Keywords

1.1 Introduction

Nowadays, there is increasing evidence concerning the physiological, nutritional, and medicinal benefits to human health from various natural products, as well as the possible harmful effects of the use of certain synthetic products. The market for bioactive natural ingredients is among the most attractive at the moment, and natural lipophilic compounds of various structures and functionalities provide excellent molecules for the production of active ingredients for functional foods and food additives (e.g., preservatives, flavours, or colourants).

Extracts from natural sources play an important role, and the different parts of plants such as seeds, leaves, flowers, berries, bark, and roots represent the major sources of natural extracts, although some extracts may be obtained from animal sources.

In order to address the need for safer and healthier products and to compete in meeting consumer expectations, manufacturers must adopt the most suitable procedure for producing the natural ingredients. Conventional extraction involves the use of solid–liquid techniques that typically rely on organic solvents, which present several drawbacks, including toxic residues, chemical transformation of extracts, and toxic wastes. This has led to the development of more innovative approaches for the production of the desired compounds. The selection of an extraction method is dependent on the type of compound to be extracted, and it should take into account the potential for scaling up of the technology.

The aim of this chapter is to summarize and provide an overview of the main lipophilic bioactives and their characteristics, sources, and methods of extraction from natural matrices. As supercritical carbon dioxide extraction has been shown to be an effective “green” alternative for organic solvent-based extraction of non-polar compounds, supercritical fluid technology will be discussed in greater detail.

1.2 Lipophilic Bioactives

Lipophilic bioactives constitute a wide range of substances with a variety of functions, including flavour, antimicrobial, antioxidant, and several health-promoting properties. The group of lipophilic bioactives is very diverse in terms of molecular properties (e.g., molecular weights, structures, functional groups, polarities, and charge), leading to different physicochemical and physiological properties including solubility, physical state, rheology, optical properties, chemical stability, surface activity, and bioactivity (McClements et al. 2007).

The most important classes of lipophilic bioactives are briefly presented below, including their physicochemical characteristics, bioactivity, and main sources. The chemical structures of various bioactives are schematically presented in Table 1.1.

Table 1.1 Chemical structure of some bioactives [PubChem database (Kim et al. 2015)]
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1.2.1 Carotenoids

Carotenoids, the most significant and extensive group of pigments found in nature (Brunner 2005a), are a large group of lipophilic compounds that give a range of colours in foods from yellow to red. Furthermore, it has been strongly suggested that consuming carotenoid-rich foods reduces the risk of several types of disease, including cancer (Ziegler 1989; Gerster 1993; Astorg 1997), cardiovascular disease (Bendich 1994; Kohlmeier and Hastings 1995; Mayne 1996; Sesso et al. 2005; Riccioni 2009), age-related macular degeneration (Seddon et al. 1994), cataracts (Jacques and Chylack 1991; Snodderly 1995; Mayne 1996; Olmedilla et al. 2003), diseases related to low immune function (Bendich 1994; Chew 1995; Meydani et al. 1995), and other degenerative diseases (Stahl and Sies 2005; Perera and Yen 2007).

Carotenoids are polyenes consisting of 3–13 conjugated double bonds and sometimes six carbon ring structures at one or both ends of the molecule (Cavalcanti et al. 2013). They can exist in cis and trans forms, but cis isomers are less stable than the trans forms due to stoichiometric conformation. Thus, the majority of natural carotenoids are in the all-trans configuration (McClements et al. 2007). Based on their structure, carotenoids are divided in two classes: (i) carotenes, which are pure polyene hydrocarbons containing only carbon and hydrogen atoms, and include acyclic lycopene and bicyclic β- and α-carotene; and (ii) xanthophylls, containing oxygen in the form of hydroxyl (lutein), epoxy (violaxanthin), and oxo (canthaxanthin) groups (Sajilata et al. 2008). The most abundant pigments in nature are β-carotene, lycopene, lutein, and zeaxanthin (de Paz et al. 2012).

Carotenoids are found in a large variety of natural sources: vegetables, animals, bacteria, yeast, and microalgae. About 90 % of the carotenoids in the human diet and body are β- and α-carotenes, which are commonly found in yellow-orange vegetables and fruits; α-cryptoxanthin is present in orange fruits, lutein is provided by dark green vegetables, and lycopene is obtained from tomatoes and their by-products (Rao and Rao 2007).

The carotenoids are the main dietary source of vitamin A precursors and antioxidants (de Paz et al. 2012). Although β-carotene is the main compound with pro-vitamin A activity, any carotenoid with at least one unsubstituted β ring, such as α-carotene or β-cryptoxanthin, has the added advantage of being able to be converted to vitamin A. Even though lycopene is a carotenoid with no pro-vitamin A activity, it is an important antioxidant and free radical scavenger (Pingret et al. 2013). Processed foods are frequently fortified with carotenoids such as lycopene to increase their nutritive value and/or enhance their attractiveness (Wang and Weller 2006). The major sources of lycopene are ripe tomatoes, and tomato products and by-products (skins and seeds) (Burton-Freeman and Reimers 2011).

1.2.2 Polyunsaturated Fatty Acids (PUFAs)

The polyunsaturated fatty acids (PUFAs) contain more than one double bond in their structure. PUFAs can be classified into various groups by their chemical structure: methylene-interrupted polyenes, conjugated fatty acids, and other polyunsaturated fats. The methylene-interrupted polyenes comprise the ω-3 essential fatty acids (hexadecatrienoic acid, α-linolenic acid, stearidonic acid, etc.), ω-6 fatty acids (linoleic acid, γ-linolenic acid, eicosadienoic acid, etc.), and ω-9 fatty acids (oleic acid, erucic acid, mead acid, etc.). The conjugated fatty acids have two or more conjugated double bonds, for example, linoleic acids (rumenic acid) and linolenic acids (β-calendic acid). Pinolenic acid and podocarpic acid are examples of other PUFAs (McClements et al. 2007).

There is an extensive body of scientific literature supporting the positive effects of ω-3 fatty acids on human health. Important natural sources of ω-3 PUFA are marine organisms (fish, seafood, algae, and other marine sources) directly or indirectly fed from marine phytoplankton, the primary producer of ω-3 in the trophic chain (Rubio-Rodriguez et al. 2010), and selected seed plants and other marine sources (Sahena et al. 2009b). Consumption of ω-3 fatty acids can reduce the risk of heart disease (Hooper et al. 2006) and high blood pressure (Lungershausen et al. 1994), prevent blood clots, protect against cancer, and alleviate depression (Von Schacky et al. 1999). With regard to ω-6 fatty acids, they are often used to develop pharmaceutical drugs and for the treatment of atherosclerosis, asthma (Hodge et al. 1998; Oddy et al. 2004), arthritis (Geusens et al. 1994), vascular disease, thrombosis (Yamashita et al. 2005), immune-mediated inflammatory disorders (Fritsche 2006), and cancer (Cunnane 2003; Simopoulos 2002, 2008). Unlike ω-3, the ω-6 and ω-9 fatty acids are not classified as essential fatty acids, as they can be synthesized by the human body from unsaturated fatty acids. Among all compounds, particular attention has been focused on concentrates of EPA (all-cis-5,8,11,14,17-eicosapentaenoic acid) and DHA (all-cis-4,7,10,13,16,19-docosahexaenoic acid) due to their pharmaceutical value (Cavalcanti et al. 2013).

1.2.3 Tocopherols and Tocotrienols

Tocopherols constitute a class of chemical compounds that includes various methylated phenols. Tocopherols are extremely valuable compounds because of their vitamin bioactivity and antioxidant capacity (Lee et al. 2000). They are abundant in seeds and leaves of plants. γ-Tocopherol is present at high concentrations in seed oils (olive, sunflower, corn, and soybean) and α-tocopherol in leaf lettuce (Cavalcanti et al. 2013).

Vitamin E includes both tocopherols and tocotrienols that occur in groups of four (α, β, γ, δ) lipophilic antioxidants synthesized by photosynthetic organisms. The isomer with the highest vitamin E activity is α-tocopherol, and it has become an important additive in numerous food products. However, although it has a number of biological properties, it can cause indigestion, thus affecting its bioavailability in the intestine. Many biological functions related to tocopherol consumption have been identified, including relief of stress (Liu 2005) and premenstrual symptoms (London et al. 1983), prevention of cellular damage (Marubayashi et al. 1986), improved blood circulation (Kunisaki et al. 1998), tissue regeneration (Andıran et al. 2000), and intermittent claudication (Haeger 1974). Additionally, the antioxidant activity of tocopherols is associated with inhibition of membrane lipid peroxidation and the elimination of reactive oxygen species (Folmer et al. 2009).

1.2.4 Phytosterols

Phytosterols and phytostanols , the saturated form of phytosterols, are steroidal compounds similar to cholesterol but of plant origin. They vary only in their carbon side chains and/or presence or absence of a double bond (Ostlund 2002). Vegetable oils and vegetable oil-containing products are rich sources of phytosterols (Cavalcanti et al. 2013). Large-scale isolation of phytosterols is conducted mainly in two major raw materials, vegetable oils and tall oil (Fernandes and Cabral 2007).

The most common phytosterols in the human diet are β-sitosterol (65 %), campesterol (30 %), and stigmasterol (3 %). Among phytostanols, the most common in the human diet are sitostanol and campestanol, which together constitute about 5 % of dietary phytosterols (Cavalcanti et al. 2013). They are known to reduce low-density lipoprotein (LDL) serum cholesterol levels (Jones et al. 1997), and thus foodstuffs containing phytosterols are widely used as a dietary therapeutic option for reducing plasma cholesterol and the risk of atherosclerosis (Moghadasian and Frohlich 1999). Grain products, vegetables, fruits, and berries are not as rich in phytosterols as vegetable oils, but they can also be significant sources due to their high consumption, reaching 150–450 mg/day (Cavalcanti et al. 2013).

Phytosterols play a major role in several areas, including pharmaceuticals (production of therapeutic steroids), nutrition (anti-cholesterol additives in functional foods, anti-cancer properties), and cosmetics (due to their anti-inflammatory activity, which is an interesting property for anti-ageing products).

1.2.5 Squalene

Squalene (2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexaene) is a biosynthetic precursor to all steroids (He et al. 2001). Squalene is a symmetrical 30-carbon polyprenyl compound containing six prenyl units. Shark liver oil (Squalus spp.) is considered the richest source of squalene, and it is an intermediate metabolite in the synthesis of cholesterol. In humans, about 60 % of dietary squalene is absorbed and distributed in all tissues, being one of the major components in human epidermis lipids. Its major function is acting as an antioxidant, protecting the skin from lipid peroxidation (Briganti and Picardo 2003).

It is hypothesized that the lower risk of various cancers associated with high olive oil consumption could be due to the presence of squalene (Smith 2000). Research has also suggested that squalene has chemopreventive effects against colon cancer (Rao et al. 1998), and it has been recognized as having cholesterol-lowering effects (Smith et al. 1998; Kelly 1999) (Khor and Chieng 1997).

The traditional source of squalene is primarily shark (Centrophorus squamosus) and whale (Physeter macrocephalus) liver oils (Jahaniaval et al. 2000).

1.2.6 Essential Oils

Essential oils contain an important group of bioactive organic compounds that are responsible for the aroma and are involved in the defence mechanisms of many plants. EOs contain 85–99 % volatile and 1–15 % non-volatile components. The volatile constituents are a mixture of terpenes, terpenoids, and other aromatic and aliphatic constituents of low molecular weight. Terpenes are made from combinations of several five-carbon-base (C5) units called isoprenes. The main terpenes are the monoterpenes (C10) and sesquiterpenes (C15). Terpenoids are terpenes containing oxygen. Monoterpenes, formed from the coupling of two isoprene units, are the most representative molecules, constituting 90 % of the EOs. Aromatic compounds are derived from phenylpropane. Aldehydes (cinnamaldehyde), alcohols (cinnamic alcohol), phenols (eugenol), methoxy derivatives (anethole, estragole), and methylenedioxy compounds (myristicin, apiole) are examples of their aromatic components (Sánchez-González et al. 2011).

These compounds can be stored in several organs, such as flowers (orange, bergamot), leaves (lemongrass, eucalyptus, and menthe), bark (cinnamon), wood (sandalwood, rosewood), rhizomes (curcuma, ginger), fruits (star anise, fennel), and seeds (nutmeg) (Pereira and Meireles 2010).

Essential oils generally have a broad spectrum of bioactivity owing to the presence of several active ingredients that work through various modes of action. Known for their antiseptic—i.e., bactericidal (Friedman et al. 2002), virucidal (Garcia et al. 2003), and fungicidal (Ranasinghe et al. 2002)—and medicinal properties and their fragrance, they are used in embalmment, for food preservation, as antimicrobial (Burt 2004) and analgesic remedies, sedatives, anti-inflammatory (Silva et al. 2003) and spasmolytic (Sadraei et al. 2001) agents, and as a local anaesthetic (Bakkali et al. 2008). These properties have made essential oils an attractive candidate for use as ingredients in cosmetics, food, and pharmaceutical products (Pereira and Meireles 2010).

1.3 Methodologies for Extraction of Lipophilic Bioactives

Various extraction procedures have been used for the recovery of bioactive compounds from natural matrices. These include both conventional and non-conventional methods.

1.3.1 Conventional Extraction Techniques

Soxhlet extraction, hydro-distillation, and maceration with an alcohol–water mixture or hot fat are conventional techniques applied for the extraction of nutraceuticals from natural matrices using heat and/or agitation (Wang and Weller 2006; Azmir et al. 2013).

The Soxhlet extractor was first proposed in 1879 by Franz Ritter von Soxhlet, a German chemist, and it has since been widely adopted as a standard technique and the primary reference for assessing the performance of new extraction alternatives (Wang and Weller 2006; Azmir et al. 2013). With the exception of the extraction of thermolabile compounds, Soxhlet extraction is a generally well-established method, with performance exceeding that of other conventional extraction techniques. Briefly, a small amount of dry sample is placed in a thimble filter, which is then placed in a thimble holder above a distillation flask containing the solvent of interest. When the liquid reaches the overflow level, a siphon aspirates the solution passing through the thimble holder and unloads it back into the distillation flask, carrying extracted solutes into the bulk liquid. In the solvent flask, solute is separated from the solvent using distillation. The solute is left in the flask, and fresh solvent condensed by reflux after evaporation is passed back into the plant solid bed. The operation is repeated until complete extraction is achieved (Wang and Weller 2006; Azmir et al. 2013). The main criticism with this method is the length of time required to complete the process (Luque de Castro and Priego-Capote 2010).

Hydro-distillation is another traditional method for extraction of bioactive compounds and essential oils from plants. With this technique, organic solvents are not used, and it can be performed without drying the plant materials. There are three types of hydro-distillation: water distillation, water and steam distillation, and direct steam distillation (Palma et al. 2013). In water distillation, the solid matrix is sustained and packed in a still compartment and immersed in the boiling water or floating on it, depending on its density. In the water and steam distillation method, steam is also directly injected into the plant sample. Hot water and steam extract and release the bioactive compounds from the plant tissue. Indirect cooling by water condenses the vapour mixture of water and oil. The condensed mixture flows from the condenser to a separator, where oil and bioactive compounds automatically separate from the water. Lastly, in direct steam distillation, the solid matrix is supported on a perforated grid or screen inserted above the bottom of the still, but it is not in direct contact with the liquid water. The saturated steam flows up through the extracted solids, evaporating and gathering the volatile components (Palma et al. 2013), and the mixture is then condensed and separated as described above.

Hydro-distillation involves three main physicochemical processes: hydro-diffusion, hydrolysis, and decomposition by heat. This precludes its use for the extraction of thermolabile compounds, because some volatile compounds may be lost (Reverchon and Senatore 1992).

Maceration has become a popular and inexpensive way to extract essential oils and bioactive compounds, and it generally consists of several steps. First, the plant materials are ground into small particles to increase the surface area for proper mixing with the solvent. Next, an appropriate solvent, called menstruum, is added in a closed vessel. The liquid is then strained off, and the solid residue is pressed to recover as much occluded solution as possible. The strained and pressed liquids are mixed and separated from impurities by filtration (Palma et al. 2013). In order to facilitate extraction, occasional shaking can be used, increasing the diffusion and the removal of concentrated solution from the sample surface, while carrying fresh menstruum inward for higher extraction yields (Azmir et al. 2013).

1.3.2 Non-conventional Extraction Techniques

The major drawbacks of conventional extraction procedures include long extraction time, low extraction selectivity, thermal decomposition of thermolabile compounds, and evaporation of large amounts of solvent (Azmir et al. 2013). To overcome these limitations, new and promising techniques have been explored.

1.3.2.1 Supercritical Fluid Extraction

The use of supercritical fluids (SCFs) is often highlighted as an important strategy within green chemistry to replace harmful organic solvents and to enable green sustainable technologies (Leitner and Poliakoff 2008). The critical point is characteristic of each substance and it is schematically illustrated in Fig. 1.1. The state of a substance is called supercritical when temperature and pressure exceed the critical values.

Fig. 1.1
figure 1

Phase diagram for a pure component (Brunner 2005b)

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Going over the liquid-vapour line of a pure substance, the rise in temperature and pressure have different effects in each phase. When the temperature of a substance reaches its critical value, the densities of the gas and the liquid phases become identical, and the liquid is no longer distinguishable from the gas. At this point, only one phase exists and is now described as an SCF, whose properties range between those of gases and liquids, as shown in Table 1.2. Furthermore, SCFs are characterized by gas-like viscosities and solvating properties of a wide range of various organic solvents.

Table 1.2 Orders of magnitude of main physical properties of SCFs [adapted from (Brunner 2005b)]
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The most important feature of SCFs is that in the supercritical region (Fig. 1.1), minor variations in pressure and/or temperature lead to significant variation in density, which causes rapid changes in thermodynamic and transport properties.

Commensurate with the goal of environmentally benign processing is the use of liquefied or supercritical carbon dioxide (SC-CO2) for non-polar to moderately polar solutes. Environmentally benign carbon dioxide in its supercritical state has demonstrated significant potential for the development of a wide range of alternative processes that totally or partially eliminate the need for some of the most commonly used organic solvents. In addition to this factor, the rapid mass transfer properties associated with the lower viscosity of SCF carbon dioxide can lead to more time-efficient production capabilities for various types of important industrial processes (King and Srinivas 2009).

The extraction of compounds from natural sources is the most widely studied application of SCFs. In fact, supercritical fluid extraction (SCFE) has significant advantages over conventional solvent methods : it is an environmentally friendly technique, it enhances extraction efficiency and selectivity, and avoids the expensive post-processing of the extracts for solvent elimination. For the above-mentioned -reasons, carbon dioxide (CO2) is the most frequently used SCFE solvent. Extracts from supercritical processing with SC-CO2 can be regarded as all natural, and the products allowed for food applications may have GRAS (generally recognized as safe) status. In addition to the advantages related to non-toxicity and mild operating conditions, the extraction system provides a light- and oxygen-free environment, thereby minimizing degradation and preserving their bioactivity and antioxidant properties.

Typical SCFE systems for solid and liquid extraction are presented in Figs. 1.2 and 1.3, respectively. Basically, the system consists of pumps for delivering solvent and modifiers (co-solvents) throughout the system and for raising the pressure of the recycled solvent, a high-pressure extractor, a pressure reduction valve, heat exchangers, compressors, and one or more separators in which the extract is collected and the solvent (e.g., CO2) is depressurized and removed (Fig. 1.2) (Shi et al. 2012). A small amount of co-solvent (ethanol, water, etc.) increases the ability of SC-CO2 to dissolve polar compounds (Reverchon and De Marco 2006).

Fig. 1.2
figure 2

Schematic diagram of a supercritical fluid extraction system (Solid-scFluid extraction) used to fractionate bioactive components. 1 CO2 cylinder; 2 Co-solvent; 3 CO2 and co-solvent pumps; 4 Heat exchanger; 5 Extraction vessel; 6 Separators; 7 Back pressure valves; 8 Extracts outlet

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Fig. 1.3
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Schematic diagram of a supercritical fluid extraction system (Liquid-scFluid extraction) used to fractionate bioactive components. 1 Feed; 2 CO2 cylinder; 3 CO2 and liquid pumps; 4 Heat exchangers; 5 Extraction column; 6 Separators; 7 Back pressure valves; 8 Extracts outlet; 9 Raffinate

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Column fractionation can be done with a column operating in two different modes: cross-current and counter-current. The latter is the most popular. In such a counter-current separation process, the components are distributed between the solvent (extract phase) and the liquid (raffinate phase) which flow in a counter-current manner through the column, reducing the amount of solvent needed and increasing throughput. Counter-current processing with SCFs extends the possibilities of separation processes like distillation, absorption and liquid–liquid extraction to the isolation and purification of components of low volatility, allowing separation of components with very similar properties (Fig. 1.3).

The industry has recently focused on “fractional separation”, where the natural materials are extracted under relatively severe pressure and temperature conditions to remove all the desired components. The resulting fluid extract is then passed through a series of two, three, or four separator vessels in which the operating parameters (temperatures and pressures) in each vessel are set to selectively precipitate one specific component of interest. This can create a range of unique fractions with new application potential.

The past 20 years have seen an expansion of the “critical fluid” technology platform with respect to using or combining multiple types of unit operations and compressed fluids in both their sub- and supercritical states. For example, combining SCFE with fractionation methods using SCFs, and/or reaction chemistry in critical fluid media can produce a variety of extracts or products (King 2000).

Table 1.3 summarizes the advantages, disadvantages, parameters to control, and ease of scale-up of SCFE, in particular with carbon dioxide.

Table 1.3 Advantages, disadvantages, parameters to control and ease of scale-up of SCFE
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In summary, SC-CO2 is suitable for applications in which (i) processing costs are not a limiting factor, (ii) conventional solvent extraction is restricted by environmental regulations, (iii) consumer demand or health considerations play an important role, (iv) a “natural” label on the product is required, and/or (v) the application of traditional extraction techniques does not preserve the bioactive properties of the product. The major disadvantage of SCFE is the expensive equipment. With regard to the process itself, the drawbacks are the high pressure required and the associated hazards, which have hindered wider application.

SCFE has been commonly used for the extraction of flavours, essential oils, seed oils, antioxidants, and bioactives from several natural sources. Some of these applications have been reviewed extensively (Reverchon and De Marco 2006; Herrero et al. 2006a; Cheah et al. 2006; Díaz-Reinoso et al. 2006; Sahena et al. 2009a; Catchpole et al. 2009), and are summarized in Table 1.4.

Table 1.4 High-added-value compounds extracted with supercritical fluid extraction (Reverchon and De Marco 2006; Herrero et al. 2006a; Cheah et al. 2006; Díaz-Reinoso et al. 2006; Sahena et al. 2009a; Catchpole et al. 2009; Adil et al. 2007, 2008; Ibáñez et al. 2000; Nobre et al. 2009)
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1.3.2.2 Other Non-conventional Extraction Techniques

An overview of other non-conventional extraction techniques, including ultrasound-assisted extraction, microwave-assisted extraction (MAE), and pressurized liquid extraction (PLE), is presented in Table 1.5, along with the principles, main advantages, limitations, and information regarding the scaling up of each technology.

Table 1.5 Conventional solvent extraction and Non-conventional extraction techniques: principle of the method, advantages, disadvantages, parameters to control, and scale-up
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1.4 Extraction of Lipophilic Bioactives

1.4.1 Carotenoids

Carotenoid extraction has traditionally been performed using organic solvents that are associated with the known problems of low selectivity and adverse environmental impact. For these reasons, the use of SCF has been explored for their extraction. Because carotenoids show very low or moderate solubility in SC-CO2, depending on the molecular structure (molecular weight and polarity), suitable modifiers (co-solvents) have been employed to enhance the solubility of the target compounds. The most frequently used co-solvent is ethanol, since its presence (in traces) in final products does not compromise their use in food, nutraceutical, and pharmaceutical applications (Reverchon and De Marco 2006). Vegetables oils have also been used as potential modifiers for the extraction of carotenoids (Longo et al. 2012).

SCFE of lycopene, a carotenoid with relevant antioxidant activity and protective effects against coronary heart disease and cancer, was performed using pure CO2 with the modifiers (co-solvents) ethanol, olive oil, and hazelnut oil (Ciurlia et al. 2009; Perretti et al. 2013; Vasapollo et al. 2004; Shi et al. 2009; Baysal et al. 2000), and high recovery yields were obtained. Ultrasound-assisted extraction with organic solvents (petroleum ether, acetone, n-hexane, ethanol, and ethyl acetate) has also been explored (Konwarh et al. 2012; Eh and Teoh 2012; Lianfu and Zelong 2008). Table 1.6 summarizes studies involving lycopene extraction from tomato and tomato by-products.

Table 1.6 Extraction of lycopene and β-carotene from tomato and tomato by-products using SC-CO2 extraction and UAE
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In other works, astaxanthin, β-carotene, and other carotenoids have been extracted by SC-CO, MAE, ultrasonic-assisted extraction (UAE), and PLE from microorganisms and small marine animals that produce high levels of carotenoids. These studies are summarized in Table 1.7, and extraction yields, when available, are presented.

Table 1.7 Extraction of carotenoids from selected microorganisms and marine organisms by SC-CO2, UAE, MAE and PLE
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1.4.2 Fatty Acids

Several researchers have investigated SC-CO2 extraction of oils rich in ω-3 fatty acids, and have demonstrated that this technology has a negligible environmental impact, and is thus also a potential tool for functional foods and nutraceuticals applications. For example, Cheung et al. (1998a) compared the yields of ω-3 fatty acids extracted from brown seaweed (Sargassum hemiphyllum) using SC-CO2 (P = 24.1–37.9 MPa; T = 313/323 K) versus Soxhlet extraction with chloroform/methanol as control. Extraction conditions of 37.9 MPa/313 K and 37.9 MPa/323 K gave the highest lipid yield, which was comparable to that of the Soxhlet method (Fig. 1.4), and the highest concentrations of total and individual ω-3 fatty acids, which were significantly higher than with solvent extraction (Fig. 1.5).

Fig. 1.4
figure 4

Original lipid extracted (mg/g of dry weight) from S. hemiphyllum using SC-CO2 and Soxhlet solvent extraction (Cheung et al. 1998b)

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Fig. 1.5
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Extraction yield of ω-3 fatty acids (mg/g of dry weight) from S. hemiphyllum using CO2 and Soxhlet solvent extraction (Cheung et al. 1998b)

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Amiguet et al. (2012) recently investigated the potential of northern shrimp by-products as a source of omega-3 polyunsaturated fatty acids (ω-3 PUFAs). Supercritical CO2 extraction (SCFE) of shrimp by-products, at 35 MPa and 313 K, generated a deep red oil, rich in ω-3 PUFAs, specifically 7.8 ± 0.06 % eicosapentaenoic acid (EPA) and 8.0 ± 0.07 % docosahexaenoic acid DHA).

The SCFE of the omega-3-rich oil contained in hake by-products of the fish industry were studied by Rubio-Rodriguez et al. (2008) and the results are presented in Fig. 1.6. Extraction conditions of 25 MPa/313 K gave the highest lipid yield, which was comparable to that of the Soxhlet method (Fig. 1.6), and the highest concentrations of total fatty acids (FA), monounsaturated FA (MUFAs), polyunsaturated FA (PUFAs), EPA + DHA, ω-3, and ω-6 fatty acids, which were significantly higher than with the solvent extraction.

Fig. 1.6
figure 6

Fatty acid profile of hake oil extracted with SC-CO2 and with hexane as determined by AOAC method (Rubio-Rodríguez et al. 2008)

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High-value seed oils are those that contain one or more PUFAs with desirable bioactivity. The most commonly extracted oils are those rich in γ-linoleic acid (GLA or C18:3 ω-3) and α-linoleic acid (ALA or C18:3 ω-6). They are traditionally produced by the extraction of hexane from ground seeds. Despite the efficiency of this extraction method, however, solvent elimination after extraction and possible thermal degradation of the oil represent major problems. Therefore, several authors have proposed the substitution of the traditional process with SC-CO2 extraction of oil from seeds. In fact, the extraction of these oils by SC-CO2 has reached the commercial stage, and the most commonly studied seeds are evening primrose, borage, blackcurrant, kiwifruit, hemp, and rose hip (Catchpole et al. 2009).

Wang et al. (2011) recently studied the fatty acid composition of tea (Camellia sinensis L.) seed oil extracted using optimized SC-CO2. The objectives of this study were to employ SC-CO2 to extract oil from tea seed and to determine the fatty acid composition of the extracted oil and its antioxidant activity. The authors then compared the extracts obtained by SC-CO2 and by Soxhlet extraction. The total yield of tea seed oil by Soxhlet extraction was 25.3 ± 1.0 %, significantly lower than that (29.2 ± 0.6 %) using SC-CO2 under optimal conditions.

The fatty acid (FA) profiles of tea seed oil extracted by SC-CO2 and Soxhlet extraction (SE) were analysed by GC, and the results were similar (Fig. 1.7).

Fig. 1.7
figure 7

Comparison of main fatty acid content of tea seed oil by different methods [adapted from (Wang et al. 2011)]

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The tea seed oil extracted by SC-CO2 showed much stronger DPPH radical scavenging ability than the oil extracted by Soxhlet.

Table 1.8 presents additional studies that explored the use of SC-CO2 technology to recover fatty acids from plants, fish, algae, and fruits. non-conventional extraction techniques, namely MAE, UAE, and PLE, with hexane, ethanol, water, and acetyl lactate have been revealed as alternative technologies for isolating fatty acids and fatty acid methyl esters (FAME) from natural sources (Table 1.8).

Table 1.8 Extraction of Fatty-acids by SC-CO2, UAE, MAE and PLE
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1.4.3 Tocopherols and Tocotrienols

The extraction of vitamin E from natural sources has attracted increasing interest due to the high antioxidant activity associated with this family of compounds. Moreover, natural vitamin E has been demonstrated to be more effective than a synthetic form (Friedrich 1987). Several natural matrices have been used as sources of vitamin E and tocopherols, and SCFE has been highlighted as one of the most promising methodologies for recovery of these compounds.

Wheat germ is an important source of vitamin E, and some studies have confirmed that SCFE (with CO2) can achieve extraction yields for tocopherols similar to traditional hexane extraction (Molero and Martinez de la Ossa, 2000). In particular, Ge et al. (2002a) demonstrated that the extract derived from SCFE using the best operating conditions (P = 15 MPa; T = 213 K; CO2 flow rate 1.5 L/min) presented higher quality (free fatty acids, 12.4 %; tocopherol content, 416.7 mg tocopherol/g wheat germ oil). Similarly another study performed with wheat germ showed higher extracted amounts of vitamin E and its isomers than those obtained using traditional methods (with n-hexane or chloroform/methanol mixtures) (Fig. 1.8). Thus, the extraction process using CO2 may be economically competitive with conventional processes , as it shortens the oil refinement steps significantly and avoids the solvent distillation stage, both of which are very costly in terms of energy consumption.

Fig. 1.8
figure 8

Yield comparison of vitamin E and its isomers extracted by SCFE-CO2 (P = 27.6 MPa; T = 313 K) with those by conventional solvent extraction (Ge et al. 2002b)

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Table 1.9 summarizes published works regarding the extraction of tocopherols through SC-CO2 and other non-conventional extraction techniques, namely MAE, UAE, and PLE.

Table 1.9 Extraction of tocopherols and tocotrienols by SC-CO2, UAE, MAE and PLE
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1.4.4 Phytosterols

Plant-derived sterols in tissues and oil seeds can be isolated by solvent extraction with hexane, chloroform, diethyl ether, and acetone. However, these extraction procedures require large amounts of organic solvents, which are often expensive and potentially harmful. Extraction with supercritical carbon dioxide fluid constitutes an alternative that is advantageous, at least in terms of environmental impact (Lu et al. 2007).

For instance, King and Dunford (2002a) described a two-step supercritical fluid fractionation (SFF) method for obtaining phytosterol-enriched triglyceride fractions from rice bran and soybean oil deodorizer distillates (DD). The method comprised an extraction at 13.6 MPa and 318 K as a first step in order to remove the free fatty acids (not desirable in edible oils), while in a second step, the sterol ester-enriched triglyceride fractions were collected at 20.4 MPa and 353 K. With this method it was possible to obtain oil fractions with ~20 and 30 % (w/w) sterol content from rice bran oil and soybean oil DD, respectively, with relatively low free fatty acid content (Fig. 1.9) (King and Dunford 2002b).

Fig. 1.9
figure 9

Comparison of sterol and lipid composition of final products obtained using a two-step SFF method (rice bran oil SFF and soybean oil SFF) with rice bran oils DD and soybean oil DD as feeds of SFF (King and Dunford, 2002a)

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The two-step SFF method demonstrated improvement over conventional methods used to obtain phytosterol-enriched products.

Huang et al. (2007) extracted β–sitosterol, stigmasterol, and ergosterol from Anoectochilus roxburghii. Under optimal conditions, i.e., pressure of 25 MPa, temperature of 318 K, and using ethanol as modifier, the concentrations of β-sitosterol, stigmasterol, and ergosterol in the extract were found to be 2.89, 3.56, and 2.96 (g/g %), respectively (Fig. 1.10).

Fig. 1.10
figure 10

Comparison of concentration of sterols obtained by SCFE and Soxhlet extraction [adapted from (Huang et al. 2007)]

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The concentrations of β-sitosterol, stigmasterol, and ergosterol in the extract were 4.7, 4.0 and 4.0 times higher, respectively, when SCFE was used compared with Soxhlet extraction. SCFE produced higher yields of sterols than did Soxhlet extraction.

Table 1.10 presents published works involving the extraction of phytosterols through SC-CO2, MAE, UAE, and PLE.

Table 1.10 Extraction of phytosterols by SC-CO2, UAE, MAE and PLE
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1.4.5 Squalene

The importance of squalene in the cosmetic and pharmaceutical industries has driven the development of new technologies, such as counter-current SCFE, for the purification of squalene from various sources. The primary source of squalene is shark liver oil, but it can also be found in olive oil by-products.

The recovery of squalene from shark liver oil by SCFE has been reported in the literature (Catchpole et al. 2000a, b). This raw material also contains many triacylglycerols, alkoxyglycerols, sterol esters, and pristine squalene. Therefore, extraction process conditions are needed that allow the highest solubility of only squalene, in order to maximize the purity and yield of the extract. This is also the case when olive oil DD containing free fatty acids and methyl and ethyl esters are used as raw materials.

Al-Darmaki et al. (2012) recently studied the extraction and recovery of squalene using SC-CO2 as solvent from palm fatty acid distillate (PFAD). The process was carried out on a counter-current glass bead-packed column. The operating conditions investigated were pressure and temperature, which varied from 10 to 20 MPa and from 313 to 353 K, respectively (Al-Darmaki et al. 2012).

Table 1.11 presents three studies related to successful isolation of squalene-rich extracts from shark liver oil and olive oil by-products.

Table 1.11 Extraction of squalene from animal and vegetable sources
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1.4.6 Essential Oils

Essential oils have become a target for the recovery of natural bioactive substances. According to Fornari et al. (2012), since 2000, nearly 4000 articles have been published in which “essential oil” or “volatile oil” appeared as keyword, and around 3000 of these also included the word “bioactive” or “bioactivity” in the article text (Fornari et al. 2012). Moreover, essential oils and their extraction have been reviewed extensively ( Moyler 1993; Chen and Spiro 1994; Kerrola 1995; Reverchon 1997; Burt 2004; Lucchesi et al. 2004; Edris 2007; Mustafa and Turner 2011; Mason et al. 2011; Xu et al. 2011a; Sovová 2012; Abad et al. 2012; Fornari et al. 2012; Kokolakis and Golfinopoulos 2013; Capuzzo et al. 2013). For this reason, an overview of selected publications regarding the extraction of essential oils using different methodologies is presented instead of a resume table.

In industrial practice, essential oils are obtained by steam distillation; in laboratory practice, steam distillation and hydro-distillation are used. However, processing temperature is a drawback in the extraction of thermally labile compounds (Pereira and Meireles 2010). In order to overcome this, SCFs have been used successfully for the recovery of these substances, and in some cases, comparisons with conventional methods have been performed.

Chamomile flower heads, which are widely used in the cosmetic, pharmaceutical, and food industries due to their biological activity (anti-spasmodic, anti-inflammatory, and antimicrobial properties), were extracted using SC-CO2 technology (Kotnik et al. 2007). Various pressures (10, 15, and 25 MPa) and temperatures (303 and 313 K) were assessed, with the highest extraction yield achieved at 25 MPa and 313 K. The results were further compared with those obtained using Soxhlet extraction, steam distillation, and maceration (Fig. 1.11), and SC-CO2 was found to offer considerable advantages over the others. Although the yield was lower, the extract obtained with SC-CO2 had much higher content of the active compounds matricine and bisabol (Fig. 1.12). In addition, using SC-CO2, the matricine content was able to be increased almost threefold by using two-step separation procedures. It is also important to note that the process was successfully implemented at a pilot scale.

Fig. 1.11
figure 11

Extraction Yield (%) of chamomile flower head with SCFE and conventional methods [adapted from (Kotnik et al. 2007)]

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Fig. 1.12
figure 12

Content of active components in chamomile extracts obtained by SCF extraction at 25 MPa, 313 K, with SCFE and conventional methods [adapted from (Kotnik et al. 2007)]

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The same was observed in a study conducted by Moura et al. (2012), which evaluated SCFE of essential oils using ethanol and isopropyl alcohol as co-solvents. The global yield obtained with conventional techniques, including Soxhlet extraction, low-pressure solvent extraction (LPSE), ultrasound, and hydro-distillation, were compared with the global yield of SCFE (Moura et al. 2012). Essential oils were detected in extracts from all extraction methods used in this study (Fig. 1.13). Although higher global yields were obtained with conventional extraction methods, SCFE resulted in better recovery of essential oils.

Fig. 1.13
figure 13

Global yield and relative intensity of essential oils in extracts of P. guajava leaves from different extraction processes [adapted from (Moura et al. 2012)]

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In another work, three methods—hydro-distillation, PLE, and SCFE—were optimized and compared for the extraction of volatile compounds from Cyperus rotundus. The study showed the highest extraction efficiency with PLE, while SCFE had the best selectivity for the extraction of β-cyperone and α-cyperone (Tam et al. 2007).

The extraction of lavender essential oil was investigated using hydro-distillation, SC-CO2, and hexane extraction techniques, and results were compared in terms of yield, chemical composition, and antimicrobial and antioxidant activity. SC-CO2 produced a yield of 6.7 % (dry weight), which was comparable to that of solvent extraction (7.6 %) but significantly higher than that of hydro-distillation (4.6 %). The chemical composition of the oils showed considerable variation among extraction methods. Hexane extraction produced oils with the presence of waxes, colour pigments, and albuminous materials with semi-solid consistency, while hydro-distillation extracts showed evidence of thermal degradation. The SC-CO2 extract had an aroma most closely resembling the starting material, showed negligible thermal degradation, and exhibited significantly higher antioxidant activity than the hydro-distillation and hexane extracts. Antimicrobial activity was higher in oils produced by SC-CO2 and hydro-distillation than by hexane extraction. The results of this study demonstrate that SC-CO2 is a promising technique for the extraction of lavender essential oil (Danh et al. 2013).

In a study by Okoh et al. (2010), Rosmarinus officinalis essential oil extracts obtained by hydro-distillation and microwave extraction were compared in terms of yield and antibacterial activity. Briefly, the total yield of the volatile fractions obtained through hydro-distillation and microwave extraction were 0.31 and 0.39 %, respectively. With regard to the minimum inhibitory concentration (MIC) of oils obtained by the two methods, results showed greater activity against microorganisms in the oil obtained by microwave extraction than that obtained by hydro-distillation. This observation was explained by the fact that the microwave-extracted oil contained more oxygenated compounds, and that this class of compounds has been proven to possess strong antibacterial and antifungal activity. This result shows that the composition of essential oils may vary depending on the method of extraction used (Okoh et al. 2010).

In a study by Herzi et al. (2013), the extraction of two species of Eucalyptus by SC-CO2 and hydro-distillation were compared in terms of yield, chemical composition, and antioxidant activity. Superior yields were obtained using SC-CO2. The antioxidant activity of the extracts was also assessed using the two methods, and promising radical scavenging activity was observed in the SC-CO2 extracts. In this work, SC-CO2 demonstrated important advantages compared to hydro-distillation, including faster extraction, improved yield, and extracts with high antioxidant quality (Herzi et al. 2013).

1.5 Process Optimization for Targeting Bioactivity

Over the last few years, special focus has been given to the design and optimization of extraction processes for the recovery of mixtures of bioactive molecules with potential health benefits, rather than the isolation of pure and single compounds. In this field, the recovery of anticancer-bioactive-rich fractions from natural sources has attracted the attention of the scientific community in their efforts to develop new natural chemotherapeutic agents. For this propose, high-pressure processes, including SCFE, were the most common recovery technologies applied. For example, in the work performed by Serra et al. (2010) a high-pressure fractionation process was optimized to isolate compounds with antiproliferative effects against colon cancer, including perillyl alcohol from sweet cherries (“Saco” variety). The methodology employed comprised a first step with SC-CO2 followed by a second step using mixtures of CO2 and ethanol. The effect of SC-CO2 pre-treatment and the influence of the ethanol concentration in the solvent mixture composition was studied in relation to extract yield, phenolic content, and antiproliferative effects in human colon cancer cells. The product derived from CO2:ethanol (90:10 v/v) extraction exhibited the highest antiproliferative activity, likely due to the presence of perillyl alcohol and high polyphenol content in the resulting extract (Fig. 1.14).

Fig. 1.14
figure 14

Antiproliferative effect in HT29 colon cancer cell line of different cherry extracts produced by high-pressure technology (P = 250 bar, T = 323 K) (results obtained after 96 h of treatment with 0.5 mg/mL of extract). Comparison of bioactive effect with phenolic content (filled circle)) and presence of perillyl alcohol (dark filled bar-well-defined zone detected; light filled bar-less-defined zone detected; unfilled bar-none detected) [adapted from Serra et al. (2010)]

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This methodology was later applied to other sweet cherry varieties, as well as to plum and peach pomaces, in order to evaluate its effectiveness on the isolation of bioactive fractions with anticancer properties from these natural sources (Silva 2013). Results showed that all extracts inhibited colon cancer cell growth and exhibited similar terpenes profiles (Fig. 1.15), evidencing the selectivity of this extraction methodology.

Fig. 1.15
figure 15

Characterization of cherry, peach and plum extracts obtained using high-pressure technology (P = 250 bar, T = 323 K, CO2 + EtOH 10 %). a Antiproliferative effect on HT29 cell line after 24 h of treatment- IC50 values; b Terpene profile detected by TLC (Legend: 1 perillyl alcohol standard; 2 Linalool standard; 3 Brucks cherry; 4 Sweet Hearth cherry; 5 plum; 6 peach) [adapted from Silva (2013)]

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In a study performed by Vicente et al. (2013) SCFs technology was explored for producing rosemary extracts of different composition and their anticancer effects in human liver carcinoma cells. Several extraction conditions employed produced different extract composition in terms of carnosic acid and volatile compounds, namely 1,8 cineole, camphor, borneol, verbenone, and bornyl acetate. Results showed that the conditions P = 15 MPa, 5 % (w/w) of ethanol as co-solvent and t = 180 min obtained a fraction with an effective composition of carnosic acid and volatile compounds demonstrating the highest antiproliferative potential in cancer cells (Fig. 1.16). Among compounds, carnosic acid was shown to have a crucial effect on growth inhibition conferred by rosemary extract. Moreover, other substances comprising the volatile oil fraction may synergize with the rosemary compound in its anti-tumoural action.

Fig. 1.16
figure 16

Comparison of the antiproliferative effect in HepG2 cells of rosemary extracts obtained by supercritical fluid technology with composition in terms of carnosic acid and volatile compounds. Rosemary extracts were obtained using supercritical fluid technology under different pressure conditions and percentages of co-solvent (ethanol); (IC50 values were obtained after 48 h of treatment) (Vicente et al. 2013)

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Together, these studies demonstrate the importance not only of developing methods for isolating individual compounds, but also of optimizing processes for separating fractions rich in synergistic mixtures of bioactivity.

1.6 Summary

Lipophilic compounds of various structures have shown promising bioactivity. In this chapter, conventional and new alternative techniques for the recovery and isolation of high-value lipophilic bioactives have been reviewed, with particular emphasis on supercritical fluid extraction.