**4. Supercritical fluid extraction (SFE) of antioxidant compounds from plant matrices**

When a new extract from a natural source is tested, the most important aspects to take into account are the extraction method and the type of solvent used, as this will affect the antioxidant properties. Several extraction methods for the selective extraction from plant matrices such as *R. officinalis* were identified in the scientific literature [71].

Thus, the bioactive compound extraction has been considered one of the most important steps in the approach of obtaining or recovering bioactive compounds. Conventional extractions have been the most used technology for these compound recovery. It is based on the extraction power of different solvents and the application of high temperatures, promoting mass transfer. However, there are drawbacks associated with conventional extraction processes such as the use of large amounts of organic solvents, toxic to human health and the environment, extraction time, and the use of high temperatures that can degrade the thermosensitive compounds [6, 8]. They motivated the search for environmentally safe extraction techniques such as microwave-assisted extraction (MAE), ultrasonic-assisted extraction (UAE), pressurized liquid extraction (PLE), and supercritical fluid extraction (SFE) [36, 78]. SFE has already been studied to obtain antioxidant compounds from natural sources [24, 33, 77, 79]. **Table 1** presents the antioxidant activity values of different plant extracts obtained with SFE, involving the plants under study (*C. sicyoides* and *R. officinalis*).

#### **4.1 SFE procedure**

A solvent is considered a supercritical fluid when the pressure and temperature of the system are above its critical point. This point is defined as the highest temperature and pressure at which a substance can exist in equilibrium between the liquid and vapor phases. Above its critical temperature (Tc) and critical pressure (Pc), the supercritical fluid can be considered as an expanded liquid or as a compressed gas, whose density (ρ) is relatively high and consequently has a high solvency power. This effect gives the solvent a certain degree of selectivity, in addition to allowing easy separation of the solvent from the solute, which can be achieved by a simple system depressurizing, resulting in products totally solventfree and without thermal degradation of the compounds of interest, due to low operating temperatures [35, 38].

**325**

**Table 2.**

(ρc) = 0.468 g/cm3

*Antioxidant and Biological Activity of* Cissus sicyoides and Rosmarinus officinalis *Extracts*

**determination**

**Antioxidant capacity**

g of extract/g of DPPH

Trolox/mg extract

DPPH μg/μg dry extract

dry extract

μg.ml<sup>−</sup><sup>1</sup>

DPPH 404.81 ± 2.78 EC50:

μg.ml<sup>−</sup><sup>1</sup>

mg/ml

DPPH 2.13 ± 0.24 EC50:

60°C/400 bar CO2 + ethanol DPPH >200 EC50 (μg/ml) Antioxidant [86]

**Biological Activity Refs.**

Antioxidant [84]

Antioxidant [59]

Antioxidant [85]

Antioxidant [87]

Antioxidant and anti-inflammatory [29]

[21]

[37]

**)**

Neuroprotective and anti-inflammatory

effect

Antioxidant, antibacterial, and antifungal

**Solvents Method of** 

*R. officinalis* 40°C/300 bar CO2 DPPH 12.85 ± 0.10 IC50:

100°C/350 bar CO2 DPPH 0.23 ± 0.01 IC50:

50°C/300 bar CO2 ORAC 1.9 ± 0.10 μmol

35°C/400 bar CO2 DPPH 359 mg TE/100 g

40°C/300 bar CO2 DPPH 103.28 EC50: of

One of the most commonly used solvents in SFE is carbon dioxide (CO2) because

apolar and has an ideal behavior for thermosensitive compound extraction [34, 37]. In addition to the supercritical CO2 (Sc-CO2), there are other substances that are

Due to its low polarity, Sc-CO2 presents a limitation to dissolve polar molecules. However, this disadvantage can be solved by the addition of polar solvents, called modifiers or cosolvents, which modify the supercritical fluid polarity and, consequently, improve the extraction of polar fractions rich in bioactive substances, such as phenol compounds related to high antioxidant activity [37, 38]. Methanol is the solvent most used as a modifier for various plant matrices, but it is toxic and

**Fluid Tc (°C) Pc (bar) ρc (g/cm3**

Nitrous oxide (N2O) 36.5 71.0 0.457 Ethane (C2H6) 32.2 48.8 0.203 Propane (C3H8) 96.7 42.5 0.220 Propylene (C3H6) 91.9 46.2 0.230 Benzene (C6H6) 289.0 48.9 0.302 Toluene (C7H8) 318.6 41.1 0.290 Ammonia (NH3) 132.5 112.8 0.240 Water (H2O) 374.2 220.5 0.272

*Critical properties of some substances used as solvents in supercritical extraction processes.*

), nontoxic, non-flammable, affordable, chemically inert, and

its critical points are moderate (Tc = 31.1°C, Pc = 73.8 bar, and critical density

*Presentation of the antioxidant activity values of different plant extracts obtained with SFE, involving the* 

also used as supercritical fluids, as shown in **Table 2**.

*DOI: http://dx.doi.org/10.5772/intechopen.83733*

ethanol

55°C/100 bar CO2 + 20% of ethanol

*plants under study (C. sicyoides and R. officinalis).*

**Plants Extraction** 

*Mangifera indica L.*

*Eugenia uniflora L.*

*Raphanus sativus L.*

*Piper nigrum L*

**Table 1.**

**conditions**

*C. sicyoides* 40°C/400 bar CO2 + 10% of

*Antioxidant and Biological Activity of* Cissus sicyoides and Rosmarinus officinalis *Extracts DOI: http://dx.doi.org/10.5772/intechopen.83733*


#### **Table 1.**

*Antioxidants*

synthetic preservatives [17].

study (*C. sicyoides* and *R. officinalis*).

operating temperatures [35, 38].

**4.1 SFE procedure**

**plant matrices**

literature [71].

In relation to the antibacterial activity, *R. officinalis* essential oils obtained by hydrodistillation exhibited antibacterial activity against *Escherichia coli*, *Salmonella typhi*, *S. enteritidis*, and *Shigella sonnei*; this activity was associated with the oil ability to reduce DPPH radical formation (CI50 = 3.82 μg/ml) [61]. However, the antibacterial and antifungal activities of *R. officinalis* leaf extracts obtained by SFE extraction were confirmed, and the extracts showed antibacterial activity against Gram-positive bacteria (*Staphylococcus aureus* and *Bacillus cereus*) and Gram-negative bacteria (*Escherichia coli* and *Pseudomonas aeruginosa*) and antifungals against *Candida albicans*. Obtaining *R. officinalis* extracts by SFE has been shown to be a promising extraction with respect to its incorporation into various foods, cosmetics, and pharmaceuticals products that a natural aroma, color, and antioxidant/antimicrobial additive are desired. These properties are also necessary for the food industry in order to find possible alternatives to

**4. Supercritical fluid extraction (SFE) of antioxidant compounds from** 

When a new extract from a natural source is tested, the most important aspects to take into account are the extraction method and the type of solvent used, as this will affect the antioxidant properties. Several extraction methods for the selective extraction from plant matrices such as *R. officinalis* were identified in the scientific

Thus, the bioactive compound extraction has been considered one of the most important steps in the approach of obtaining or recovering bioactive compounds. Conventional extractions have been the most used technology for these compound recovery. It is based on the extraction power of different solvents and the application of high temperatures, promoting mass transfer. However, there are drawbacks associated with conventional extraction processes such as the use of large amounts of organic solvents, toxic to human health and the environment, extraction time, and the use of high temperatures that can degrade the thermosensitive compounds [6, 8]. They motivated the search for environmentally safe extraction techniques such as microwave-assisted extraction (MAE), ultrasonic-assisted extraction (UAE), pressurized liquid extraction (PLE), and supercritical fluid extraction (SFE) [36, 78]. SFE has already been studied to obtain antioxidant compounds from natural sources [24, 33, 77, 79]. **Table 1** presents the antioxidant activity values of different plant extracts obtained with SFE, involving the plants under

A solvent is considered a supercritical fluid when the pressure and temperature of the system are above its critical point. This point is defined as the highest temperature and pressure at which a substance can exist in equilibrium between the liquid and vapor phases. Above its critical temperature (Tc) and critical pressure (Pc), the supercritical fluid can be considered as an expanded liquid or as a compressed gas, whose density (ρ) is relatively high and consequently has a high solvency power. This effect gives the solvent a certain degree of selectivity, in addition to allowing easy separation of the solvent from the solute, which can be achieved by a simple system depressurizing, resulting in products totally solventfree and without thermal degradation of the compounds of interest, due to low

**324**

*Presentation of the antioxidant activity values of different plant extracts obtained with SFE, involving the plants under study (C. sicyoides and R. officinalis).*

One of the most commonly used solvents in SFE is carbon dioxide (CO2) because its critical points are moderate (Tc = 31.1°C, Pc = 73.8 bar, and critical density (ρc) = 0.468 g/cm3 ), nontoxic, non-flammable, affordable, chemically inert, and apolar and has an ideal behavior for thermosensitive compound extraction [34, 37]. In addition to the supercritical CO2 (Sc-CO2), there are other substances that are also used as supercritical fluids, as shown in **Table 2**.

Due to its low polarity, Sc-CO2 presents a limitation to dissolve polar molecules. However, this disadvantage can be solved by the addition of polar solvents, called modifiers or cosolvents, which modify the supercritical fluid polarity and, consequently, improve the extraction of polar fractions rich in bioactive substances, such as phenol compounds related to high antioxidant activity [37, 38]. Methanol is the solvent most used as a modifier for various plant matrices, but it is toxic and


#### **Table 2.**

*Critical properties of some substances used as solvents in supercritical extraction processes.*

#### *Antioxidants*

**Figure 5.**

*Scheme of the SFE procedure of plant matrices.*

different from ethanol, which is an environmentally safe solvent being a good choice for SFE processes, and can be used in the extraction of natural products [80, 81]. Water is also a very attractive cosolvent for natural product extraction due to its high polarity, which considerably increases the polarity of Sc-CO2 [79].

For antioxidant compound extraction and recovery by SFE, several vegetable matrices were used, such as seeds, fruits, leaves, flowers, rhizomes, roots, fruit peels, and tree branches. The SFE process consists basically in the extraction of soluble compounds present in the solid matrix by a supercritical solvent and then separates these compounds from the solvent after depressurizing the system. In order to achieve an efficient and adequate extraction, several factors must be taken into account, having a careful control of the operating conditions and process step optimization [35, 36, 82].

Initially, the raw material must pass through a pretreatment stage before being fed into the fixed bed extractor; this procedure is performed to prepare the solid particles, allowing a greater efficiency to be achieved in the extraction process [83]. As shown in **Figure 5**, after the raw material is collected, one of the first stages of its pretreatment is the solid matrix moisture reduction, for example, drying leaves in an oven with air circulation. Generally, the plant matrix moisture should not exceed 14% (wet basis). Another important step is the moisture content determination by the distillation method of the Jacobs immiscible solvent, with the purpose of knowing if the quantity of water in the sample is adequate for the supercritical extraction process. The sieving stage is applied to standardize and determine the average particle size of the solid particles. The real and apparent density and bed porosity determination is also very important as they affect the particles packaging in the extraction vessel and consequently the solvent flow and the mass and heat transfer processes [35, 82].

After a suitable pretreatment, the solid matrix is placed in an extraction vessel forming a fixed bed. Depending on the compounds of interest, the supercritical solvent (Sc-CO2) or solvent + cosolvent is fed by the solvent pump and/or cosolvent into the extraction vessel, where it continuously flows through the fixed bed and dissolves the extractable components from the solid matrix. The mixture of solutes that is removed from the solid matrix is called extract. In the separation step, the mixture formed by the solvent extraction + extract leaves the vessel and feeds the separator (collection flask) where the mixture is separated by rapid reduction of

**327**

**Author details**

**Acknowledgements**

**Conflict of interest**

content and writing of the manuscript.

scholarship.

Pará, Brazil

provided the original work is properly cited.

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

Marcilene Paiva da Silva, Flávia Cristina Seabra Pires, Ana Paula de Souza e Silva,

Mar Salazar (Process Number: 1777277) thanks CAPES for the doctorate

The authors have no conflict of interest to declare and are responsible for the

LABEX/FEA (Faculty of Food Engineering), Federal University of Para, Belém,

Marielba de los Angeles Rodriguez Salazar\*, Glides Rafael Olivo Urbina,

Priscila do Nascimento Bezerra, Vânia Maria Borges Cunha,

\*Address all correspondence to: marielba434@hotmail.com

Sérgio Henrique Brabo de Sousa and Raul Nunes de Carvalho Jr

*Antioxidant and Biological Activity of* Cissus sicyoides and Rosmarinus officinalis *Extracts*

pressure (ambient pressure). The extract precipitates in the separator, and the

The identification of new natural antioxidant compounds is of great interest to the food, pharmaceutical, and cosmetic industry in order to find possible alternatives to synthetic antioxidants. In this way, plants such as *C. sicyoides* and *R. officinalis* have been extensively studied for their antioxidant activity. The *C. sicyoides* extract obtained by SFE has a neuroprotective and anti-inflammatory effect; these effects are associated with the presence of phenolic compounds and the high antioxidant activity in the extract. *R. officinalis* extract is antibacterial, antifungal, anti-inflammatory, and effective, associated with the presence of carnosic acid, carnosol, rosmarinic acids, and hesperetin. It has been corroborated that these plants contain chemical compounds that exhibit the capacity of FRSs and reduce the onset of different diseases. Finally, obtaining extracts from plant matrices using environmentally safe extraction technology such as SFE represents a great opportunity to obtain bioactive compounds.

*DOI: http://dx.doi.org/10.5772/intechopen.83733*

solvent is removed from the system [38, 83].

**5. Conclusion**

*Antioxidant and Biological Activity of* Cissus sicyoides and Rosmarinus officinalis *Extracts DOI: http://dx.doi.org/10.5772/intechopen.83733*

pressure (ambient pressure). The extract precipitates in the separator, and the solvent is removed from the system [38, 83].
