Biorefining of industrial hemp (Cannabis sativa L.) threshing residues into cannabinoid and antioxidant fractions by supercritical carbon dioxide, pressurized liquid and enzyme-assisted extractions

Biorefining of industrial hemp (Cannabis sativa L.) threshing residues into cannabinoid and antioxidant fractions by supercritical carbon dioxide, pressurized liquid and enzyme-assisted extractions

Food Chemistry xxx (xxxx) xxx–xxx Contents lists available at ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodchem Biore...

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Food Chemistry xxx (xxxx) xxx–xxx

Contents lists available at ScienceDirect

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: Hemp threshing residues High-pressure extraction Enzyme-assisted extraction Cannabinoids Antioxidants

C. sativa threshing residues were biorefined by consecutive supercritical carbon dioxide (SFE-CO2) pressurised liquid (PLE) and enzyme-assisted extractions (EAE). SFE-CO2 at optimised parameters yielded 8.3 g/100 g of lipophilic fraction containing 0.2 and 2.2 g of cannabidiol and cannabidiolic acid per 100 g of threshing residues, respectively. The recovery of cannabinoids from plant material was > 93%. PLE gave 4.3 and 18.9 g/100 g of flavonoid-containing polar extracts, while EAE added 20.2% (w/w) of water-soluble constituents and increased 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 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 preferable 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 obtained mainly via cold pressing or hydrocarbon solvent (Latif & Anwar, 2009) and, more recently, supercritical carbon dioxide (SFE-CO2) extraction (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 (FloresSanchez & Verpoorte, 2008a, 2008b; Andre, Hausman, & Guerriero, 2016). A large number of studies demonstrated health promoting and



medicinal properties of phytocannabinoids. Among them, Δ9-tetrahydrocannabinol (Δ9-THC) is a well-known natural psychotropic compound; therefore, today only the approved cultivars of C. sativa accumulating 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 modulating 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 compounds 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 important nutrients, e.g. phytochemical antioxidants; it was recently demonstrated for different hemp seed meal fractions (Pojić et al., 2014), inflorescences (Da Porto, Decorti, & Natolino, 2014), kernels and seed

Corresponding author. E-mail address: [email protected] (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

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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 Pressure, MPa

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Optimal

10 30 30 10 30 10 30 30 10 30 50 10 50 30 30 50 30 50 30 50 conditions: 46.5

SFE-CO2 extract yield

CBD yield

Temperature, °C

Time, min

g/100 g DW1

mg/g extract

35 70 52.5 52.5 52.5 70 52.5 52.5 70 52.5 52.5 35 70 52.5 35 70 52.5 35 52.5 35

60 90 90 90 60 120 90 90 60 90 90 120 60 120 90 120 90 60 90 120

1.76 ± 0.03b 7.93 ± 0.08cd 7.62 ± 0.09cd 0.63 ± 0.00a 7.65 ± 0.08cd 0.27 ± 0.01a 7.90 ± 0.13cd 7.61 ± 0.05cd < 0.00a 7.56 ± 0.00cd 8.25 ± 0.19d 2.51 ± 0.02b 9.63 ± 0.26e 7.80 ± 0.17cd 7.30 ± 0.13c 10.36 ± 0.31e 7.50 ± 0.16cd 7.39 ± 0.21cd 7.63 ± 0.05cd 7.51 ± 0.04cd

64.18 25.75 19.13 –nd 18.31 –nd 18.46 21.00 –nd 20.89 21.41 44.22 24.15 20.16 23.80 21.52 20.89 19.42 20.07 18.28

70

120

8.30 ± 0.01d

24.72 ± 0.31cd

± 2.98e ± 1.31d ± 0.73ab ± 0.72a ± 3.61ab ± 0.72abcd ± ± ± ± ± ± ± ± ± ± ±

0.17abcd 0.21 abcd 1.94e 0.89bcd 0.96abcd 0.54bcd 0.84abcd 0.17abcd 0.89abc 0.60abc 0.68a

CBDA yield g/100 g DW1 0.11 0.20 0.15 –nd 0.14 –nd 0.15 0.16 –nd 0.16 0.18 0.11 0.23 0.16 0.17 0.22 0.16 0.14 0.15 0.14

± 0.01ab ± 0.00ef ± 0.01bcd ± 0.01abc ± 0.03bcd ± 0.01cd ± ± ± ± ± ± ± ± ± ± ±

0.00c 0.00de 0.01a 0.01f 0.01cd 0.00de 0.01f 0.00cd 0.01abcd 0.01cd 0.01abc

0.21 ± 0.00ef

mg/g extract 157.6 185.6 232.5 –nd 236.2 –nd 231.6 233.3 –nd 237.8 231.5 203.8 204.1 221.1 236.2 223.7 239.3 179.9 230.4 209.1

± 6.0a ± 8.3ab ± 0.5bcd ± 14.0bcd ± 9.9bcd ± 1.5bcd ± ± ± ± ± ± ± ± ± ± ±

3.1cd 2.4bcd 7.9b 7.3b 12.5bc 12.1bcd 14.1bc 1.0cd 9.2ab 9.9bc 7.6b

261.4 ± 2.2d

g/100 g DW1 0.28 1.47 1.77 –nd 1.81 –nd 1.83 1.78 –nd 1.80 1.91 0.51 1.97 1.73 1.72 2.32 1.80 1.33 1.76 1.57

± 0.01a ± 0.06bc ± 0.00de ± 0.11de ± 0.08de ± 0.01de ± ± ± ± ± ± ± ± ± ± ±

0.02de 0.02ef 0.02a 0.07ef 0.10cde 0.09cde 0.15f 0.01de 0.07b 0.08de 0.06bcd

2.17 ± 0.02f

: 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).

1

2. Materials and methods

hulls (Chen et al., 2012). Therefore, there is an obvious scientific and industrial interest in utilising such by-products or waste more efficiently. Soluble bioactive substances are usually isolated by the conventional solvent extraction, however such method, as a rule, may recover 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 technologies has become very attractive. For instance, SFE-CO2 has been recognised as a good alternative to soli-liquid extraction with hydrocarbon solvents (e.g. hexane, petrol ether) for the isolation of lipophilic compounds. SFE-CO2 does not require the removal of toxic solvent residues 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-assisted 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 technology for the isolation of valuable phytocannabinoids and antioxidant fractions from industrial hemp threshing residues via consecutive application 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 byproducts into novel bioactive ingredients with functional food, nutraceutical and pharmaceutical applications.

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 material 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 controlled manually by the micro-metering valve and kept at 2–3 SL/min during all experiments. Other important extraction parameters, pressure (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 collected in the glass bottles, weighed ( ± 0.001 g) and stored at −20 °C. 2

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The solid residue after SFE-CO2 was collected and kept in a dry, wellventilated place prior to the analysis.

hexoses and maltose for dihexoses) were used for quantification (Supplementary Material).

2.3. Pressurised liquid extraction (PLE)

2.7. In vitro antioxidant activity assessment

PLE was performed from hemp material residue remaining after SFE-CO2 as described by Povilaitis et al. (2015) in an accelerated solvent 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, PLEEtOH/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 residues, 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.

Total phenolic content (TPC), ferric reducing antioxidant power (FRAP), ABTS%+/DPPH% scavenging capacities and oxygen radical absorbance 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, Rochester, NY) and FLUOstar Omega reader (BMG Labtech, Offenburg, Germany), respectively. Antioxidant capacity of solid substances was determined 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, respectively 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 comparing the retention times with those of the standards, accurate masses, using literature sources and free chemical databases (Supplementary Material).

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 cellulolytic enzyme mixture Viscozyme® L was added to reach the enzyme/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 centrifugation 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 fractions) were freeze-dried (−50 °C, 0.5 mbar), weighed ( ± 0.001 g) and stored at −20 °C.

2.9. Statistical analysis Extraction experiments and phytochemical composition analysis were performed in duplicate; cannabinoid and sugar content – in triplicate; 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).

2.5. Cannabinoid analysis by HPLC-DAD

3. Results and discussion

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).

Multistep extraction scheme (Fig. S1, Supplementary Material) was designed and tested for biorefining of harvesting by-products of industrial hemp, which consisted of a mixture of leaves, floral bracts, flower fragments and immature seeds, in order to evaluate the possibilities 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 separating 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, antioxidant 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.

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

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yield, g/100 g DW) and RFIII (CBDA yield, g/100 g DW) is summarised by the analysis of variance (ANOVA) presented in Table 2. The adequacy of the model, as it may be judged from the total determination coefficient R2 (RFI: 0.9979; RFII: 0.9756; RFIII: 0.9829), indicates reasonable fit of the models to the experimental data. Model analysis also showed good agreement between the adjusted and predicted coefficients of determination (R2): 0.9960 and 0.9763 (RFI); 0.9537 and 0.8247 (RFII); 0.9675 and 0.8563 (RFIII), respectively. Calculated adequate precision (Press) values of 71.693 (RFI), 23.885 (RFII) and 24.314 (RFIII), which compare the range of the predicted values to the average prediction error at the experimental design points (desirable signal to noise ratio > 4), indicate that the signal is adequate and the model can be used to navigate the design space. Quadratic regression model analysis for SFE-CO2 yield (Table 2) showed that the model was significant according to the Student test (p < 0.05) with a calculated Fvalue of 525.26; consequently, ‘lack of fit’ was not significant relative to the pure error (p = 0.1053). The results obtained showed that P, τ, T, PT interaction and second-order term of P2 were significant on the total SFE-CO2 extract yield (RFI) in the following order: P (p < 0.0001) > P2 (p < 0.0001) > PT (p < 0.0001) > τ (p = 0.0105) > T (p = 0.0232). For RFII and RFIII the model itself with F-values of 44.47 and 63.82 for CBD and CBDA yield, respectively, and the factors P, PT and P2 were significant, as for SFE-CO2 extract yield (RFI). However, in this case significant ‘lack of fit’ values were obtained for RFII (CBD yield; p < 0.0125) and RFIII (CBDA yield; p < 0.0002). It may be assumed that the changes of CO2 properties at different P-T levels have more pronounced effect on the cannabinoid solubility and extraction efficiency, as compared to the total SFE-CO2 yield; this may partially explain the significant ‘lack of fit’ of the model in the case of RFII and RFIII. For example, remarkably lower yields of the total extract, as well as the yields of CBD and CBDA were obtained at the lowest applied P = 10 MPa and T = 35 °C; after increasing T > 50 °C phytocannabinoids were not found in the extracts at all (Table 1). It may be explained by the remarkable decrease of CO2 density at lower pressure levels by increasing temperature, resulting in a weaker diffusivity and solvating power (Kryževičiūtė et al., 2016). At P > 30 MPa, the increase of T had positive effect both on the total SFECO2 yield and the yields of phytocannabinoids, reaching maximum values at 40–50 MPa and 70 °C, most likely due to the so-called “enhanced solubility effect”, which occurs when the increasing vapour pressure of solute outweighs the decreasing solvating power of CO2 (Kraujalis & Venskutonis, 2013; Da Porto et al., 2014). Similar observations regarding the varying molar solubility of non-psychotropic CBD and cannabigerol as well as psychoactive THC and cannabinol in supercritical CO2 at different temperature (313–334 K) and pressure (11.3–21.1 MPa) ranges were previously reported by Perrotin-Brunel et al., 2010. Second order polynomial regression model, describing relationship between dependent and independent variables (P, T, τ), is given in the equations (1−3). Predicted values were calculated using a second order polynomial equation and compared with experimental values in Fig. S2 (Supplementary Material).

Table 2 Analysis of variance of the regression parameters for response surface quadratic model for SFE-CO2 extract, CBD and CBDA yields (g/100 g DW) from C. sativa threshing residues. Source

SS

RF I: SFE-CO2 extract Model Pressure (P, MPa) Temperature (T, °C) Time (τ, min) PT Pτ Tτ P2 T2 τ2 Residual Lack of fit Pure error Corrected Total

yield (g/100 g DW): 195.26 9 21.70 144.17 1 144.17 0.30 1 0.30 0.41 10.33 3.613 · 10−3 2.113 · 10−3 25.47 0.048 0.16 0.41 0.32 0.095 195.68

RF II: CBD yield (g/100 g DW): Model 0.082 Pressure (P, MPa) 0.048 Temperature (T, 3.610 · 10−5 °C) Time (τ, min) 4.000 · 10−7 PT 0.020 Pτ 2.813 · 10−5 Tτ 1.250 · 10−7 P2 0.011 T2 3.555 · 10−3 τ2 5.682 · 10−5 Residual 2.045 · 10−3 Lack of fit 1.857 · 10−3 Pure error 1.873 · 10−4 Corrected Total 0.084 RF III: CBDA yield (g/100 g DW): Model 10.47 Pressure (P, MPa) 6.90 Temperature (T, 0.011 °C) Time (τ, min) 0.056 PT 0.59 Pτ 0.016 Tτ 1.953 · 10−3 P2 1.51 T2 0.026 τ2 0.014 Residual 0.18 Lack of fit 0.18 Pure error 3.176 · 10−3 Corrected Total 10.65

df

MS

F-Value

p-Value

525.26 3490.40 7.16

< 0.0001* < 0.0001* 0.0232*

1 1 1 1 1 1 1 10 5 5 19

0.41 10.33 3.613 · 10−3 2.113 · 10−3 25.47 0.048 0.16 0.041 0.064 0.019

9.88 250.05 0.087 0.051 616.57 1.16 3.89

0.0105* < 0.0001* 0.7735** 0.8256** < 0.0001* 0.3074** 0.0767**

3.35

0.1053**

9 1 1

9.092 · 10−3 0.048 3.610 · 10−5

44.47 232.86 0.18

< 0.0001* < 0.0001* 0.6832**

1 1 1 1 1 1 1 10 5 5 19

4.000 · 10−7 0.020 2.813 · 10−5 1.250 · 10−7 0.011 3.555 · 10−3 5.682 · 10−5 2.045 · 10−4 3.715 · 10−4 3.747 · 10−5

1.956 · 10−3 97.33 0.14 6.114 · 10−4 56.03 17.39 0.28

0.9656** < 0.0001* 0.7185** 0.9808** < 0.0001* 0.0019* 0.6096**

9.91

0.0125*

9 1 1

1.16 6.90 0.011

63.82 378.38 0.63

< 0.0001* < 0.0001* 0.4456**

1 1 1 1 1 1 1 10 5 5 19

0.056 0.59 0.016 1.953 · 10−3 1.51 0.026 0.014 0.018 0.036 6.352 · 10−4

3.05 32.27 0.88 0.11 82.92 1.44 0.75

0.1111** 0.0002* 0.3693** 0.7501** < 0.0001* 0.2574** 0.4053**

56.38

0.0002*

* : significant; **: not significant; df: degree of freedom; F: Fisher value.; MS: mean square; RF: response factor; SS: sum of square.

3.1. Optimization of SFE-CO2 parameters for hemp lipophilic fraction and its main phytocannabinoids SFE-CO2 has been proved as an effective green technology for the isolation of high added value products from botanicals. Therefore, it was selected as a 1st step for valorising hemp threshing residues. So far as the effectiveness of SFE-CO2 depends on several process parameters, 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 the two most important lipophilic bioactive constituents, CBD and CBDA. The model selected 20 experimental sets, their characteristics and results are listed in Table 1. It may be observed that process parameters had remarkable effects on the selected responses: the total 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 157.6–239.3 mg/g of CBDA (0.3–2.3 g/100 g DW). Model evaluation for RFI (extract yield, g/100 g DW), RFII (CBD

YieldSFE − CO2 (RFI ) = 7.58 + 3.80 × P + 0.17 × T + 0.20 × τ + 1.14 × (PT )−0.021 × (Pτ ) + 0.016 × (Tτ )−3.04 × (P 2) + 0.13 × (T 2) + 0.24 × (τ 2)

(1)

YieldCBD (RFII ) = 0.15 + 0.069 × P−0.0019 × T −0.0002 × τ + 0.05 × (PT )−0.001875 × (Pτ ) − 0.0000125 × (Tτ )−0.065 × (P 2) + 0.036 × (T 2) + 0.004545 × (τ 2) (2)

YieldCBDA (RFIII ) = 1.75 + 0.38 × P + 0.034 × T + 0.075 × τ + 0.27 × (PT ) + 0.045 × (Pτ ) − 0.016 × (Tτ )−0.74 × (P 2)−0.098 × (T 2) + 0.071 × (τ 2) 4

(3)

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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.

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 phytocannabinoids are obtained during 60 min; however, further increase of SFE-CO2 extract, CBD and CBDA yields by 8%, 14% and 9%, respectively, was recorded after prolonging extraction time to 120 min at

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 5–8fold 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

Fig. 1. (continued)

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Fig. 1. (continued)

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 cannabinoids and their separation from other hemp fractions. The differences of the solubility of four phytocannabinoids in supercritical CO2 were reported to be dependent on their chemical structures, volatility and melting points (Perrotin-Brunel et al., 2010).

P = 30 MPa within T interval of 52.5 and 70 °C. Considering all responses, the following optimal conditions for obtaining 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 determination of optimal parameters in the selected range of variables. For comparison, previously reported CBD content in commercially available 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 SFECO2 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

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, MartinDiana, & 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 acetonesoluble 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 efficiency of PLE at the longest applied time (75 min) was further tested at 6

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reaction pathway (Hossain et al., 2011). Small increase in the yield (by 11%) and TPC (by 16%) was obtained at 100 °C and 75 min of extraction, while at 70 °C these values were by 10 and 17% lower, as compared to 45 min PLE. Based on these results, 100 °C and of 45 min 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 4.3 g/100 g DW of starting material prior SFE-CO2; Table 4) with the TPC recovery of 5.5 mg GAE/g DW. PLE-Ac yields at various parameters (Table 2) are in the ranges of the previously reported yields (Chen et al., 2012) obtained with 50–100% acetone from hemp seed residues of C. sativa cultivars Bama (kernels: 0.5–8.4%; hulls: 0.2–2.4%) and Yunma No. 1 (kernels: 0.5–9.7%; hulls: 0.3–2.5%). The residues remaining after PLE-Ac at preferable conditions were further extracted by PLE-EtOH/H2O, which was the 3rd step in biorefining hemp threshing residue. Three solvents were prepared by mixing ethanol and distilled water at the ratios of 1:4 (25% EtOH), 1:1 (50% EtOH) and 4:1 (75% EtOH). Chen et al. (2012) showed that EtOH/H2O mixtures produce remarkably higher (> 5-fold) extraction yields from hemp seed kernel and hulls. In our study (Table 3), extract yields and TPC were similar both for 50 and 75% EtOH; they were on average 21.7 g/100 g DW and 123.9 mg GAE/g (26.9 mg GAE/g DW). When EtOH concentration was 25%, the total extract yield was by 35–41% higher; however, TPC in such extract was by 41% lower than in case of other applied EtOH concentrations. Considering that TPC recovery from plant DW was almost similar, it is evident that at the highest H2O concentration in the mixture, the compounds reacting with Folin-Ciocalteus reagent (TPC) are diluted with other, neutral water soluble substances. Therefore, the highest EtOH/H2O ratio (4/1 v/v) was selected for PLE-EtOH/H2O at 100 °C for 45 min, amounting 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; Table 4) with TPC recovery of 26.9 mg GAE/g DW.

Table 3 PLE-Ac and PLE-EtOH/H2O optimization and values of observed responses for the extraction of polar constituents from C. sativa threshing residues after SFE-CO2 (46.5 MPa, 70 °C, 120 min). PLE parameters

PLE extract yield

TPC

g/100 g DW

mg GAE/g extract

mg GAE/g DW

1

PLE-Ac : 15 min (3 × 5 min) 30 °C 70 °C 100 °C 130 °C 45 min (3 × 15 min) 30 °C 70 °C 100 °C 130 °C 75 min (3 × 25 min) 70 °C 100 °C

0.66 1.79 3.45 6.61

± ± ± ±

0.13a 0.25b 0.17c 0.18e

92.77 123.0 131.1 152.2

± ± ± ±

3.31a 0.8bc 10.5c 4.1d

0.61 ± 0.02a 2.20 ± 0.01b 4.45 ± 0.36d 10.05 ± 0.27g

1.20 3.36 4.73 7.74

± ± ± ±

0.05ab 0.16ce 0.14d 0.38f

100.8 120.4 115.8 148.3

± ± ± ±

2.5a 5.5bc 2.2bc 5.4d

1.20 ± 0.03a 3.98 ± 0.18d 5.48 ± 0.10e 11.40 ± 0.42h

3.00 ± 0.12c 5.26 ± 0.39d

PLE-EtOH/H2O2: 100 °C, 45 min (3 × 15) EtOH/H2O, 1:4 v/v 35.05 ± 0.78b EtOH/H2O, 1:1 v/v 20.71 ± 0.44a EtOH/H2O, 4:1 v/v 21.58 ± 0.59a

97.16 ± 6.45a 120.6 ± 8.6bc

2.90 ± 0.20c 6.34 ± 0.45f

73.04 ± 1.23a 125.0 ± 3.7b 124.7 ± 2.5b

25.61 ± 0.43a 25.88 ± 0.76ab 26.90 ± 0.53b

1

: PLE-Ac extract yields and TPC values were expressed g/100 g DW and mg GAE/g DW of sample after optimized SFE-CO2 (46.5 MPa, 70 °C, 120 min); 2: PLE-EtOH/H2O extract yields and TPC values were expressed g/100 g DW and mg GAE/g DW of sample after optimized PLE-Ac (10.3 MPa, 100 °C, 45 min); Ac: acetone EtOH: ethanol; PLE: pressurized liquid extraction; TPC: total phenolic content. Different superscript letters within the same column of individual PLE-Ac or PLE-EtOH/H2O treatments indicate significant differences (one way ANOVA and Tukey’s test, p < 0.05).

70 and 100 °C, since the yields at 30 °C were rather small, while at 130 °C the extract acquired yellow colour and sweet odour notes already after 15 min of PLE; it may be attributed to the degradation and/ or unfavourable chemical interactions between various endogenous extract constituents at the elevated temperatures, e.g. via the Maillard

3.3. Enzyme assisted extraction of PLE residues (EAE-Viscozyme) 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 antioxidant capacity

Extracts

Crude plant material and solid residues after extraction

SFE-CO2

PLE-Ac

PLE-EtOH/H2O

EAE-Viscozyme

Prior SFE-CO2

After SFE-CO2

After PLE-Ac

After PLEEtOH/H2O

After EAEViscozyme

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 mg/g DW1 8.17 ± 0.05d

115.8 ± 2.2b 5.02 ± 0.09c

124.7 ± 2.5c 23.52 ± 0.47f

6.38 ± 0.21a 1.29 ± 0.04a

–na 35.49 ± 1.37h

–na 28.91 ± 0.54g

–na 11.15 ± 0.47e

–na 4.18 ± 0.32bc

–na 2.95 ± 0.11b

TEACFRAP, mg TE/g: mg/g extract 71.49 ± 0.80b mg/g DW1 5.44 ± 0.06a

352.2 ± 15.8c 15.25 ± 0.56c

457.1 ± 10.9d 86.20 ± 2.77g

17.40 ± 1.59a 3.52 ± 0.27a

–na 80.12 ± 0.94f

–na 68.39 ± 2.87e

–na 53.84 ± 2.86d

–na 10.69 ± 0.38b

–na 8.27 ± 0.38ab

TEACDPPH, mg TE/g: mg/g extract 72.51 ± 1.39e mg/g DW1 2.52 ± 0.11c

48.31 ± 1.62c 2.09 ± 0.07bc

58.99 ± 2.00d 11.13 ± 0.34e

2.99 ± 0.08a 0.60 ± 0.02a

–na 41.88 ± 0.96h

–na 38.11 ± 0.48g

–na 29.21 ± 1.33f

–na 4.28 ± 0.23d

–na 1.02 ± 0.05ab

TEACABTS, mg TE/g: mg/g extract 1025 ± 13c mg/g DW1 77.99 ± 1.01d

1060 ± 89c 45.89 ± 3.16c

898.3 ± 67.1b 169.4 ± 12.2f

45.91 ± 1.25a 9.27 ± 1.24a

–na 189.6 ± 9.3g

–na 199.9 ± 10.4g

–na 110.7 ± 5.4e

–na 24.84 ± 0.37b

–na 20.12 ± 0.39b

TEACORAC, mg TE/g mg/g extract 469.5 ± 21.9c mg/g DW1 35.68 ± 1.65b

282.4 ± 18.9b 12.23 ± 0.82ab

1088 ± 109d 205.2 ± 20.6d

54.30 ± 3.36a 10.97 ± 0.68ab

–na 174.8 ± 21.4cd

–na 146.3 ± 13.8c

–na 183.5 ± 21.3d

–na 21.24 ± 1.91b

–na 2.36 ± 0.13a

Yield g/100 g DW1

: 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 superscript 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).

1

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antioxidant potential indicators of extraction residues by 18–50% and 19–71%, respectively. After the final EAE step of biorefining the antioxidant 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.

EtOH/H2O at the selected preferable parameters was further processed by EAE using a mixture of cellulolytic enzymes Viscozyme L. The following 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 recalculated 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 extracted glucose from the Viscozyme-treated sample was by 94% higher as compared to the enzyme-untreated plant material, while disaccharide maltose was not detected in the control sample at all. Previously, five different enzyme preparations were tested to assist coldpressing 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.

3.5. 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 characterization 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 cannabinolic acids are the major phytocannabinoids in fiber-type hemps; they are biosynthesized enzymatically via the oxydocyclization of predominantly cannabigerolic acid by their respective synthases (FloresSanchez & 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-MB231 breast cancer cell migration with promising therapeutic properties in breast cancer treatments (Takeda et al., 2012). The chromatographic profiles of PLE fractions showed that quantified 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 luteolin. 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, luteolin7-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

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/hydrogen transfer-based assays (TPC, FRAP, DPPH%/ABTS%+ radical scavenging) and ORAC assay, which applies biologically relevant radical 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 extracts 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 measured for ethanol, methanol, acetone and aqueous extracts from kernels and hulls of C. sativa cultivars Bama and Yunma No 1; however, antioxidant 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 reduced 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 material prior extractions. Thus, up to 19% of the initial antioxidant activity 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

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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.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

Peak area1, arbitrary units/g DW × 109

UPLC/ESI-Q-TOF

Compound2

RT (min)

MS [M−H]− m/z

Formula[M-H]

SFE-CO2

PLE-Ac

PLE-EtOH/H2O

EAE-Viscozyme

EAE-Control

0.3 0.3 0.3 0.4 0.5 0.8 2.0 2.0 2.0 2.0 2.0 2.1 2.2 2.2 2.2 2.2 2.2 2.3 2.4 2.6 2.7 3.4 4.9 4.9 5.6 6.0 6.6 6.8 7.0 8.3 8.4

341.1067 387.1144 439.0763 193.0354 133.0142 191.0197 311.0770 341.0875 371.0982 431.1924 623.1607 609.1458 281.0663 355.1029 447.0926 593.1561 639.1558 577.1563 461.0724 295.0459 445.0771 459.0923 289.1437 373.2009 297.1541 311.1685 325.1841 357.2067 359.2228 357.2056 367.2643

C10H9N14O C13H23O13 C17H11N8O7 C6H9O7 C4H5O5 C6H7O7 C14H15O8 C15H17O9 C16H19O10 C20H31O10 C23H11N24 C27H29O16 C13H13O7 C16H19O9 C21H19O11 C27H29O15 C28H32O17 C27H29O14 C21H17O12 C13H11O8 C21H17O11 C22H19O11 C17H21O4 C22H29O5 C9H17N10O2 C9H23N6O6 C10H25N6O6 C22H29O4 C22H31O4 C22H29O4 C25H35O2

– – – – – – – – – – – – – – – – – – – – – – 5 3 281 661 265 619 121 91 107

– – – – – – – – – – – – – – – – – – – – – – – – 50 123 51 63 31 17 18

3 3 33 – – – 6 8 7 5 6 16 6 3 4 16 2 7 46 – 32 6 – – – – – 17 – – –

– – – 19 16 33

– – – 2 11 24









– – 5 – –

– – 4 – –

– – – – – – –

– – – – – – –

Not identified Not identified Not identified Glucuronic acid Malic Acid Citric acid Not identified Caffeic acid glucoside Dihydro ferulic acid glucoside Not identified Not identified Rutin/ kaempferol-O-sophoroside Not identified Feruloylglucose Luteolin/ Kaempherol glucoside Luteolin/kaempherol-rutinoside Flavonoid glycoside Apigenin rutinoside Luteolin/kaempherol glucuronide Succinic acid Apigenin glucuronide Flavonoid glycoside Acetyl cannabispirol Resolvin Not identified Not identified Not identified Cannabidiolic acid Cannabigerolic acid Cannabichromene acid 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 EAEViscozyme; 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.

4. General conclusion

Felina and Futura. Similar phenolic composition was shown by FloresSanchez 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 either kaempferol-O-sophoroside or quercetin rutinoside. The presence of kaempferol-O-sophoroside in the pollen of C. sativa was discussed previously (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 scavenging 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 identified in the soluble EAE supernatants by matching molecular ion formulas 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).

In total, 51.7 g of extractable substances were recovered from 100 g of hemp threshing residues. High pressure extraction techniques contributed 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 PLEEtOH/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 PLEEtOH/H2O extract. The combined SFE-CO2, PLE and EAE reduced initial 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.

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