nation of optimal parameters in the selected range of variables. For
comparison, previously reported CBD content in commercially avail-
able hemp seed oils was 4–236 mg/kg and hemp-leaf containing herbal
teas 26–60 mg/kg (Lachenmeier, Kroener, Musshoff, & Madea, 2004;
Petrović, Debeljak, Kezić, & Džidara, 2015).
To evaluate the efficiency of CBD and CBDA recovery their amounts
were determined in the solid starting plant material and the residue
after SFE-CO2 (Table S1; Supplementary Material). Thus, prior to SFE-
CO2 the concentration of CBD and CBDA was 0.1 and 2.4 g/100 g DW,
respectively, while in the extraction residues it was only 0.003 and
0.2 g/100 g DW. Consequently, the recovery of CBD and CBDA was
almost 100% and ∼93%, respectively, which is in agreement with the
CBDA recovery (92%) calculated from its concentration in SFE-CO2
extract. It was observed that higher amount of CBD was recovered by
SFE-CO2 than it was present in the raw hemp threshing residue and
therefore the ratio of CBDA:CBD decreased from 17:1 in plant material
prior SFE-CO2 to 10:1 in SFE-CO2 extract. It may be assumed that these
changes were caused by the decarboxylation of some CBDA portion
during extraction. In general, SFE-CO2 was proved to be a very efficient
solvent-free process for the recovery of phytocannabinoids from the
industrial hemp harvesting and processing by-products.
Previously SFE-CO2 was used mainly for the extraction of hemp seed
oil (Da Porto, Voinovich, et al., 2012; Da Porto, Decorti, et al., 2012; Da
Porto et al., 2015; Tomita et al., 2013; Aladić et al., 2015) These studies
also concluded that extraction pressure had a major positive effect on
the total hemp seed oil yield. SFE-CO2 was also successfully applied for
separating waxy fractions and volatile oil from hemp inflorescences by
using comparatively low extraction pressure and temperature (10 and
14 MPa, 40 °C) and two separators operating at 7 MPa/25 °C and 5
6
Food Chemistry xxx (xxxx) xxx–xxx
MPa/15 °C (Da Porto et al., 2014). However, to the best of our
knowledge, other hemp oil and fibre industry by-products have not
been valorised by SFE-CO2 previously and this is the first report on CCD
and RSM-based optimization, which is focused on the recovery of
bioactive CBD and CBDA from hemp threshing residues. The results
obtained may serve for further studies in developing SFE-CO2 processes
and parameters for a more selective extraction of individual cannabi-
noids and their separation from other hemp fractions. The differences of
the solubility of four phytocannabinoids in supercritical CO2 were re-
ported to be dependent on their chemical structures, volatility and
melting points (Perrotin-Brunel et al., 2010).
3.2. Pressurised liquid extraction (PLE) of SFE-CO2 residues
Plant material residue after SFE-CO2 was further subjected to PLE
with acetone and ethanol/water mixtures in order to isolate insoluble in
supercritical CO2 substances, mainly consisting of higher polarity
compounds with potential antioxidant capacity. PLE was demonstrated
as an efficient alternative to conventional solid-liquid extractions for
remarkably faster extraction of a broad spectrum of antioxidants such
as phenolic acids, flavonoids and other polyphenols using low-boiling
temperature solvents such as acetone and alcohols or their mixtures at
elevated pressures and temperatures (Hossain, Barry-Ryan, Martin-
Diana, & Brunton, 2011; Povilaitis et al., 2015). Both acetone and
ethanol can be used in compliance with good manufacturing practice
and no maximum residue limits in the foodstuffs are set by the EU
Directive 2009/32/EC.
Firstly, several parameter sets in the range of T = 30–130 °C and
τ = 15–70 min were tested for measuring their effects on PLE extract
yield and total phenolic content (TPC) (Table 3). The yields of acetone-
soluble components after first 15 min of extraction were in the range of
0.7–6.6 g/100 g DW, while TPC values of the extract obtained were
0.6–10.1 mg GAE/g DW (92.8–152.2 mg GAE/g extract). After 45 min
of PLE, remarkable increase in total extraction yield (by 80–87%) and
TPC values (by 81–97%) was achieved at T < 70 °C, while PLE at
T = 100–130 °C during 45 min gave by 17–41% higher yields and by
13–33% higher TPC values compared to 15 min extraction. The effi-
ciency of PLE at the longest applied time (75 min) was further tested at
V. Kitrytė et al. Food Chemistry xxx (xxxx) xxx–xxx

Table 3 reaction pathway (Hossain et al., 2011). Small increase in the yield (by
PLE-Ac and PLE-EtOH/H2O optimization and values of observed responses for the ex- 11%) and TPC (by 16%) was obtained at 100 °C and 75 min of ex-
traction of polar constituents from C. sativa threshing residues after SFE-CO2 (46.5 MPa,
traction, while at 70 °C these values were by 10 and 17% lower, as
70 °C, 120 min).
compared to 45 min PLE. Based on these results, 100 °C and of 45 min
PLE parameters PLE extract yield TPC were selected as preferable parameters for PLE-Ac, providing the yields
of 4.7 g/100 g DW from solid residues remaining after SFE-CO2 (or
g/100 g DW mg GAE/g extract mg GAE/g DW 4.3 g/100 g DW of starting material prior SFE-CO2; Table 4) with the
1
PLE-Ac : TPC recovery of 5.5 mg GAE/g DW. PLE-Ac yields at various para-
15 min (3 × 5 min) meters (Table 2) are in the ranges of the previously reported yields
30 °C 0.66 ± 0.13a 92.77 ± 3.31a 0.61 ± 0.02a (Chen et al., 2012) obtained with 50–100% acetone from hemp seed
70 °C 1.79 ± 0.25b 123.0 ± 0.8bc 2.20 ± 0.01b residues of C. sativa cultivars Bama (kernels: 0.5–8.4%; hulls: 0.2–2.4%)
100 °C 3.45 ± 0.17c 131.1 ± 10.5c 4.45 ± 0.36d
130 °C 6.61 ± 0.18e 152.2 ± 4.1d 10.05 ± 0.27g
and Yunma No. 1 (kernels: 0.5–9.7%; hulls: 0.3–2.5%).
45 min (3 × 15 min) The residues remaining after PLE-Ac at preferable conditions were
30 °C 1.20 ± 0.05ab 100.8 ± 2.5a 1.20 ± 0.03a further extracted by PLE-EtOH/H2O, which was the 3rd step in bior-
70 °C 3.36 ± 0.16ce 120.4 ± 5.5bc 3.98 ± 0.18d efining hemp threshing residue. Three solvents were prepared by
100 °C 4.73 ± 0.14d 115.8 ± 2.2bc 5.48 ± 0.10e
mixing ethanol and distilled water at the ratios of 1:4 (25% EtOH), 1:1
130 °C 7.74 ± 0.38f 148.3 ± 5.4d 11.40 ± 0.42h
75 min (3 × 25 min) (50% EtOH) and 4:1 (75% EtOH). Chen et al. (2012) showed that
70 °C 3.00 ± 0.12c 97.16 ± 6.45a 2.90 ± 0.20c EtOH/H2O mixtures produce remarkably higher (> 5-fold) extraction
100 °C 5.26 ± 0.39d 120.6 ± 8.6bc 6.34 ± 0.45f yields from hemp seed kernel and hulls. In our study (Table 3), extract
PLE-EtOH/H2O2: yields and TPC were similar both for 50 and 75% EtOH; they were on
100 °C, 45 min (3 × 15) average 21.7 g/100 g DW and 123.9 mg GAE/g (26.9 mg GAE/g DW).
EtOH/H2O, 1:4 v/v 35.05 ± 0.78b 73.04 ± 1.23a 25.61 ± 0.43a When EtOH concentration was 25%, the total extract yield was by
EtOH/H2O, 1:1 v/v 20.71 ± 0.44a 125.0 ± 3.7b 25.88 ± 0.76ab
35–41% higher; however, TPC in such extract was by 41% lower than in
EtOH/H2O, 4:1 v/v 21.58 ± 0.59a 124.7 ± 2.5b 26.90 ± 0.53b
case of other applied EtOH concentrations. Considering that TPC re-
1
: PLE-Ac extract yields and TPC values were expressed g/100 g DW and mg GAE/g DW of covery from plant DW was almost similar, it is evident that at the
sample after optimized SFE-CO2 (46.5 MPa, 70 °C, 120 min); 2: PLE-EtOH/H2O extract highest H2O concentration in the mixture, the compounds reacting with
yields and TPC values were expressed g/100 g DW and mg GAE/g DW of sample after Folin-Ciocalteus reagent (TPC) are diluted with other, neutral water
optimized PLE-Ac (10.3 MPa, 100 °C, 45 min); Ac: acetone EtOH: ethanol; PLE: pressur- soluble substances. Therefore, the highest EtOH/H2O ratio (4/1 v/v)
ized liquid extraction; TPC: total phenolic content. Different superscript letters within the
was selected for PLE-EtOH/H2O at 100 °C for 45 min, amounting
same column of individual PLE-Ac or PLE-EtOH/H2O treatments indicate significant
differences (one way ANOVA and Tukey’s test, p < 0.05). 21.6 g/100 g DW of PLE extract from hemp threshing residues after
PLE-Ac (or 18.9 g/100 g DW of starting material prior SFE-CO2;
70 and 100 °C, since the yields at 30 °C were rather small, while at Table 4) with TPC recovery of 26.9 mg GAE/g DW.
130 °C the extract acquired yellow colour and sweet odour notes al-
ready after 15 min of PLE; it may be attributed to the degradation and/ 3.3. Enzyme assisted extraction of PLE residues (EAE-Viscozyme)
or unfavourable chemical interactions between various endogenous
extract constituents at the elevated temperatures, e.g. via the Maillard At the final biorefining step, solid residue remaining after PLE-

Table 4
Total phenolic content (TPC), ferric reducing antioxidant power (FRAP), DPPH%, ABTS%+ and ORAC scavenging properties of non-polar (SFE-CO2) and polar (PLE and EAE) extracts and
solid residues, obtained from C. sativa threshing residues after consecutive SFE-CO2 (46.5 MPa, 70 °C, 120 min), PLE-Ac (10.3 MPa, 100 °C, 45 min), PLE-EtOH/H2O (10.3 MPa, 100 °C,
45 min, EtOH/H2O 4/1 v/v), and EAE (E/S 6% v/w, 40 °C, pH 3.5, 7 h).

In vitro Extracts Crude plant material and solid residues after extraction
antioxidant
capacity SFE-CO2 PLE-Ac PLE-EtOH/H2O EAE-Viscozyme Prior SFE-CO2 After SFE-CO2 After PLE-Ac After PLE- After EAE-
EtOH/H2O Viscozyme

Yield
g/100 g DW1 8.30 ± 0.01b 4.33 ± 0.12a 18.86 ± 0.51c 20.20 ± 0.23d 100.0 91.70* 87.37** 68.51*** 48.31****

TPC, mg GAE/g:
mg/g extract 107.4 ± 0.6b 115.8 ± 2.2b 124.7 ± 2.5c 6.38 ± 0.21a –na –na –na –na –na
mg/g DW1 8.17 ± 0.05d 5.02 ± 0.09c 23.52 ± 0.47f 1.29 ± 0.04a 35.49 ± 1.37h 28.91 ± 0.54g 11.15 ± 0.47e 4.18 ± 0.32bc 2.95 ± 0.11b

TEACFRAP, mg TE/g:
mg/g extract 71.49 ± 0.80b 352.2 ± 15.8c 457.1 ± 10.9d 17.40 ± 1.59a –na –na –na –na –na
mg/g DW1 5.44 ± 0.06a 15.25 ± 0.56c 86.20 ± 2.77g 3.52 ± 0.27a 80.12 ± 0.94f 68.39 ± 2.87e 53.84 ± 2.86d 10.69 ± 0.38b 8.27 ± 0.38ab

TEACDPPH, mg TE/g:
mg/g extract 72.51 ± 1.39e 48.31 ± 1.62c 58.99 ± 2.00d 2.99 ± 0.08a –na –na –na –na –na
mg/g DW1 2.52 ± 0.11c 2.09 ± 0.07bc 11.13 ± 0.34e 0.60 ± 0.02a 41.88 ± 0.96h 38.11 ± 0.48g 29.21 ± 1.33f 4.28 ± 0.23d 1.02 ± 0.05ab

TEACABTS, mg TE/g:
mg/g extract 1025 ± 13c 1060 ± 89c 898.3 ± 67.1b 45.91 ± 1.25a –na –na –na –na –na
mg/g DW1 77.99 ± 1.01d 45.89 ± 3.16c 169.4 ± 12.2f 9.27 ± 1.24a 189.6 ± 9.3g 199.9 ± 10.4g 110.7 ± 5.4e 24.84 ± 0.37b 20.12 ± 0.39b

TEACORAC, mg TE/g
mg/g extract 469.5 ± 21.9c 282.4 ± 18.9b 1088 ± 109d 54.30 ± 3.36a –na –na –na –na –na
mg/g DW1 35.68 ± 1.65b 12.23 ± 0.82ab 205.2 ± 20.6d 10.97 ± 0.68ab 174.8 ± 21.4cd 146.3 ± 13.8c 183.5 ± 21.3d 21.24 ± 1.91b 2.36 ± 0.13a

1
: g/100 g, mg GAE or TE/g DW of plant material prior optimized SFE-CO2; –na: not applicable; *: Calculated as: 100 − Extract Yield [SFE-CO2]; **: Calculated as: 100 − Extract Yield
[SFE-CO2 + PLE-Ac]; ***: Calculated as: 100 − Extract Yield [SFE-CO2 + PLE-Ac + PLE-EtOH/H2O]; ****: Calculated as: 100 − Extract Yield [SFE-CO2 + PLE-Ac + PLE-EtOH/H2O
+ EAE-Viscozyme]; Ac: acetone; EAE: enzyme-assisted extraction; EtOH: ethanol; PLE: pressurized liquid extraction; SFE-CO2: supercritical carbon dioxide extraction. Different su-
perscript letters within the same line for individual in vitro antioxidant activity assessment assays indicate significant differences one way ANOVA and Tukey’s test, p < 0.05).

7
V. Kitrytė et al.
EtOH/H2O at the selected preferable parameters was further processed
by EAE using a mixture of cellulolytic enzymes Viscozyme L. The fol-
lowing previously optimized EAE parameters were used: E/S ratio 6%
v/w, 40 °C, pH 3.5, 7 h (Oktay Basegmez et al., 2017). The total EAE
yield was 29.5 g/100 g DW of PLE residue or 20.2 g/100 g, when re-
calculated for the initial plant material prior SFE-CO2 (Table 4), which
was ∼2-fold higher as compared to the control (no added enzyme). The
total amount of hexoses and dihexoses in EAE supernatants were
13.0 g GLU (glucose units) and 0.8 g MAU (maltose units) per 100 g of
PLE residue (Table S2, Supplementary Material). The content of ex-
tracted glucose from the Viscozyme-treated sample was by 94% higher
as compared to the enzyme-untreated plant material, while dis-
accharide maltose was not detected in the control sample at all. Pre-
viously, five different enzyme preparations were tested to assist cold-
pressing of hemp seed oil; enzyme pre-treatment enhanced oil recovery
from 6 to 23%, with the maximum value obtained for Viscozyme L
sample (Latif & Anwar, 2009). It was suggested that Viscozyme L, as a
multi-enzyme complex consisting of a wide range of carboxylases,
promotes hemp seed cell-wall breakdown and extractability of its
structural components to a higher extent, as compared to other enzyme
preparations tested.
3.4. In vitro antioxidant activity assessment of extracts and solid residues
As recommended for the representative in vitro antioxidant activity
evaluation of plant extracts (Prior, Wu, & Schaich, 2005), electron/hy-
drogen transfer-based assays (TPC, FRAP, DPPH%/ABTS%+ radical
scavenging) and ORAC assay, which applies biologically relevant ra-
dical source (Prior et al., 2003), were used. As the solid residues after
various steps of biorefining process may still retain active constituents,
their antioxidant potential was assessed by using the so-called
QUENCHER approach (Gökmen et al., 2009). Since TPC and TEAC
values of extracts obtained at the 4th step of biorefining (EAE) did not
depend on Viscozyme treatment only the data for enzyme-treated
sample are reported.
The following antioxidant capacity values per gram of extract and
starting material prior SFE-CO2 were obtained (Table 4): TPC:
6.4–124.7 mg GAE/g extract (1.3–23.5 mg GAE/g DW); TEACFRAP:
17.4–457.1 mg TE/g extract (3.2–86.2 mg TE/g DW); TEACDPPH:
3.0–72.5 mg TE/g extract (0.6–11.1 mg TE/g DW); TEACABTS:
45.9–1060 mg TE/g extract (9.3–169.4 mg TE/g DW); TEACORAC:
54.3–1088 mg TE/g extract (11.0–205.2 mg TE/g DW). In total, all ex-
tracts contributed to 38.0 mg of GAE and 110.4 (FRAP), 16.3 (DPPH%),
302.6 (ABTS%+) and 264.1 (ORAC) mg of TE per 1 g of hemp threshing
residue, while the impact of each individual fraction on the recovery of
antioxidants was decreasing in a following manner (in comparison to
the most active fraction): PLE-EtOH/H2O > SFE-CO2 (2–16-fold
lower) > PLE-Ac (4–17-fold lower) > EAE-Viscozyme (18–2-fold
lower). Previously DPPH% and ABTS%+ scavenging capacity was mea-
sured for ethanol, methanol, acetone and aqueous extracts from kernels
and hulls of C. sativa cultivars Bama and Yunma No 1; however, anti-
oxidant activity was expressed in extract IC50 values, which were in the
ranges of 0.01–4.6 mg/mL (Chen et al., 2012). N-trans-caffeoyltyramine
and cannabisin B extracted with 60% EtOH from hemp seed hulls were
reported in this study as important DPPH% scavengers and effective
inhibitors of LDL oxidation. Also, these compounds were more recently
obtained with 80% MeOH from hemp meal fractions of > 250 μm
particle sizes (Pojić et al., 2014).
It may be observed that antioxidant potential of solid residues re-
duced after each biorefining step (Table 4); for instance, the highest
TPC (35.5 mg GAE/g DW) and TEAC values (41.9–189.6 mg TE/g DW)
in QUENCHER assay in most cases were found for the raw plant ma-
terial prior extractions. Thus, up to 19% of the initial antioxidant ac-
tivity of plant material was lost after removing the lipophilic fraction by
means of SFE-CO2. PLE-Ac and PLE-EtOH/H2O, which were applied for
the higher polarity constituents, resulted in a further decrease of
8
Food Chemistry xxx (xxxx) xxx–xxx
antioxidant potential indicators of extraction residues by 18–50% and
19–71%, respectively. After the final EAE step of biorefining the anti-
oxidant potential of the residue decreased less remarkably, by 2–12%.
In summary, the multistep biorefining, which includes SFE-CO2, PLE
and EAE, reduced TPC, FRAP, DPPH%, ABTS%+ and ORAC values of C.
sativa threshing by-products by 92, 90, 98, 89 and 99%, respectively.
The results confirm that biorefining scheme was very effective for the
recovery of the main bioactive phytocannabinoids and antioxidants
from hemp threshing waste.
Pearson correlation coefficients (0.6125–0.9284 with p < 0.05)
between different antioxidant activity indices of non-polar (SFE-CO2)
and polar (PLE and EAE) extracts as well as the solid residues after
consecutive SFE-CO2, PLE-Ac, PLE-EtOH/H2O and EAE-Viscozyme
(Table S3, Supplementary Material) indicate the presence of a strong
positive correlation between TPC and antioxidant capacity indicators in
the following decreasing order: ABTS > FRAP > DPPH > ORAC.
3.5. Preliminary phytochemical characterisation of extracts
Phytochemical composition of the products isolated from C. sativa
threshing residue by the consecutive SFE-CO2, PLE-Ac, PLE-EtOH/H2O
and EAE was analysed by UPLC-QTOF-MS. Retention times, accurate
masses, molecular ion [M-H] formulas and peak areas (in arbitrary
units/g DW of starting plant material) of major extract constituents are
reported in Table 5. Concerning the variability of the data, the relative
standard deviations of peak areas were < 5%. Tentative characteriza-
tion of compounds was achieved via comparison of the experimental
accurate mass measurements and predicted molecular formula with
previously reported compounds in the literature and Metlin database. In
all cases the maximum allowed difference between the theoretical and
the experimental accurate mass did not exceed 3 ppm.
As given in Table 5, the SFE-CO2 extract is distinguished by the
presence of cannabinoids. Based on previous reports and experimental
data, peaks eluting at 6.8, 8.2 (m/z = 357.20) and 7.0 (m/
z = 359.2228), with deprotonated molecular formulas of C22H29O4 and
C22H31O4, could be ascribed to cannabidiolic, cannabichromenic and
cannabigerolic acids, respectively. Both cannabichromenic and canna-
binolic acids are the major phytocannabinoids in fiber-type hemps; they
are biosynthesized enzymatically via the oxydocyclization of pre-
dominantly cannabigerolic acid by their respective synthases (Flores-
Sanchez & Verpoorte, 2008a). CBDA is also a precursor for other major
non-psychotropic cannabinoid CBD, demonstrating a wide range of
bioactivities and pharmaceutical effects (Mechoulam et al., 2002). The
reports on CBDA properties are rather scarce; however, it was shown
that CBDA acts a selective inhibitor of cyclooxygenase-2 and MDA-MB-
231 breast cancer cell migration with promising therapeutic properties
in breast cancer treatments (Takeda et al., 2012).
The chromatographic profiles of PLE fractions showed that quanti-
fied phytocannabinoids were present in PLE-Ac at the 5.4–9.8-fold
lower concentrations than in SFE-CO2 extract. CBDA was the only
cannabinoid, which was still found in PLE-ETOH/H2O extract. These
results verify that the major portion of phytocannabinoids is recovered
at the 1st step of hemp threshing residue biorefining by SFE-CO2.
However, higher polarity constituents, mainly phenolic compounds and
flavonoid glycosides were found in PLE-ETOH/H2O extracts. It may be
assumed that these compounds are at least partially responsible for the
significant antioxidant activities of PLE-ETOH/H2O extract in all in vitro
assays (Table 4).
Regarding individual phenolic constituents, the peaks 15, 16 and 19
could be attributed to glycosylated forms of either kaempferol or lu-
teolin. The peaks eluting at 2.3 and 3.7 min with an accurate masses of
577.1563 and 445.0771 could be ascribed to apigenin rutinoside and
apigenin glucuronide, respectively. Vanhoenacker, Van Rompaey, De
Keukeleire, and Sandra (2002) reported that orientin, vitexin, luteolin-
7-O-β-d-glucuronide and apigenin-7-O-β-d-glucuronide were the major
flavonoids in the leaves and flowers of the THC-free hemp cultivars
V. Kitrytė et al. Food Chemistry xxx (xxxx) xxx–xxx

Table 5
Characterization of individual compounds by means of UPLC/ESI-QTOF analysis in non-polar (SFE-CO2) and polar (PLE and EAE) extracts, obtained from C. sativa threshing residues after
consecutive SFE-CO2 (46.5 MPa, 70 °C, 120 min), PLE-Ac (10.3 MPa, 100 °C, 45 min), PLE-EtOH/H2O (10.3 MPa, 100 °C, 45 min, EtOH/H2O 4:1 v/v), and EAE (E/S 6% v/w, 40 °C, pH
3.5, 7 h).

Peak No. UPLC/ESI-Q-TOF Peak area1, arbitrary units/g DW × 109 Compound2

RT (min) MS [M−H]− m/z Formula[M-H] SFE-CO2 PLE-Ac PLE-EtOH/H2O EAE-Viscozyme EAE-Control

1 0.3 341.1067 C10H9N14O – – 3 – – Not identified

2 0.3 387.1144 C13H23O13 – – 3 – – Not identified
3 0.3 439.0763 C17H11N8O7 – – 33 – – Not identified
4 0.4 193.0354 C6H9O7 – – – 19 2 Glucuronic acid
5 0.5 133.0142 C4H5O5 – – – 16 11 Malic Acid
6 0.8 191.0197 C6H7O7 – – – 33 24 Citric acid
7 2.0 311.0770 C14H15O8 – – 6 Not identified
8 2.0 341.0875 C15H17O9 – – 8 Caffeic acid glucoside
9 2.0 371.0982 C16H19O10 – – 7 Dihydro ferulic acid glucoside
10 2.0 431.1924 C20H31O10 – – 5 Not identified
11 2.0 623.1607 C23H11N24 – – 6 Not identified
12 2.1 609.1458 C27H29O16 – – 16 – – Rutin/ kaempferol-O-sophoroside
13 2.2 281.0663 C13H13O7 – – 6 Not identified
14 2.2 355.1029 C16H19O9 – – 3 Feruloylglucose
15 2.2 447.0926 C21H19O11 – – 4 Luteolin/ Kaempherol glucoside
16 2.2 593.1561 C27H29O15 – – 16 – – Luteolin/kaempherol-rutinoside
17 2.2 639.1558 C28H32O17 – – 2 Flavonoid glycoside
18 2.3 577.1563 C27H29O14 – – 7 – – Apigenin rutinoside
19 2.4 461.0724 C21H17O12 – – 46 – – Luteolin/kaempherol glucuronide
20 2.6 295.0459 C13H11O8 – – – 5 4 Succinic acid
21 2.7 445.0771 C21H17O11 – – 32 – – Apigenin glucuronide
22 3.4 459.0923 C22H19O11 – – 6 – – Flavonoid glycoside
23 4.9 289.1437 C17H21O4 5 – – Acetyl cannabispirol
24 4.9 373.2009 C22H29O5 3 – – Resolvin
25 5.6 297.1541 C9H17N10O2 281 50 – – – Not identified
26 6.0 311.1685 C9H23N6O6 661 123 – – – Not identified
27 6.6 325.1841 C10H25N6O6 265 51 – – – Not identified
28 6.8 357.2067 C22H29O4 619 63 17 – – Cannabidiolic acid
29 7.0 359.2228 C22H31O4 121 31 – – – Cannabigerolic acid
30 8.3 357.2056 C22H29O4 91 17 – – – Cannabichromene acid
31 8.4 367.2643 C25H35O2 107 18 – – – Tocotrienol

1
: Expressed as arbitrary units/g DW of starting plant material × 109, taking into account yields (g/100 g DW: 8.30 for SFE-CO2; 4.33 for PLE-Ac; 18.86 for PLE-EtOH/H2O; 20.2 for EAE-
Viscozyme; 9.4 for EAE-Control), sample concentration (1 mg/mL) and injection volume (1 μL); 2: tentatively identified. Ac: acetone; EAE: enzyme-assisted extraction; EtOH: ethanol;
PLE: pressurized liquid extraction; SFE-CO2: supercritical carbon dioxide extraction.

Felina and Futura. Similar phenolic composition was shown by Flores-
Sanchez and Verpoorte (2008b) for Kompoliti and Fasamo fiber-type
hemps. The peak eluting at 2.1 min with a mass of 609.1461 and a
deprotonated molecular formula of C27H29O16 could correspond to ei-
ther kaempferol-O-sophoroside or quercetin rutinoside. The presence of
kaempferol-O-sophoroside in the pollen of C. sativa was discussed pre-
viously (Ross et al., 2005). Andre et al. (2016) in their recent review
reported that approximately 20 flavonoids (mainly O-glycosides of
apigenin, luteolin and kaempferol) can be present in the plants of the
genus Cannabis, some of them with a well-established radical scaven-
ging capacity and chelating properties both in vitro and in vivo and
plausible cancer chemopreventive properties (Heim,
Tagliaferro, & Bobilya, 2002; Chen & Chen, 2013). It should be noted
that the concentrations of various phytocannabinoids and flavonoids
largely depend on many factors such as hemp genotype, vegetation
period, anatomical part and plant tissue type, cultivation, harvesting,
storage and processing conditions (Flores-Sanchez & Verpoorte, 2008a,
2008b).
Glucuronic, malic, citric and succinic acids were tentatively iden-
tified in the soluble EAE supernatants by matching molecular ion for-
mulas of C6H9O7 (193.0354 m/z), C4H5O5 (133.0142 m/z), C6H7O7
(191.0197 m/z) and C13H11O8 (295.0459). Although the same organic
acids were found both in the enzyme-treated and control samples, a
significant peak area increase of those compounds was obtained after
the treatment with Viscozyme (Table 5).
4. General conclusion
In total, 51.7 g of extractable substances were recovered from 100 g
of hemp threshing residues. High pressure extraction techniques con-
tributed to the major portion (61%) of all extracted constituents, while
enzyme-assisted extraction gave the remaining 39%. Therefore, under
the optimized SFE-CO2 (46.5 MPa, 70 °C, 120 min), 8.3 g/100 g DW of
lipophilic fraction was obtained, recovering > 93% of initial CBD and
CBDA amount from plant material. PLE-Ac (100 °C, 45 min) and PLE-
EtOH/H2O (100 °C, 45 min, EtOH/H2O 4:1 v/v) yielded respectively
4.3 and 18.9 g/100 g DW of flavonoid-containing polar fractions.
Further PLE residue treatment with cellulolytic enzyme Viscozyme L
additionally released 20.2 g of hydrophilic constituents, increasing the
release of mono- and disaccharides up to 94%. Antioxidant capacity of
non-polar and polar fractions was in the range of 1.3–23.5 mg GAE/
g DW and 0.6–205.2 mg TE/g DW, with the highest activities of PLE-
EtOH/H2O extract. The combined SFE-CO2, PLE and EAE reduced in-
itial total phenolic content, reducing power and radical scavenging
capacity of plant material by 90–99%. Thus, it can be concluded that
the suggested biorefining scheme is an efficient way to recover the
major portion of non-polar and polar bioactive constituents with in vitro
antioxidant properties from hemp threshing residues.
Acknowledgement

This research was funded by JSC Agropro, grant no. 8743.

9
V. Kitrytė et al. Food Chemistry xxx (xxxx) xxx–xxx

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