ObtainingAntioxidants from Botanic Matrices Applying Novel Extraction Techniques

Botanic matrices are abundant sources of antioxidants which have the capacity to avoid the lipid oxidation of food and present remarkab le health benefits. The natural antioxidants might be obtained applying many extraction techniques. Satisfactory results of obtaining extracts with antioxidant properties and high yields using modern extraction techniques are shown by recent studies. The selection of the suitable technique depends on the desired class of substances to be extracted. In this overview, the advances reached in scientific researches involving natural antioxidants are presented. The advantages and potential applicat ions of four novel ext raction techniques: Supercritical Flu id Extraction, Pressurized Liquid Extraction, Microwave Assisted Extraction and Ultrasound Assisted Extraction are discussed, considering the characteristics of the target compounds. These techniques reduce the solvent consumption and abridge the extraction time. Consequently, the process productivity is increased.


Introduction
The interest in the prevention of chronic d iseases, such as cancer, has led to modificat ions in the nutritional composition of foods in the last years. These modified foods are classified as functional foods because they contain, besides the basic nutrition, co mponents that provide health benefits [1]. The continued ingestion of food supplemented with antio xidant substances cause inhibitory effects on the proliferation o f carcinogenic cells in human beings [2][3][4]. These effects appear because the antioxidant substances are able to perform so me functions, as free radical scavenging, peroxide deco mposition, suppression of singlet oxygen, enzy matic inhibit ion [1] and increasing the levels of endogenous defences [5].
In general, there are two categories of antio xidants: natural and synthetic. The natural antio xidants comprise a wide variety of substances found in the nature, such as polyphenols (flavonoids and phenolic acids), terpenoids and vitamins E and C. The phenolic co mpounds present high antioxidant capacity in b iologic and food systems, especially the flavonoids group [6].
Th e iden t ificat ion o f the ch emical co mp os ition o f extractsfro m several botanic mat rices that present agents with potential antio xidant properties has been the focus of 2.1. 1

. Flavonoids
Flavonoids are secondary metabolites of low mo lecular massproduced by plants, which belong to the class of phenolic compounds and present high antioxidant activity [7]. They act as antioxidants because they have many active sites to scavenge free radicals [8]. The flavonoids are divided into groups according to their chemical structures, as flavones and isoflavones, flavanones, flavonols, flavanols and anthocyanins (Table 1).
Living organis ms have an o xidation-reduction system necessary to keep the level of generated free radicalsconstant. The format ion of free radicals in higher levels than the ideal induces cellular o xidative stress that leads to lipid peroxidation of the cellu lar memb rane, which can cause degenerative diseases and aging [9]. Therefore, scientific investigations search for solutions to avoid the cellular oxidation by the supplementation of antio xidant co mpounds in food. There are evidence that flavanols, flavanones and anthocyanidins have beneficial effects to the memory, perception and neurodegeneration [10].
Hemodialysis patients face an elevated risk of cancer, ascribed in part to increased oxidative stress. Anthocyanins, present at high amounts in red fru its, show a good efficacy on the reduction of the oxidative damage in these individuals through the decrease on the risks of DNA o xidation and on the lipid and protein pero xidation [11].
Saponarin, an antio xidant belonging to the flavones group and that was recently found in barley leaves, inhibited the malonaldehyde fo rmation. In a normal reaction, malonaldeh yde is formed fro m o xidized lip ids on the skin surface by ultraviolet irradiation [12].
Naringin, a dietetic flavanone, is an effective antioxidant for the prevention of oxidative stress and for the protection against liver carcinogenesis in rats [13]. In human beings, phenolic extracts obtained from apple and grape containing flavonoids showed potential protection of lung cells exposed to the oxidative stress [14].
Proanthocyanidins ext racted fro m blueberry (Vaccinium angustifolium) are ab le to reduce the cognitive function loss by the protection against the deficient Ca 2+ recovery and moderate o xidative / inflammatory stress signalling [15]. Flavonoids extracted fro m fennel (Foeniculum vulgare) present antitumoral effect by modulat ing the lip id peroxidat ion [16]. Flavonoids extracted fro m carob (Ceratonia siliqua), mostly the miricetin, caused biochemical changesin rats physiologic system, suggesting protection of liver and renal cells by the capacity of free radicals scavenging [17].
Flavanols extracted fro m lychee (Litchi chinensis) were supplemented on the diet of 20 healthy ma le long-distance runners. The decrease of the cellular o xidative stress and the reduction of the tissue damage caused by high-intensity e xerc ise training were observed [18].

Non-flavonoids
The non-flavonoids compounds are phenolic acids which present a functional carbo xy l g roup, divided into benzo ic and cinnamic acids derivatives [7]. Hydro xycinnamic acids are present in many foods, such as coffee, yerba mate, apple and plu m (Table 1) [19]. The phenolic acids present importa nt biologic and pharmacologic properties, part icularly on cancer prevention [20].
Caffeic acid, a hydro xycinnamic acid found in high concentrations in fruits and coffee beans, induces the apoptosis of human breast cancer cells [21]. The use of this phytochemical for the protection against disturbances of the antioxidant defense system has been tested as the possible mechanis m whereby botanic compounds slow down the skin aging process. Pretreatment of skin cells with caffeic acid prior UVA (ultrav iolet A) irradiat ion inhibits cytotoxicity, induction of metalloprotease-1 (enzy me responsible for the damage caused on collagen) and free radicalsgeneration [22].
Ferulic acid is a powerfu l phenolic antio xidant and photo-protector obtained from p lants such as corn, rice, tomato, peanut, apple, orange and pineapple. Ferulic acid decreases the absorption of UVB (ultrav iolet B) radiat ion on human epidermis and inhibits the fo rmation of tu mors, because this compound blocks the secretion of cytokines generated after the skin is exposed to the UVB radiat ion [23].

Ter penoi ds
Terpenoids are classified according to the number of carbon atoms in their chain.β-carotene and lycopene are tetraterpenoids (carotenoids formed by 40 carbons) [24]. The main vegetal sources of carotenoids and their mo lecular structures are listed in Table 1.
Scientific ev idences link the antio xidant properties of carotenoids with their beneficial effect against chronic diseases. Annatto (Bixa orellana) ext ract, constituted by carotenoids with bixin as major co mpound, was identified as a potential therapeutic agent for modulation of the equilibriu m of reactive o xygen and nit ric o xide species, two substances that induce diabetes [25]. Annatto constituents were also studied as toxic agents against a wide variety of tumor cells. Cis-bixin has the capacity of inhibiting the enzy mes associated with the oxidative stress [26].
Experimental assays point out that lycopene can protect the organism against damages caused by the exposure to tobacco [27], [28], and it is beneficial on the treat ment of acute and chronic pancreatitis by reducing intracellular free radicals [29]. β -carotene can help the prevention of prostate cancer [30]and gastric carcino ma [31].

Vitamin E
The supplementation with v itamin E in the d iet of 180hea lthy elderly people during 4 months apparently alleviates the oxidative stress by improving the erythrocyte me mbrane flu idity and by reducing the erythrocyte hemolysis [32]. Vitamin E tested in rats inhib ited the formation of o xygen reactive species, decreased the level of lip id pero xide, increased the levels of glutathione and lipid pero xidation enzy mes and presented the capacity to prevent the mitochondrial apoptosis [33].

Modern Techniques for Extraction of Antioxidants
Antioxidants are substances that, in lo w concentrations, inhibit or prevent the oxidation of other substances [83]. Many foods still contain synthetic antioxidants in their formulat ions, as Butylated Hydroxytoluene (BHT) and Butylated Hydro xyanisole (BHA ), because they are thermally stable and of low cost. However, experimental investigations show that BHT and BHA are carcinogenic and cytotoxic above 500 pp m [84]. The maximu m reco mmended BHT daily intake is 0.125 mg/kg of body mass and the maximu m reco mmended BHA daily intake is 0.5 mg/kg of body mass. In the European Union the use of BHT and BHA in food prepared for babies and young children is not allo wed [85].
Botanic matrices are abundant sources of nutraceutical compounds. The natural antio xidants, belonging to the GRA S (Generally Recognized As Safe) group of FDA (Food and Drug Admin istration), are extracted fro m herbs or plants and are commonly phenolic co mpounds that present health benefits, as the prevention of diabetes, cancer, hypertension, asthma and in fections [93], [94].
The separation or the isolation of the target compounds fro m their orig inal matrix is the method used to obtain these antioxidant substances. Conventional extraction techniques (steam distillat ion and Soxh let extract ion, for instance) possess some drawbacks due to the use of high temperatures and/or high amounts of organic solvents; another limitation is that the steam distillat ion process can be used only to obtain volatile oils (mostly terpenes). These conventional techniques are being substituted by novel techniques, as supercritical flu id ext raction (SFE) [95][96][97][98]; pressurized liquid extraction (PLE) [90], [99], [100]; microwave assisted extraction (MA E) [101][102][103]; and ultrasound assisted extraction (UA E) [104], [105]. The choice of the suitable technique depends on: the desired class of co mpounds to be extracted; the structural characteristics of the botanic matrix (fru its, stems, seeds, leaves, root, flowers, etc.); the quality and yield required for the extract; the process conditions (temperature, pressure, etc.) and the economic feasibility for scaling up the process.
For instance, SFE using pure CO 2 is mo re appropriate for extracting nonpolar co mpounds as terpenoids, tocopherols and sitosterols [106], while PLE is more appropriate for extracting polar antio xidants as the phenolic compounds: anthocyanins [107] and flavonols [108]] using solvents with high polarity. MAE is an extraction technique indicated when the botanic matrix contains large amounts of water, because the water is responsible for the absorption of the energy generated by the microwaves. This energy disrupts the cells and facilitates the release of chemical constituents [109]. The UAE technique is also suitable for obtaining antioxidants. The characteristic of UAE is the reduced solvent consumption [110], the possibility of processing several samples in the same equipment and the short extraction time [109].
The solvent selection for the ext raction is based on some factors, as: physicochemical properties, availab ility, cost and toxicity. The choice of the ideal solvent should consider its selectivity, as well the solute solvating capacity, interfacial tension, viscosity, stability and reactivity [109].

Historical Aspec ts
The application of supercrit ical technology in obtaining bioactive compounds has evolved over the past decade. However, the divulgation of the investigations related to this area in patent form started in the early of 1970 when the first patent comprising a process for recovering caffeine fro m green coffee using carbon dioxide in supercritical conditions was registered by Zosel [111]. In 1981, another patent was published dealing with the decaffeination of coffee [112]. Fro m that date to now, over than 300 patents were registered and are available at "Web of Knowledge" database. One of these recent patents comprises a useful method for preparing carotenoid microcapsules with a controllable iso meric rat io applying a supercritical fluid at high temperatures [113]. In the same way, another invention utilizes olive by-products for the isolation and separation of tocopherols with supercritical fluids [114]. In 1997, a inovative study was published dealing with the effects of ultrasound on mass transfer in SFE [115]. This coupled system has been investigated currently in the extraction of lutein esters from marigold [116] and of o il fro m adlay seed [117].
The number of scientific investigations published after the year 2000 co mprising modern ext raction techniques for obtaining antioxidants has significantly risen by 2012. Figure 1 shows this tendency, whereby searches in "Scopus" and "Web of Knowledge" database inserting the terms "SFE and antioxidant" returned together more than 780 documents in the year 2012, while less than 80 documents covering the same subject were published in the year 2000. The same search procedures were used for PLE, MAE and UAE. These novel techniques also present an important evolution in the scientific scenario, mainly in the last five years where the number of publications related to MAE and UA E in Scopus, for instance,increased from 150 and 102 to 551 and 393, respectively. The participations of SFE, PLE, MAE and UAE in obtaining natural antio xidants referent to the overall techniquesin 2003 have been 5.9%, 1.0%, 1.4% and 0.9%, respectively. In 2012, these relative participations have increased to 9.5%, 4.3%, 7.5% and 5.4%, respectively. Therefore, the contribution of both techniques in 2003 has been 9.2%, wh ile in 2012 it has been 26.7%, a lmost 3 times higher than ten years ago.

Supercritical Flui d Extracti on (SFE)
Extraction of bioactive compounds with conventional solvents is characterized by low selectiv ity and may require high temperature [118]. Because of these limitations, SFE has some characteristics that justify its use for obtaining natural antio xidants. CO 2 , the solvent mostly used in SFE, presents critical temperature of only 304 K, wh ich allo wsits use for the ext raction of thermo sensitive (thermolabile) compounds.Several phytochemicals show high solubility in CO 2 around supercritical conditions (304 K/ 7.4 MPa).
Bioactive compounds extracted from botanic matrices by SFE technique present a pronounced reproduction of the sensory characteristics of the raw material when co mpared to conventional techniques. The thermal degradation and the decomposition of thermolab ile substances are strongly reduced, since the SFE procedure occurs at low temperatures and in the absence of o xygen and light. This feature is especially useful in the extraction of antio xidants, because it guarantees the conservation of their functionalproperties [11 9]. Moreover, SFE is mo re selective than the conventional extraction techniques, and it is suitable for obtaining solvent-free products [118].
Extraction of antio xidants with supercrit ical CO 2 requires the use ofhigh pressures. At this condition, the co-extraction of other undesirable co mpounds, as waxes and oleoresins, might happen. When the co-ext raction of these compounds cannot be avoided, several separator vessels can be displayed in series, operating at different temperature and pressure conditions, to fractionate the extract [120].
Belonging to the Lamiaceae family, rosemary (Rosmarin us officinalis) is a plant with powerfu l antio xidant agents. Carnosic acid (CA) and carnosol are the major phenolic diterpenes present in rosemary ext racts obtained by SFE, as shown by Kuo et al. [121]. The CA content obtained in the referred study was approximately 110 mg/g extract, resulting in IC 50 of 7.47 µg/cm 3 . The IC 50 is the concentration of extract or act ive co mpound needed to inhibit 50% of oxidation of a defined substance, which can be determined by the DPPH (2,2-diphenyl-1-picrylhydrazy l) test. An IC 50 of 7.47 µg/cm 3 is very attractive, because low concentrations of CA present significant effects on the free rad icals scavenging. At the concentration of 80 µg/cm 3 , CA presented inhibition of84.1% of the lipid pero xidation, wh ile the synthetic BHT antio xidant inhibited 80.8% of the lip id peroxidation.
The results obtained by Kuo et al. [121] corroborate the studies carried out by Vicente et al. [89] using rosemary leaves; the authors obtained high CA content in the extract in 1 h of extract ion by SFE. The antio xidant activity o f the extract increased with the ext raction time because the volatile oil is depleted from the vegetal matrix at the beginning of the process, and the phenolic co mpounds, which present higher antioxidant activity, are only later extracted.
The interest in SFE has been increasing in the last years, which is shown by the several studies found in literature dealing with this topic (Figure 1). One of SFE features is that the raw material must be dried prior to the extract ion with supercritical CO 2 . The water decreases the efficiency of this technique by limiting the contact between the CO 2 and nonpolar solutes [120]. The water present in the solid material may also compete with CO 2 to dissolve the solute, which affects the mass transfer rate. Considering these aspects, the drying of the raw material at ideal conditions, without causing degradation of the bioactive compounds, is required [109]. Supercrit ical CO 2 is a solvent appropriate to ext ract nonpolar solutes. Co mpounds presenting high molecular mass, as flavonoids, are poorly soluble in pure CO 2 . In such case, the addition of a polar cosolvent to the CO 2 , to form a mixtu re with it in ideal proportions, improves the solubility of polar organic co mpounds. Thus, the mixture CO 2 + cosolvent can increase the mass transfer rate. The solubility improvement in the supercritical reg ion is associated with mo lecular interactions, mostly hydrogen bonds [128].
Ethanol and water are the most appropriatesolvents to be applied in the food industry. Ethanol is widely used to increase the efficiency of the extraction of phenolic acids and flavonoids, and is easily removed fro m the final product by distillat ion [120], [133]. The cosolvents commonly used to extract antio xidant compounds are listed in Table 2. The interactions between solute and solvent in the SFE process can be studied considering a solution of infin ite dilution because the solubility of the bioactive compounds is low in supercritical CO 2 , and, moreover, usually the solution is far fro m saturation. This fact makes the equilibriu m study simp le in systems where only CO 2 is used as solvent. On the other hand, the crit ical temperature and pressure change when cosolvent is added depending on its fraction in the mixtu re. As an examp le, for the system CO 2 + ethanol the critical point moves fro m 310.6 K and 7.77 MPa for 0.0044 mo l% ethanol to 410.3 K and 15.17 MPa for 0.403 mol% ethanol [134]. The temperatures above 330 K are not recommended for processing thermo sensitive compounds, the existence of two phases inside the extractor is co mmon when using high fractions of cosolvent. Despite the fact that the process can be performed using such system, in this caseit presents a behaviour that cannot be predicted by the models used for CO 2 alone.Therefore, when using cosolvents the system is much more co mp lex than for CO 2 alone and each case should be individually studied. Increasing the cosolvent fraction above 50% changes the process to PLE, where ethanol is the solvent and CO 2 is the cosolvent, when the latter is used.
Another recent trend concerning cosolvents is using vegetable oils as modifiers. In the extraction of carotenoids by SFE, it not only allo ws recovering compounds that have low solubility in CO 2 , but it also leads to the production of carotenoid-rich vegetable oils. Canola, o live, hazelnut, sunflower seed, soybean and rapeseed oils have been used for this purpose, among others [135].

Pressurized Li qui d Extraction (PLE)
The PLE technique is an alternative that has been recently used to obtain bioactive co mpounds; it uses an aqueous or organic solvent at high pressure and/or temperature by circulat ing the solvent through the sample. High pressure is not the most important feature in PLE process. In fact, most often the purpose of raising the pressure is to keep the solvent in the liquid phase. Designations for PLE can be found in literature, as accelerated solvent extract ion (ASE), pressurized hot solvent extraction (PHSE), pressurized water extraction (PWE), high pressure solvent extraction (HPSE) and subcritical solvent extract ion (SSE), among others. Antioxidants can be obtained using solvents at temperatures above their boiling point. For these reasons, a generic term is used: "superheated solvent ext raction" (SHSE) [140]. Liquid carbon dioxide cannot be used in this case because its critical temperature is lo w (at about 304 K) co mpared to the temperatures used in PLE. So, at pressures and temperatures above the critical point of CO 2 the process is called SFE.
Flavonoids, catechins, anthocyanins, flavanones, and flav ones are some o f the phenolic co mpounds which were obtained using PLE [107], [108], [141][142][143][144]. King andGrabiel [145], in their patent, demonstrated the potential of PLE technique for extracting polyphenols fro m fru its and vegetables wastes. Also, a method for extract ion of phenols fro m grape skins by ASE using ethanol-water mixtures is found in literature [146].
In PLE, high temperature is usually attractive, because it improves the extraction yield. The increase in temperature modifies the solvent dielectric constant and the solute solubility in the solvent [109]. Studies show that polyphenols extracted at temperatures above 363 K are unstable and can suffer pronounced thermal degradation, although the quantity of antioxidants extracted is high at elevated temperatures [107], [147], [148].
Co mparative assays using PLE and conventional extraction techniques were carried out for obtaining the three ma jor flavones (hesperitin, nobiletin and tangeretin) present in tangerine peels (Citrus reticulata). The flavones were efficiently extracted by PLE, reaching higher yields than the conventional methods. Additionally, the ext raction time was lower for PLE [141].
The recovery of phenolic compounds from oregano leaves (Origanum vulgare) by PLE was tested by Miron et al. [149]. The operational conditions used in the experimental assays were temperatures of 323 K, 373 K, 423 K and 473 K, and different proportions of ethanol/water as solvents. The extracts obtained using PLE with 100% of water in batch mode applying an 11 cm 3 ext ractor at 323 K and 10 M Pa presented the highest amount of phenols and the highest antioxidant activity. Under these conditions, the total phenols content was 184.9 mg GA E/g extract, where GA E means "gallic acid equivalents". The antio xidant activ ity was established as the amount of ext ract necessary to reduce the DPPH concentration in 50%, resulting in IC 50 of only 6.98 µg/cm 3 . When pure ethanol was used as solvent at temperature of 373 K, the total phenols content was only 102.2 mg GA E/g ext ract and the IC 50 was 11.5 µg/cm 3 . These results suggest that the solvent and the temperature influenced both the extract yield and its quality [149].
Recovery of anthocyanins and phenolic co mpounds from jabuticaba (Myrciaria cauliflora) was studied applying PLE and Low-Pressure Solvent Extraction (LPSE). Similar yields were obtained using both techniques. However, the PLE technique was attractive because it resulted in a rapid p rocess (≈ 9 min ) and it allowed low solvent consumption. The content of anthocyanins and total phenols in PLE extract were 2.15 and 1.66 t imes higher, respectively, than their content in LPSE extract. Moreover, PLE extract resulted in cost of manufacturing 40 times lower than LPSE extract due to the short processing time [150].
PLE technique is usually appropriate for obtaining antioxidants from lignocellulosic materials. So me of the effects achieved under temperatures of hydrothermal treatment (for instance above 493 K) are listed as [120]: i) solubilizat ionof acid-solublelignin; ii) hydrolytic depolymerizat ion of hemicellu lose into compounds of highmolecular mass (soluble fibers); iii) ext ractionof lipophilic co mpounds; iv) ext raction of lignans; v) extract ion of non-saccharides as terpenes, fatty acids and monomeric phenols. The phenolic compound vanillin was the major component with antioxidant activity found in barley husks subjected to non-isothermal auto-hydrolysis in aqueous med iu m [151]. The solubilized portion obtained in the auto-hydrolysis of pine (Pinus radiata) using several tests (DPPH radicalscavenging, hydroxy l radical scavenging, Trolo x equivalent antio xidantcapacity, b-carotene bleaching and reducing power) presented specific antio xidant activity 40 t imes higher than BHT, 25 times h igher than α-tocopherol, 8 t imes higher than caffeic acid, 3.5 t imes higher than BHA and 3 times higher than gallic acid [152].
PLE d iffers fro m conventional techniques because PLE uses high temperature and pressure in extract ive process, which may be conducted in semi-continuous (dynamic) or batch (static) modes. A wide range of temperature might be applied; it usually varies between 293 K and 473 K. The pressure commonly used varies between 3 MPa and 20 MPa [153]. Therefore, PLE is a suitable technique for extracting several solutes, both polar and nonpolar. PLE has been used as an alternative for obtaining antio xidant substances with high molecular mass derived from the hemicellulose frag mentation [120].

Microwave Assisted Extracti on (MAE)
MAE is another innovative technique that has been prese nting special interest. Microwaves consist of nonionizing electro magnetic energy with a frequency from 0.3 GHz to 300 GHz that is applied directly to the raw material. They transmit energy wh ich penetrates into the biologic matrix and interacts with polar mo lecules, mostly water, generating heat; the heat expands and disrupts the vegetal cell, favoring the extraction of intracellular phytochemical co mpounds. MAE is a technique frequently used for extract igthermolab i le co mpounds [109].
Extraction of phenolic co mpounds from cherry (Prunus cerasus)pulp was performed using the MAE method in batch mode by Simsek et al. [103]. Epicatechin (flavanol) was the major phenolic co mpound extracted, and its concentration was higher when using the MAE technique than when using the conventional technique. The antioxidant efficiency of MAE ext ract was 28.32 mg DPPH/g sample [103]. Antioxidants present in deoiled rosemary (Rosmarinus officinalis) leaves were ext racted by MAE using ethanol and water as solvents. Carnosic acid and carnosol were responsible for most of the antioxidant activity found in the extract. The IC 50 values using water and ethanol as solvents were 22.8 µg/cm 3 and 41.0 µg/cm 3 , respectively [154].
Onion (Allium cepa) varieties are rich in quercetin (flavonol). Their flavonoids content and antioxidant activity were evaluated using MAE as the extract ion technique. The red onion variety exhib ited the highest antioxidant activity. According tothe DPPH test, the IC 50 was 17.09 mg/c m 3 and the total amount of quercetin extracted was 134.7 mg/ 100 g dried samp le [155].
In a recent study, the antioxidant activity of polyphenol compounds present in pomegranate (Punica granatum) peels was evaluated using the MAE technique with water as solvent. Pronounced polyphenols yields were reached (210.4 mg GA E/g extract). The IC 50 was 14.53 µg/cm 3 (DPPH test), confirming an elevated antio xidant capacity of pomegranate e xtract [156]. Figure 2 showsthe free radical scavenging capacity of the po megranate ext ract evaluated by the DPPH test.The phenolic co mpounds extracted by MAE have activity equivalent to the synthetic BHT antio xidant in concentrations over 40 µg/cm 3 . At this concentration, the antioxidant activity of pomegranate extract and BHT were over 90%, indicat ing MAE as a potential alternative for obtaining natural antioxidants.
The conventional solid-liquid extract ion can generate undesirable residues with products. In addit ion, the extract cansuffer o xidative transformations during the solvent removal step [119]. Several scientific investigations report MAE applicability for obtaining natural antioxidants, without generating undesirable residues, as the extraction of polyphenols from peanut skins [157], whole to mato (Solanum spp.) [101], grape (Vitis vinifera) seeds [158], sweet potato (Ipomoea batatas) leaves [159] and bean (Phaseolus vulgaris L.) [160]. In general, the antio xidant activity of extracts obtained by MAE is higher than the activity of extracts obtained by conventional techniques, because the micro wave treat ment does not cause any deterioration of antioxidant properties of the extract [161]. MAE process is short, usually fro m 2 min to 40 min. This fact makes MAE an attractivetechnique, since for thermolab ile co mpounds long extraction times can result in degradation and consequent antioxidant capacity loss [101].
The results obtained using MAE are satisfactory because the microwaves cause molecular mot ion due to the migration of ionic species and dipole rotation. Therefore, the micro waves effect is proportional to the mediu m dielectric constant and to the solid matrix. In addition, the increase of the process temperature improves the solvent penetration [109].
Considering these aspects, the solvent selection should be made taking into account its dielectric constant. Polar mo lecules and ionic solutions have permanent dipole mo ment and strongly absorb the energy of microwaves. Therefore, ethanol, methanol and water are ideal solvents, while hexane and toluene have low dielectric constants and are not recommended for MAE [109], [162].

Ultrasound Assisted Extracti on (UAE)
Most of ext raction techniques consist of the manipulat ion of the solvent physical properties to reduce its superficial tension, to increase the solute solubility and to improve the mass transfer rate; in some cases, these manipu lations also induce changes in the solvent polarity [109].
The UAE technique consists in using mechanic vibrat ions caused by sound waves with frequencies higher than 20 kHz. Sound waves are intrinsically different fro m electro magnetic waves, because the latter can propagate through the vacuum, while sound waves need a physical med iu m to propagate. The mechanic vibrat ions cause expansion and compression cycles in the mediu m, creating bubbles which collapse and cause cavitation, instantly creating a h igh local pressure and 1 This figure is under the terms of the creative commons attributions license. intense local heating. These fast changes induce disruption and thinning of the cell membranes, consequently increasing the mass transfer rate of o rganic substances fro m the solid matrix to the solvent [109], [163].
The advantages of UAE technique include the simplicity of the equip ment and the possibility of using different solvents for the extraction, includ ing water-ethanol mixtu res [163]. In food and pharmaceutical fields, UA E is used to extract several bioactive co mpounds fro m botanic matrices, as flavonoids [104], [164], [165], polyphenols [166], [167], alkaloids [168], terpenoids [169] and anthocyanins [105], [170]. The imp rovement of extract ing bioactive compounds when applying ultrasounds is attributed to the mass transfer rate increase due to the solvent cavitation induced by the ultrasounds wave passing through the med iu m [154].
An efficient extraction procedure for recovering antioxidant compounds from jabuticaba (Myrciaria cauliflora) skins was proposed in literature: 10 min of UAE + conventional agitated bed extract ion (ABE). This combination maximized the extraction of polyphenols and resulted in extracts with h igh antio xidant activity. At 30 min of reaction (based on the couple oxidation of β -carotene and linoleic acid), the antio xidant activity of the ext racts obtained using UAE + A BE was over 85%, wh ile the ABE process presented extracts with antio xidant activity under 65%. Furthermore, UA E + A BE was the best option fro m the economic point of v iew, because the extract obtained by this combined technique presented the lowest cost of manufacturing (US$ 387.2/kg of crude extract) [61].
Considerable concentrations of rosmarinic acid (6.36 mg/c m 3 ) and total phenols (8,790 ppm GA E) were obtained fro m deoiled rosemary (Rosmarinus officinalis) leaves applying the UA E technique and water as solvent. The high concentration of antio xidants in the extract resulted in high antioxidant activity and the IC 50 was 23.6 µg/cm 3 . The IC 50 was measured by DPPH test [154].
In ext raction of leek (Allium porrum) stem by UA E, 69.5 mg GA E/g extractwas obtained. The antioxidant activity of the extract was compared to the standard antioxidants: vitamin C and BHT. The IC 50 value of the ethanolic extract was 61.1 µg/ cm 3 . Although theIC 50 value of the ethanolic e xtract was higher than the IC 50 of vitamin C (IC 50 = 10.6 µg/cm 3 ) and BHT (IC 50 = 39.2 µg/cm 3 ), the inhib ition composition of the ethanolic extract was very low. Thus, the leek extracts can be used in the industry as efficient agents against oxidation [171].
A certain amount of water (40% -60%) should be added to the solvent in order to obtain satisfactory yields in UA E, because water increases the extraction of flavonoids and other polar compounds. Water addition increases the med iu m relative polarity and facilitates the propagation of the ultrasonic waves [110]. Using UA E with water as solvent was efficient fo r reach ing specific hydro xylat ion of polyphenols and carotenoids in order to increase their bioactivity [172].
Ultrasound is also a broad method that can be done not only with solvent at at mospheric pressure. The co mbination of UAE followed by re-extraction of obtained extract by SFE was performed aiming to concentrate diterpenes present in sage extract. The diterpenes are generally considered to be responsible for antio xidant activity of the extracted compounds [173].
The coupled system of h igh-intensity ultrasound + SFE is an efficient manner of enhancing mass transfer in ext raction processes. In this sense, a supercritical CO 2 ext raction of o il fro m part iculate almonds was performed using power ultrasonic transducer with a frequency of 20 kHz. The process performance was evaluated, showing that this system conducted to a 30% increased yield [174]. The sa me procedure was used for obtaining extract fro m ginger. In the presence of ultrasound within the supercritical mediu m, both the ext raction rate and the y ield increased. Generally, the initial stage of extraction, wh ich is controlled by the external mass transfer, is not affected by ultrasound. Nevertheless, in the subsequent stage of ext raction, wh ich is controlled by the internal mass transfer, the u ltrasound allows an imp rovement in the yield [175]. Recently, extracts of malagueta pepper containing capsaicinoids were obtained using SFE and SFE assisted by ultrasound. The assays carried out with ultrasound presented a yield 20% h igher [176]. Table 3 shows compiled informat ion in operational conditions of the four techniques presented for ext racting antioxidants. This Table is a compendium o f the arguments discussed in the text. The distinguishing characteristics that make each technique more or less attractive for p rocessing different raw materials are also included in Table 3. For instance, SFE technique does not need cosolvent when the target phytochemicals are nonpolar (terpenoids, tocopherols and sitosterols). To extract polyphenols, on the other hand, the use of cosolvents is essential. Thus, the extract quality and yield are closely related to the extraction technique and to the process conditions. Figure 3 exemp lifies the novel extraction techniques used for obtaining antio xidants: SFE, PLE, MA E and UA E. The basic schematic d iagram of equip ment used in each technique is presented. In the case of MAE, the ext ractor must be built of a material that allows the microwaves to propagate. This material is usually glass or teflon. MAEis mostly performed in batch mode (static), while the other techniques are usually performed in semi-continuous mode (dynamic), using constant solvent flow rate in the ext raction vessel. The selection of the way to pro mote the ext ractor heating is done by the researchers; in the scheme of Figure 3, the extractors of SFE, PLE and UA E are heated using a jacket. Table 4 shows the antioxidant capacity, expressed as IC 50 , of compounds obtained from several raw materials using the four novel extract ion techniques discussed in the text. Several studies found out high antioxidant capacities of compounds in low concentrations. Certainly, the lower the IC 50 value the larger the antio xidant capacity of the extract.   The lowest values of IC 50 were found for ext racts obtained fro m rice dye (Buddleia officinalis) ( In summary, the antio xidant capacity is a function of process conditions which are related to the selected extraction technique. Process parameters of extraction influence the concentration of target co mpounds in the extract. The solvent characteristics, the extraction time and the temperature interfere in the selectivity of compounds and in the antio xidant properties of the extract. Furthermore, the origin o f the raw material emp loyed is the most influence in the antioxidant power.

Summary of the Characteristics of S FE, PLE, MAE and UAE
Almeidaet al. [122] obtained mint essential oil by SFE with CO 2 . The highest yield (2.38%, w/w) was obtained at 30 MPa and 323 K. The IC 50 in SFE ext racts obtained without using cosolvent was >250 µg/cm 3 . However,the IC 50 in SFE extracts obtained using 20% (w/w) of ethanol was 43.3 µg/cm 3 . So, the SFE with cosolvent is an efficient method for obtaining antioxidant co mpounds fro m mint (Mentha spicata L.), as carvone, cineol and pulegone. Nevertheless, the SFE process without cosolvent is not capable of recovering caffeine and phenols fro m coffee (Coffea arabica). Andradeet al. [96] reached an IC 50 of 1706 µg/cm 3 using SFE (CO 2 /20 M Pa/313 K), wh ile the IC 50 was 235 µg/cm 3 using UAE with ethanol as solvent, indicating UA E as a suitable technique for obtaining caffeine and phenols from coffee.MAE has been reported as an adequate technique for obtaining phenols with antio xidant properties fro m pomegranate (Punica granatum) [156] and phenolic diterpenes from rosemary (Rosmarinus officinalis) [154]. PLE was used for extract ing major catechins from green tea, presenting advantages such as shorter extraction time and lower energy consumption [198]

Conclusions
Obtaining extracts fro m botanic matrices usingnovel e xtr action techniques is increasing, andscientific investigations are progressively focusingon the natural antioxidants which are present in these extracts.Antioxidants have been receiving great attention because they bring benefits to the health and food fields. In this overview, the suitability of using each novel technique for obtaining different antioxidant phytochemicals, based on target compound characteristics, was emphasized.