Keywords

7.1 Introduction

The development of green extraction processes is aimed at reducing the use of organic solvents and the energy consumption through reduced reaction and processing times that will produce safe products of good quality and purity (Chemat et al. 2019). Chemat et al. (2019) have described the six principles for identifying the green extraction process (Chemat et al. 2012) and developed accordingly the good practices guidelines for each of these principles. The principles described by Chemat et al. (2012, 2019) are (1) “innovation by selection of varieties and use of renewable plant resources”, (2) “use of alternative solvents and principally water or agro-solvents”, (3) “reduction of energy consumption by energy recovery and using innovative technologies”, (4) “production of co-products instead of waste towards bio-refinery concepts”, (5) “reduction of unit operations number and development of safe, robust and controlled processes” and (6) “aim for green extract with green values and non-denatured and biodegradable extract without contaminants”. The application of supercritical carbon dioxide (SC-CO2) , a “generally recognized green solvent” (GRAS) (Rovetto and Aieta 2017; Chemat et al. 2019), for the use of the extraction of biomolecules fulfils the requirements under principle 2 and is the most common used green solvent for supercritical fluid (SF) extractions (Stuart et al. 1996; Lang and Wai 2001; Rovetto and Aieta 2017). This is also evident from the numerous review articles published in the past 20 years on the use of SFs, which have provided numerous reports on the SC-CO2 extraction applications (Table 7.1). This phenomena is attributed to the properties of the carbon dioxide as being non-flammable, non-toxic, cheap and non-corrosive (Lang and Wai 2001; Huang et al. 2012) with the SC-CO2 extraction process being highly selective, obtaining solvent free products with no development of co-products (Knez et al. 2019). Extracts obtained from a SF extraction have been commonly found to be of greater quality when compared to other methods (Fornari et al. 2012a). A wide array of published reviews discuss the supercritical extraction of bioactives (Table 7.1), which are essentially biomolecules possessing specific biological activities or functions (Fig. 7.1) with a great range of applications in industry (da Silva et al. 2016). De Melo et al. (2014) have described reported SF extractions from vegetable matrices of about 600 essays for the period 2000–2013, including modelling, operating conditions, scale-up and an economic assessment. A review by Khaw et al. published in 2017 focusses on SF extraction of bioactives from different natural sources. The review, including other green extraction methods, provides insights on the SC-CO2 extraction of bioactives and operating conditions from about 40 plant species. A summary of the reported SC-CO2 extraction parameters of bioactives, among other newer methods such as “subcritical water extraction”, “ultrasound-assisted extraction” and “microwave-assisted extraction”, specifically from marine macroalgae was reported by Cikoš et al. (2018). Gallego et al. (2019) have described reported bioactives extracted by subcritical and supercritical fluid extraction from various plant sources, seaweeds, microalgae and food by-products for the period from 2015 to 2019. Previously, Herrero et al. (2015) published a review on the same theme for the period 2006–2014. The bioactives extracted from these sources using specifically SC-CO2, with or without co-solvents, include carotenoids, anthocyanins, phenolic compounds, polyphenolics, sesquiterpenes, antioxidant compounds, polyphenols, monoterpenes, vitamin E, cannabinoids, non-polar flavonoids, lycopene, piperine, tetrahydrocannabinol, colchicine, tocols, tocopherols, polar and non-polar lipids, oils, essential oils, oleanolic acids, ursolic acids, fatty acids, chlorophyll A, ergosterol, fucosterol, fucoxanthin and triacylglycerides (Gallego et al. 2019). Michalak et al. (2017) have presented a summary of reported biologically active compounds (fucoxanthin, beta-carotene, carotenoids, astaxanthin, canthaxanthin, chlorophyll, polyphenols, fatty acids, lipids, oil, auxins, cytokinins, micro- and macro-elements) that have been obtained via SC-CO2 extraction from algal biomass. Algae extracts obtained by this method are proposed by Michalak et al. (2017) to be used in cosmetics and dietary feeds and as growth stimulants due to their components being solvent-free. Table 7.3 provides a summary of reported SC-CO2 extractions of biomolecules discussed in this chapter. The chapter intends to present recent applications of SC-CO2 extraction of selected biomolecules with potential uses in industry and provides an overview of published reports on this critical area of Green Chemistry .

Table 7.1 Published reviews on the use of supercritical fluids for the extraction of biomolecules
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Fig. 7.1
figure 1

Overview of the supercritical carbon dioxide (SF-CO2) extraction process of biomolecules (Grosso et al. 2010; Sovová 2012; Huang et al. 2012; da Silva et al. 2016; Shukla et al. 2019; Yousefi et al. 2019)

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7.2 Design and Optimization of the Supercritical Carbon Dioxide Extraction Process

In order to ensure high extraction efficiencies and to obtain high quality and purified extractions (Chemat et al. 2019; Yousefi et al. 2019), the design and the optimization strategies are critical. In particular, with SF extractions, the aim is on the reduction of extraction time, amount of solvent, energy usage, costs, waste produced and the environmental impact (Chemat et al. 2019; Yousefi et al. 2019). Figure 7.1 presents a summary of the various components of the SC-CO2 extraction process of biomolecules. Understanding the mass transfer mechanisms of the extraction process is imperative for starting the design of the extraction process (Huang et al. 2012) and in choosing the appropriate mathematical model to be applied (Kumhom et al. 2011). The development of mathematical models for the SC-CO2 extraction process was reported by various research groups (Table 7.2). These models are generally described as the models that are based on “heat transfer analogies”, models based on “differential mass balances”, empirical models and the shrinking core model (Özkal et al. 2005). Huang et al. (2012) have described several models such as the “broken and intact cell” model (BIC), hot ball diffusion (HBD) model, shrinking core (SC) model, Tan-Liou model, partitioning coefficient model and the logistic model (LM) for different SC-CO2 extraction systems (Fig. 7.1). The most widely used model for obtaining extracts via SC-CO2 extraction from plant sources is the BIC model (Huang et al. 2012). The BIC model has been found suitable for the extraction of oleoresin from marigold (Calendula officinalis) plants (López-Padilla et al. 2017). Natolino and Da Porto (2019) have applied kinetic (BIC model) and solubility (Chrastil model) modelling for the SC-CO2 extraction of pomegranate seed oil from Punica granatum L. The “shrinking core model” was applied for the isoflavone extraction (SC-CO2 and methanol) from soybean meal by Kumhom et al. (2011) and investigated the axial dispersion coefficient, effective diffusivity, solubility and the film “mass transfer coefficient”, of which the film “mass transfer coefficient” and the solubility were found to be the most significant. Sovová (2012, 2017), Sovová et al. (2016) and Sovová and Stateva (2019) have described the modelling of the SF extraction process for essential oils, lipids and carotenoids, which have been readily adapted to represent extraction curves.

Table 7.2 Reported theoretical models for supercritical fluid extraction
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Optimization targets (Fig. 7.1) are based on varying mainly the pressure, temperature and the flow rate of the SC-CO2. Co-solvents such as water, methanol, diethyl ether, ethanol, acetone, acetonitrile or dichloromethane are added at varying concentrations to observe the effect on the extraction yield and the extract composition, as these improve the solvating power of the SC-CO2 (Michalak et al. 2017; Rovetto and Aieta 2017). This is necessary for the extraction of polar compounds since SC-CO2 is a non-polar solvent and its polarity can be influenced with the use of polar modifiers (Nagavekar and Singhal 2019). Additional parameters (Fig. 7.1) that are targeted for optimization are the sample’s particle size, extraction time and solvent power (Ekinci and Gürü 2014). The effects of the solvent power of SC-CO2 are critical during any extraction process, whereby the effects on solvent power and extraction selectivity are discussed by Ekinci and Gürü (2014). Solvent density is significantly affected by changes in pressure (Derrien et al. 2018). Rovetto and Aieta (2017) investigated the effect of different pressures, flow rates and co-solvent (ethanol) on the yield of cannabinoids from Cannabis sativa L., whereby pressure and plant material had notable effects on the extraction yield.

7.3 Lipids, Volatile Oils and Oleoresins

Lipids have been extracted from various macroalgae (MA) and microalgae (MI) species using SC-CO2 such as Chlorella vulgaris (MI) (Mendes et al. 1995; Dejoye et al. 2011), Tetraselmis sp. (MI) (Li et al. 2014), Chaetomorpha linum (MA) (Aresta et al. 2005), Sargassum hemiphyllum (MA) (Cheung et al. 1998) and Hypnea charoides (MA) (Cheung 1999). Fatty acids from Arthrospira maxima (Spirulina maxima) (MI) have been extracted with SC-CO2 and with 10% ethanol (Mendes et al. 2003). Michalak et al. (2017) reported a comparative summary of the SF extraction methods and parameters used for the extraction of algae bioactives. Mainly, SC-CO2 extraction alone has been applied with some reported to have added organic solvents, water or vegetable oils to the extraction process. Biomolecules that are oxidized easily and that are heat labile are suitably extracted with SC-CO2 due to its non-oxidant nature (Michalak et al. 2017). SC-CO2 has been identified as an appropriate method to extract lipids (Li et al. 2014). A low temperature of 30 °C and a pressure of 35 MPa were found to be optimum for the SC-CO2 extraction of Moringa oleifera seed oil (75.27%) and in having the greatest solvation power (Ruttarattanamongkol et al. 2014). The solvent CO2 showed higher selectivity at the low pressure of 15 MPa towards the extraction of sterols, tocopherols and fatty acids (Ruttarattanamongkol et al. 2014). At conditions of 15 MPa and 35 °C, biomolecules β-sitosterol, campesterol, γ-tocopherol and α-tocopherol were found to be at highest concentrations of 2310.9, 1179.2, 106.8 and 230.3 mg/kg, respectively (Ruttarattanamongkol et al. 2014). SC-CO2 extraction of hemp seed oil from Cannabis sativa L. has been previously reported with oil yields of 21.50% w/w (40 °C, 300 bar, particle size of 0.71 mm) (Da Porto et al. 2012a) and 22% (300 bar and 40 °C and at 400 bar and 80 °C) obtained (Da Porto et al. 2012b). Hemp (Cannabis sativa L.) seed oil was extracted at varying temperature of 40 and 60 °C and at a constant pressure of 300 bar with a CO2 flow rate of 1.94 kg/h (Aladić et al. 2015). The yield of seed oil was not affected by changes in temperature but increased with increasing pressure (Aladić et al. 2015). The fatty acid concentration was affected by pressure, whilst extraction time did not affect the content (Aladić et al. 2015). Temperature had a varying effect on the content of the fatty acids (Aladić et al. 2015). The common vegetable oil, sunflower oil (54.37 wt%), has been extracted with SC-CO2 from the sunflower seed (particle size, 0.75 mm) with a flow rate of 10 g/min, 5% co-solvent at 400 bar and 80 °C (Rai et al. 2016). Oil from Eremanthus erythropappus (candeia wood) has been optimally extracted at 70 °C and 24 MPa with 2 ml/min flow rate and ethanol (1.3% v/v) and ethyl acetate (5% v/v) as co-solvents, yielding 2.35 wt% oil (Santos et al. 2017). The highest concentration of α-bisabolol (16.53 g/kg), a naturally occurring sesquiterpene alcohol, was obtained with 5% ethanol (Santos et al. 2017). A review on the properties (pharmacological), mechanisms of action and the applications of α-bisabolol and oils rich in α-bisabolol has been published by Kamatou and Viljoen (2010). The yield of total lipids extracted from Solidago gigantea Ait. (goldenrod) has been evaluated using a “Box-Behnken design with three variables” studied, temperature (40–80 °C), pressure (20–80 MPa) and the flow rate of CO2 (3–7 kg/h) (Wrona et al. 2019). The optimum conditions reported are temperature at 313.95 K, pressure at 68.07 MPa and CO2 flow rate of 3.18 kg/h yielding 203.32 mg stearic acid equivalent/g dry mass, with temperature having a negligible effect on the content of total lipids (Wrona et al. 2019).

Shukla et al. (2019) have applied SC-CO2 (single-step) extraction and fractionation process to obtain oleoresin enriched with gingerols and essential oil from dried ginger rhizomes. Optimum conditions reported by Shukla et al. (2019) for obtaining 28.3 wt% volatile oil and 37.97 wt% major actives were pressure at 276 bar, temperature at 40 °C and flow rate of 30 g/min for 153 min. Shukla et al. (2019) have presented a summary of reported literature on obtaining ginger extracts using SC-CO2 extraction processes. Oregano oil (Origanum vulgare L.) extracted with SC-CO2 (100 bar, 40 °C, 8 g/min ethanol) with a highest yield of 13.40% showed high antimicrobial and antioxidant activity (García-Pérez et al. 2019). The volatile compound, carvacrol (29.99%), and the fatty acids (70.9–76.8%), α-linolenic (C18:3ω3, 20.55–24.66%), palmitic (C16:0, 22.76–23.65%), oleic (C18:1ω9c, 15.19–16.63%) and linoleic acids (C18:2ω6c, 12.16–13.35%), were found at highest concentrations in the oil (García-Pérez et al. 2019). Oil has been extracted from parboiled rice bran with SC-CO2 and ethanol, and the effects of varying pressures (100, 150, 200 bar) and temperatures (40, 60, 80 °C) and ethanol to rice bran (0:1, 0.5:1, 1:1, 2:1) were analysed (Juchen et al. 2019). Conditions for one experimental run for 250 min at 200 bar, 40 °C, 45.94 g CO2/g bran and 1:1 ethanol to rice bran yielded 25.48 wt% of rice bran oil. Two sequential extractions at the same conditions yielded 26.32 wt% (Juchen et al. 2019). Roselló-Soto et al. (2019a) have compared the SC-CO2 (10–40 MPa, 40 °C) extraction with the conventional extraction (modified Folch et al. 1957) of oil extracted from the “horchata” by-products with the SC-CO2 extraction obtaining higher amounts of α-tocopherol and total phenolic compounds. The α-tocopherol concentration decreased, whilst total phenolic compounds increased with increasing pressure, respectively. The highest oil yield was obtained with conventional extraction (14.85%). Oil yield and pressure during the SC-CO2 extraction were shown to have a linear relationship (10 MPa = 0.61%; 40 MPa = 7.36%), which has been reported by several other researchers (Lasekan and Abdulkarim (2012); Rombaut et al. 2014; Koubaa et al. 2015). Pinto et al. (2018) have obtained a yield of 60.39% after SC-CO2 extraction of “bacaba oil” from Oenocarpus bacaba at temperature of 60 °C and pressure of 420 bar. A good quality bacaba-de-leque pulp oil from Oenocarpus distichus Mart. has been extracted using SC-CO2 extraction with a yield of 46% at a pressure of 270 bar and temperature of 60 °C (Cunha et al. 2019). SC-CO2 extraction (320 bar and 60 °C) has been used in the extraction of pomegranate (Punica granatum L.) seed oil that contained a higher punicic acid concentration and with a higher oxidation stability as compared to the Soxhlet extraction method (Natolino and Da Porto 2019). The extraction times with SC-CO2 (2 h) were much shorter than the period needed for Soxhlet extraction (8 h) to reach the asymptotic yield. A CO2 flow rate of 8.0 kg/h at 320 bar and 60 °C resulted in the highest yield of the seed oil (Natolino and Da Porto 2019). Fernández-Acosta et al. (2019) recently studied the effect of chemical pre-treatment, pressure, temperature, dynamic and static time and particle size on the SC-CO2 extraction of oil Avena sativa L. (oats). The oat oil yield was significantly influenced by pressure and the size of particles. The pre-treatment, temperature and particle size significantly influenced the fatty acid composition and oxygen radical absorbance capacity (ORAC) antioxidant activity. Polyphenol concentration and total phenolic content were affected by pre-treatment and temperature (Fernández-Acosta et al. 2019). Cordeiro et al. (2019) recently investigated the SC-CO2 extraction of ucuúba oil from the seeds of Virola surinamensis, a tree growing in the Amazon, with antimicrobial activity against Staphylococcus aureus. Extraction conditions applied were pressure (350 bar), temperature (40, 60 or 80 °C) and a CO2 mass flow of 7.9 × 10 kg/s (Cordeiro et al. 2019). De Cássia et al. (2016) have extracted the oil from the popular fruit (berries) açaí (Euterpe oleracea Mart.) obtained from Pará, Brazil, under varying temperature and pressure conditions. Highest oil yield was obtained at 70 °C and 490 bar. The fatty composition was affected by the varying operating conditions with significant effects reported on type and concentration of fatty acid detected (De Cássia et al. 2016). Silva et al. (2019) have evaluated the effects of pressure (350, 420, 490 bar), temperature (50, 60 or 70 °C) and geographical location on the extraction of the oil from açaí in lyophilized form. Oil yields between 49.28 (location, Anajás, 60 °C, 420 bar) and 57.06% (location, Chaves, 70 °C, 490 bar) were obtained (Silva et al. 2019) with the operating conditions not affecting the fatty acid composition within each study area. Operating conditions of 60 °C and 420 bar and 70 °C and 490 bar resulted in optimum results of antioxidant capacity, total anthocyanins and total phenolic compounds in the oil from Chaves (Silva et al. 2019). Kerrihard and Pegg (2015) reported on the suitability of the application of SC-CO2 extraction of oils containing higher concentrations of γ-linolenic acid (GLA; 18:3n-6), which have as a result higher anti-inflammatory functionalities. Scale-up experiments and validation processes for the SC-CO2 extraction of lipids and volatile oils have been investigated by several researchers with promising results of potential extraction at a commercial scale (Shukla et al. 2019; Wrona et al. 2019).

Essential oils, also referred to as volatile oils, are commonly used in traditional medicine and aromatherapy and as natural additives due to their reported antimicrobial activities (Chávez-González et al. 2016) in the food and cosmetics industry (Fornari et al. 2012a). Grosso et al. (2010) have tested five mathematical models for the modelling of the extraction of aromatic plants, fennel, coriander, cotton lavender, savoury, winter savoury and thyme by which extraction was impacted by particle size, internal mass transfer coefficient, internal diffusion, and pressure and temperature changes.

Sovová (2012) has described various published mathematical models for the extraction of essential oils from plants for a 15-year period up to 2012. Tongnuanchan and Benjakul (2014) have discussed the extraction methods, including aspects of SC-CO2 extraction among others, and the uses and bioactivities of essential oils. Yousefi et al. (2019) have compiled a review that investigates the SF extraction of essential oils from plants. The review describes conventional extraction methods as compared to the SF extraction, optimization and modelling techniques commonly applied and the effects of the various operating parameters on the supercritical extraction process (Yousefi et al. 2019). SC-CO2 has been investigated for the extraction of the volatile oil from the flower heads of Santolina chamaecyparissus L. under different operating conditions. Notably, pressure increase to 9 MPa enriched the content of sesquiterpene in the extracted Santolina chamaecyparissus essential oil (Grosso et al. 2009a). The volatile oil from Satureja montana L. extracted by SC-CO2 was reported by Grosso (2009b) to contain higher concentrations of thymoquinone (1.6–3.0%) as compared to the hydrodistillation, a conventional extraction method. Essential oils produced via SC-CO2 extraction from various herbs and plants have been reported by Fornari et al. (2012a). A cooled mill was used to crush the dried leaves (O. vulgare, Thymus vulgaris, Salvia officinalis, Rosmarinus officinalis, Ocimum basilicum, Origanum majorana) and petals (Calendula officinalis) and then sieved (200–600 μm). Essential oils were then extracted at 30 MPa and 40 °C with a flow rate of 60 g/min for 5 h (Fornari et al. 2012a). The economically important vetiver essential oil was extracted with SC-CO2 among other methods (“pressurized liquid and ultrasound-assisted methods”) from Chrysopogon zizanioides (L.) Roberty, syn. Vetiveria zizanioides (L.) Nash root with the highest yield (2.23% m/m) obtained at 20 MPa and 60 °C (Santos et al. 2019). A higher yield (2.66% (m/m)) was obtained after the addition of 5% (v/v) of the co-solvent, ethanol (Santos et al. 2019). The dominant compounds were isovalencenol (9.04% SC-CO2; 8.70% SC-CO2+ 5% EtOH 5%), khusimol (30.49% SC-CO2; 31.33% SC-CO2+ 5% EtOH 5%), zizanoic acid (8.33% SC-CO2; 6.82% SC-CO2+ 5% EtOH 5%) and α-vetivone (6.42% SC-CO2; 6.61% SC-CO2+ 5% EtOH 5%). Leaf powder (20 g, dp = 0.42 mm) of Ocimum sanctum Linn. has been used for the extraction of eugenol (2.96 mg/g dry leaves) with SC-CO2 at a flow rate of 2.5 L/min, 200 bar and 50 °C for 90 min (Chatterjee et al. 2017). Eugenol yield was affected only by pressure changes, and the kinetics of the extraction were reported to be first-order kinetics (Higuchi model) (Chatterjee et al. 2017). Eugenol (29.84%) has been extracted from clove at 40 °C and 220 bar with highest antioxidant activity of the extract found after extraction at 40 °C and 150 bar (Frohlich et al. 2019). In comparison to the Soxhlet extraction, it was revealed that the SC-CO2 was more efficient in terms of yield, antioxidant activity, sample clean-up, reaction time and temperature (Frohlich et al. 2019).

Oleoresin has been optimally extracted from Curcuma longa (conventional turmeric) and Curcuma amada (mango ginger) at 65 °C and 350 bar for 150 min and at 40 °C and 300 bar for 30 min, respectively (Nagavekar and Singhal 2019). Modifier (30% ethanol) and pre-treatment with Stargen®002 enzyme significantly improved the yield (Nagavekar and Singhal 2019).

7.4 Artemisinin

The active pharmaceutical ingredient, artemisinin, is extracted from the herbaceous plant Artemisia annua L. (sweet wormwood) and is used in pharmaceutical applications for the treatment of malaria and cancer (Rodrigues et al. 2019). Artemisinin is a sesquiterpene that is highly oxygenated with a 1,2,4-trioxane ring structure (Brown 2010). Faurant (2011) have provided a historic background on the discovery of artemisinin and market-related developments, with Brown (2010) having provided a review on the photochemistry of the plant A. annua L. and the biosynthesis of the compound artemisinin. The SC-CO2 extraction has commonly been employed in the high yield extraction of artemisinin from A. annua L. yielding high purity and clean extracts (Kohler et al. 1997; Quispe-Condori et al. 2005; Lin et al. 2006; Tzeng et al. 2007; Baldino et al. 2017; Martinez-Correa et al. 2017; Ciftzi et al. 2018; Rodrigues et al. 2019). Lapkin et al. (2006) have published a comparative analysis on existing conventional and green technologies such as SC-CO2 for the extraction of artemisinin.

Pure, non-degraded extracts of artemisinin have been obtained with a flow rate of 2 ml/min of SC-CO2/3% methanol at 50 °C and 15 MPa within 20 min (Kohler et al. 1997). Artemisinin yields of 0.62% and 0.70% have been achieved at conditions of 150 bar and 30 °C and 300 bar and 50 °C, respectively (Quispe-Condori et al. 2005). Artemisinin of high purity within a short reaction time was yielded at optimized conditions of 33 °C, 18.72 MPa, SC-CO2/16.25 wt% of n-hexane after an extraction time of 1.5 h (Lin et al. 2006). The highest purity was obtained at 60 °C and 17.34 MPa and proved to be far better than when Soxhlet extraction (hexane) was applied (Lin et al. 2006). Martinez-Correa et al. (2017) reported the suitability of a two-step extraction procedure, starting with SC-CO2 extraction (60 °C, 40 MPa), followed by further extractions with either ethanol (25 °C) or water (60 °C). Baldino et al. (2017) on the other hand reported the optimum procedure to be a one-step extraction at 40 °C and 100 bar for 600 min. Ciftzi et al. (2018) reported optimum conditions to be 33 °C and 30 MPa for yielding 1.09% (predicted yield). The cost-effectiveness of the SC-CO2 extraction process of artemisinin as compared to the conventional extraction (ethanol) was evaluated by Rodrigues et al. (2019), which determined that this process is more attractive economically and viability could be achieved if reduced cost for the raw material could be achieved. An artemisinin content of 23.4% was reached at 50 °C and 200 bar for 60 min (Rodrigues et al. 2019). A reaction time of 180 min was needed to obtain 6% of artemisinin using conventional extraction with ethanol (Rodrigues et al. 2019).

7.5 Alkylamides

Alkylamides from the dried roots of Echinacea angustifolia were extracted by Sun et al. (2002) at highest concentration as compared to the fresh roots, by which the yield was positively affected by temperature and pressure.

7.6 Phenolics, Flavonoids, Chlorophylls and Carotenoids

Phenolics and flavonoids extracted from plants via the SC-CO2 process have been reported by several researchers (Table 7.3). Ampelopsis grossedentata stems were used to optimally extract flavonoids and phenolics at 40 °C and 250 bar for 50 min with 1:3 v/v methanol/ethanol and 1:1 v/v methanol/ethanol, respectively (Wang et al. 2011). Pomelo peel was used for the extraction of flavonoids (2.37%) at 39 MPa, 80 °C, 85% ethanol for 49 min (He et al. 2012). SC-CO2 extracted flavonoids had higher scavenging activities as compared to the conventional process (He et al. 2012). Increased concentration of total phenolic content and antioxidant activity were obtained with the addition of co-solvents (ethanol and ethyl acetate) to the SC-CO2 extraction process of the oil from Eremanthus erythropappus (candeia wood) (Santos et al. 2017). Total phenolics from radishes (Raphanus sativus L.) were reported at concentrations of 1375 mg GAE/100 g and 1455 mg GAE/100 g at operating conditions of 400 bar at 35 °C and 40 °C, respectively (Goyeneche et al. 2018). Escobedo-Flores et al. (2018) extracted polyphenols from Avena sativa L. (oats) obtaining maximum yields at 55 °C and 38 MPa. An optimum concentration of 1437.57 mg/g was predicted with the generated quadratic models (Escobedo-Flores et al. 2018). The effects of particle size on the concentrations of avenanthramides and phenolics extracted from medium oat bran, whole flour (WF), low bran and fine bran have been reported by Walters et al. (2018), with larger sizes presenting a limiting factor during SC-CO2 extractions. Higher radical scavenging activities were observed with the extracts via the SC-CO2 extraction of the defatted fraction of the fine particles (Walters et al. 2018). Avenanthramides have been reported to possess strong anti-inflammatory properties (Sur et al. 2008). The yield of total phenolics and total chlorophylls extracted from Solidago gigantea Ait. (goldenrod), a medicinal plant, has been evaluated using a “Box-Behnken design with three variables” studied, temperature (40–80 °C), pressure (20–80 MPa) and the flow rate of CO2 (3–7 kg/h) (Wrona et al. 2019). The three variables studied had an effect on the yield of total phenolics and total chlorophylls with optimum conditions reported as temperature of 313.59 K and 352.22 K, pressure of 79.14 MPa and 74.59 MPa and CO2 flow rate of 3.25 kg/h and 3.00 kg/h, respectively (Wrona et al. 2019). Increased temperature resulted in a decrease in the total phenolic content, but increased when pressure was increased (Wrona et al. 2019). Alvarez et al. (2019) determined that conditions of 40 MPa and 35 °C with the co-solvent ethanol were optimum or the extraction of polyphenols and flavonoids with highest antioxidant activity. Roselló-Soto et al. (2019a) affirmed the suitability of applying SC-CO2 extraction of lipophilic phenolic compounds when compared to the conventional extraction. Isohydroxymatairesinol was extracted at highest concentrations at pressures of 30 MPa (756.22 ppb) and 40 MPa (1331.45 ppb). Increasing pressures improved the extraction of the phenolic compounds, including the antioxidant activity (Roselló-Soto et al. 2019a). A SC-CO2 extraction process to obtain an extract rich in phenolic compounds from cacao (Theobroma cacao) pod husk was developed by Valadez-Carmona et al. (2018). The yield was influenced by pressure and co-solvent percentage, and a yield of 0.52% was obtained at optimum conditions of 299 bar, 60 °C and 13.7% ethanol with high selectivity towards antioxidants (Valadez-Carmona et al. 2018).

Table 7.3 Reported publications on the supercritical carbon dioxide extraction of biomolecules
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Carotenoids are pigments and the secondary metabolites of plants and some microorganisms (Zaghdoudi et al. 2016) and can be successfully extracted using SC-CO2. Sovová et al. (2001) have published data on the effects of different temperatures and pressures with and without ethanol and vegetable oil on the solubility of β-carotene in SC-CO2. Knowledge of the solubility of biomolecules such as the carotenoids in SC-CO2 is necessary to develop appropriate SC-CO2 extraction processes. De la Fuente et al. (2006) have determined the solubility of lycopene and astaxanthin at different temperatures and pressures. Similar to the behaviour of β-carotene in SC-CO2, lycopene and astaxanthin solubility was greater with increased temperature (313–333 K) and constant pressure (30 MPa) as compared to increased pressure (30–50 MPa) at constant temperature (313 K) (de la Fuente et al. 2006). A review on the extraction of carotenoids from pumpkin (Cucurbita spp.) via the SC-CO2 process and the influence temperature and pressure, pre-treatment effects, entrainers (modifier or co-solvents) and co-matrices on total carotenoid yield and carotenoid composition has been published by Durante et al. (2014). Choudhari and Singhal (2008) have extracted lycopene, a red-coloured tetraterpenic C40 carotenoid, from Blakeslea trispora, a zygomycete, at optimized conditions of 349 bar and 52 °C for 1.1 h and using an entrainer such as acetone, yielding 92%. Astaxanthin has been extracted from the microalgae Haematococcus pluvialis with the use of olive oil and soybean oil as co-solvents achieving a yield of 51.03% and 36.36%, respectively (Krichnavaruk et al. 2008). The microalgae Haematococcus pluvialis has been reported to be one of the greatest sources of the natural occurring astaxanthin, a carotenoid with potent antioxidant activity (Shah et al. 2016).

Xanthophylls (all-trans-lutein (15.46 μg/g), all-trans-zeaxanthin (16.81 μg/g) and all-trans-β-cryptoxanthin (33.23 μg/g)) have been optimally extracted from persimmon fruits (Diospyros kaki L.) at a flow rate of 3 ml/min, 300 bars, 60 °C and 25% (w/w) ethanol for 30 min obtaining higher yields as compared to the Soxhlet extraction method (Zaghdoudi et al. 2016). Conditions of flow rate of 1 ml/min, 100 bars, 40 °C and 25% (w/w) ethanol for 30 min were better suited for the extraction of 11.19 μg/g all-trans-β-carotene (Zaghdoudi et al. 2016). Spinach by-products have been used by Derrien et al. (2018) for the optimization of SC-CO2 extraction of chlorophyll and lutein. Optimized conditions that resulted in a 72% and 50% yield of lutein and chlorophyll, respectively, were reported to be 39 MPa, 56 °C with a co-solvent of 10% ethanol for 3.6 h (Derrien et al. 2018). The SC-CO2 extraction process of carotenoids from microalgae with the use of published data was described and modelled by Sovová and Stateva (2019). The model confirmed that higher temperatures and pressures increased yield of carotenoid in oil and extraction rate due to increased solubility of carotenoid in the supercritical fluid and the reduced capacity of adsorption of the microalga (Sovová and Stateva 2019). The phase equilibrium was found to be responsible in controlling the extraction process (Sovová and Stateva 2019).

7.7 Polysaccharides

Polysaccharides have been extracted via the SC-CO2 process from the seeds of Artemisia sphaerocephala Krasch. at optimum conditions of temperature (extraction, 45 °C; separation, 56 °C), pressure (extraction, 45 MPa; separation, 10 MPa), a flow rate of 20 L/h for 2 h resulted in a yield of 18.59% (w/w) (Chen et al. 2014). The 551.3 kDa polysaccharide was composed of the monosaccharides, mannose (10.8 mg/g), rhamnose (8.78 mg/g), galactose (9.86 mg/g), glucose (16.2 mg/g), arabinose (8.48 mg/g), xylose (38.48 mg/g) and fucose (10.09 mg/g) (Chen et al. 2014). The polysaccharide extracted from Artemisia sphaerocephala Krasch. has been reported to have medicinal applications (Xing et al. 2009). A polysaccharide (2.47%) from the leaves of bamboo (Phyllostachys heterocycla) has been extracted with SC-CO2/ethanol modifier (30 ml) at optimized parameters of 50 °C and 40 MPa with a 2-h reaction time (Zou et al. 2018).

7.8 Tocopherols and Sterols

Plant cells synthesize α-, β-, γ- and δ-tocopherol, which are then stored in their seeds and leaves (Bendif et al. 2018). Tocopherols have strong antioxidant properties (Bendif et al. 2018). The content of tocopherols in extracted Cannabis sativa L. seed oil is significantly affected by temperature and pressure with higher temperatures and pressures resulting in a negative response (Aladić et al. 2015). Potential sources of tocopherol were reported by Bendif et al. (2018) to be contained in the SC-CO2 extracts obtained from Thymus munbyanus subsp. coloratus (α-tocopherol [1580 μg/g], β-tocopherol [170 μg/g], γ-tocopherol [220 μg/g], δ-tocopherol [160 μg/g]) and Thymus munbyanus subsp. munbyanus (α-tocopherol [780 μg/g], β-tocopherol [140 μg/g], γ-tocopherol [120 μg/g], δ-tocopherol [130 μg/g]). The Thymus extracts were obtained with SC-CO2 extraction with a flow rate of 2 L/min at 70 °C and 45 MPa for 210 min (Bendif et al. 2018). Sitosterol (1220 mg/kg seed) has been found to be contained in peach oil (extracted yield, 35.3 g/100 g seed) after SC-CO2 extraction from the Prunus persica seeds (0.3 mm) at optimum conditions of 200 bar, flow rate of 7 ml/min and at 40 °C after 3 h (Ekinci and Gürü 2014).

7.9 Conclusions

A wide variety of biomolecules can be efficiently extracted with the SC-CO2 extraction process. Optimization strategies are targeting to achieve parameters that can achieve high yield products with high purity and quality at less costs and environmental impacts. Research on the cost-effectiveness, economics and possible facility designs for the scaling up of the SC-CO2 extraction process is underway with some successful examples already in place. The advent of the Green Chemistry era has made it possible to explore more opportunities in the development of innovative extraction and processing technologies.