**2. Main methods of essential oil (EO) extraction**

EOs can be extracted from different plant parts and by different methodologies, which generally depend on the botanical material used, and may have a direct relation to the quality of the extracted oil. Therefore, choosing an inappropriate procedure can cause changes in its composition [18, 19]. EO extraction methods are divided into two categories: conventional methods and innovative methods. Traditional methods include hydrodistillation and steam distillation, and among the innovative ones, supercritical fluid extraction [20].

#### **2.1 Hydrodistillation**

Hydrodistillation (HD) is the most traditional, simple, and versatile technique used in the extraction of EOs [21, 22]. The basic principle of this type of extraction is azeotropic distillation (substances behave as if they were pure in relation to the boiling point), and to occur, a heating source, a container to place the vegetal biomass (for example, a volumetric flask), a condenser, and a decanter for collecting the oil and water mixture are necessary. HD is considered a multilateral method and, although simple, can be used in small or large industries because of its selectivity and low installation cost [20, 23, 24]. Hydrodistillation process is originated in alembics, however, since the third edition of the European pharmacopeia, its use along with the modified Clevenger system has been recommended, as this system enables the condensate recycling [20].

In HD, plant material, which can be any plant organ, is immersed in boiling water [19, 25]. In summary, the hydrodistillation system (**Figure 1**) consists of a container, usually a volumetric flask, which is connected to a Clevenger-type apparatus attached to a refrigeration system, with temperature ranging from 10 to

**125**

**2.2 Steam distillation**

**Figure 1.**

*Hydrodistillation system.*

*Volatile Compounds, Chemical Composition and Biological Activities of* Apis mellifera *Bee…*

15°C. The solid-liquid mixture is heated, at atmospheric pressure, until it reaches water boiling temperature, allowing the odorous molecules to evaporate along with the water, forming an azeotropic mixture, which is drawn into the condenser, where it liquefies and is collected at the end of the extraction. Due to its hydrophobic character, the oil does not mix with water, so it can be separated by decantation. After separation, the oil is completely dehydrated using anhydrous Na2SO4 [19, 26, 27]. Hydrodistillation has some drawbacks that can qualitatively and quantitatively interfere in EOs, such as prolonged extraction time and chemical changes in terpene molecules, caused by hydrolysis and cyclization reactions. These are due to exces-

Steam distillation (SD) is another traditional method for EO extraction widely used for commercialization and can be used on laboratory and/or industrial scale for its simplicity and low cost, compared to other more sophisticated methods [28, 29]. The fundamental difference between steam distillation and hydrodistillation lies in

Steam distillation is divided into two basic types: direct (or wet) steam distillation

and indirect steam distillation. In direct steam distillation (**Figure 2A**), the plant material is placed in a grid above the hot water, and steam passes through it. The leaves should be carefully distributed on the grid to allow uniform extraction and vaporization. Indirect steam distillation (**Figure 2B**) is the most common method for extraction of essential oils. In this process, no water is poured into the distillation tank. Instead, steam is directed to the tank from an external source. Volatiles are released from plant material when steam breaks the glands containing the oil molecules. From this stage, condensation and separation processes are the same [30]. Generally, the time in steam distillation extraction is reduced, which, together with the lack of contact between plant biomass and water, minimizes the chemical changes in EO's constituent molecules [20]. In addition, this technique is appreci-

Although traditional methods are still widely used in EO extraction, supercritical fluid extraction has become widespread as an alternative to conventional

sive contact time with water and loss of some polar molecules [20, 26].

the fact that, in SD, plant material is not in direct contact with water [20].

ated for generating high oil yield and being energy efficient [31].

**2.3 Supercritical fluid extraction**

*DOI: http://dx.doi.org/10.5772/intechopen.92130*

*Volatile Compounds, Chemical Composition and Biological Activities of* Apis mellifera *Bee… DOI: http://dx.doi.org/10.5772/intechopen.92130*

**Figure 1.** *Hydrodistillation system.*

*Essential Oils - Bioactive Compounds, New Perspectives and Applications*

compounds [8].

biological activities of these metabolites.

**2.1 Hydrodistillation**

condensate recycling [20].

**2. Main methods of essential oil (EO) extraction**

the innovative ones, supercritical fluid extraction [20].

In this sense, propolis may represent a natural alternative in the search for bioactive compounds [5], since the use of secondary metabolites is increasing and represents a very broad field of research that can still be explored [6]. In addition, the wide variety of natural substances that can be found in organic matrices can provide key substances for the treatment of various pathologies [7]. The main substances present in propolis are low molecular weight, nonpolar, and volatile

The chemical composition of volatile substances present in propolis is very varied.

Authors have been studying volatile compounds and their applications [11–13] and have seen how these secondary metabolites can be promising in treating various diseases, such as neurodegenerative syndromes [14, 15] and infections caused by microorganisms [16, 17]. Considering the importance of the search for volatile substances present in propolis that may be beneficial for the maintenance of human health, this work aims to perform a literature review in order to address the main

EOs can be extracted from different plant parts and by different methodologies, which generally depend on the botanical material used, and may have a direct relation to the quality of the extracted oil. Therefore, choosing an inappropriate procedure can cause changes in its composition [18, 19]. EO extraction methods are divided into two categories: conventional methods and innovative methods. Traditional methods include hydrodistillation and steam distillation, and among

Hydrodistillation (HD) is the most traditional, simple, and versatile technique used in the extraction of EOs [21, 22]. The basic principle of this type of extraction is azeotropic distillation (substances behave as if they were pure in relation to the boiling point), and to occur, a heating source, a container to place the vegetal biomass (for example, a volumetric flask), a condenser, and a decanter for collecting the oil and water mixture are necessary. HD is considered a multilateral method and, although simple, can be used in small or large industries because of its selectivity and low installation cost [20, 23, 24]. Hydrodistillation process is originated in alembics, however, since the third edition of the European pharmacopeia, its use along with the modified Clevenger system has been recommended, as this system enables the

In HD, plant material, which can be any plant organ, is immersed in boiling water [19, 25]. In summary, the hydrodistillation system (**Figure 1**) consists of a container, usually a volumetric flask, which is connected to a Clevenger-type apparatus attached to a refrigeration system, with temperature ranging from 10 to

Several compounds can be found, such as: nerolidol, α-pinene, β-pinene, cedrol, 3-methyl-2-buten-1-ol, octane, tricyclene, β-caryophyllene, spatulenol, δ-cadinene, selina-3,7(11)diene, nerolidol, benzenepropanoic acid, allyl benzyl ether, 1,8-epoxyp-menth-2-ene, γ-terpinene, mentha-3(8),6-diene, cis-sabinol, 2,3-dehydro-1,8-cineole, α-copaene, p-ethylguaiacol, β-copaene, junipene, γ-cadinene, (3e)-6-phenyl-3-hexen-2-one, p-mentha-1(7),2-dien-8-ol, 4-terpineol, β-fenchyl alcohol, sabinene, δ-3-carene, limonene, α-thujene, α-terpinene, α-terpinolene, trans-verbenol, camphene, verbenene, o-cymene, and α-phellandrene. Moreover, geographical origin and seasonality may influence this composition [9, 10].

**124**

15°C. The solid-liquid mixture is heated, at atmospheric pressure, until it reaches water boiling temperature, allowing the odorous molecules to evaporate along with the water, forming an azeotropic mixture, which is drawn into the condenser, where it liquefies and is collected at the end of the extraction. Due to its hydrophobic character, the oil does not mix with water, so it can be separated by decantation. After separation, the oil is completely dehydrated using anhydrous Na2SO4 [19, 26, 27].

Hydrodistillation has some drawbacks that can qualitatively and quantitatively interfere in EOs, such as prolonged extraction time and chemical changes in terpene molecules, caused by hydrolysis and cyclization reactions. These are due to excessive contact time with water and loss of some polar molecules [20, 26].

## **2.2 Steam distillation**

Steam distillation (SD) is another traditional method for EO extraction widely used for commercialization and can be used on laboratory and/or industrial scale for its simplicity and low cost, compared to other more sophisticated methods [28, 29]. The fundamental difference between steam distillation and hydrodistillation lies in the fact that, in SD, plant material is not in direct contact with water [20].

Steam distillation is divided into two basic types: direct (or wet) steam distillation and indirect steam distillation. In direct steam distillation (**Figure 2A**), the plant material is placed in a grid above the hot water, and steam passes through it. The leaves should be carefully distributed on the grid to allow uniform extraction and vaporization. Indirect steam distillation (**Figure 2B**) is the most common method for extraction of essential oils. In this process, no water is poured into the distillation tank. Instead, steam is directed to the tank from an external source. Volatiles are released from plant material when steam breaks the glands containing the oil molecules. From this stage, condensation and separation processes are the same [30].

Generally, the time in steam distillation extraction is reduced, which, together with the lack of contact between plant biomass and water, minimizes the chemical changes in EO's constituent molecules [20]. In addition, this technique is appreciated for generating high oil yield and being energy efficient [31].

#### **2.3 Supercritical fluid extraction**

Although traditional methods are still widely used in EO extraction, supercritical fluid extraction has become widespread as an alternative to conventional

**Figure 2.**

*Representative schemes of direct (wet) steam distillation (A) and indirect steam distillation (B) [20].*

extraction methods [32, 33]. Supercritical fluid extraction arose from the need of new techniques that could minimize chemical changes and optimize extraction time [34]. This technique is considered an innovative "green" separation process to obtain natural products, such as EOs, and presents a prominent role in food and pharmaceutical industries [35, 36]. Among many possible supercritical fluids, CO2 is the most widely used. Its critical point is reached at pressure of 72.9 atm and temperature of 31.2°C, which makes it not harmful to EO thermolabile molecules, thus preventing the chemical changes that occur in classic extraction processes [20, 36]. In addition, CO2 is an inert gas, which means it is not reactive and can be eliminated simply by pressure decrease at the extractor outlet [20].

Carbon dioxide has characteristics that justify its use as supercritical fluid, such as low viscosity, high diffusivity, and density close to that of liquids [20]. Other factors related to CO2 also help to understand the importance of using this gas as a supercritical fluid: non-toxicity, non-flammability, insipidity [20, 37], noncorrosivity, non-explosivity [35], great availability [36], and selectiveness [38]. It is also noteworthy that supercritical fluid extraction provides the purest EOs, as no trace of solvent remains after the end of the process, and no external substances are present in the extracted material [39].

In addition to providing a purer product, extraction using supercritical CO2 is also more advantageous in relation to extraction time, as it is faster than conventional methods [35]. The low viscosity and high diffusivity of the supercritical fluid increase its penetration power based on the high mass transfer rate of solutes, allowing efficient extraction of compounds in the plant material. In addition, low viscosity contributes to lower fluid transport costs [36]. The efficiency of supercritical carbon dioxide extraction is due to the fact that it is a nonpolar solvent, similar to EO's constituents [35].

Despite being a very sophisticated, advantageous, and efficient method for the production of EOs, supercritical fluid extraction has some disadvantages regarding installation costs and equipment maintenance [20], besides high energy consumption to set pressure and temperature [26].

The supercritical fluid extraction system (**Figure 3**) is basically constituted by a carbon dioxide cylinder, cooling bath, high-pressure pump, oven, extraction container, vial, air compressor, flow meter, and flux control valves [35, 40, 41].

The process begins when the CO2 contained in the cylinder is pumped into the cooling bath, in which it is liquefied and then pressurized by the high-pressure pump. The compressed CO2 is then transferred to the main extraction cell, maintaining the required process temperature. These processes guarantee the ideal

**127**

*Volatile Compounds, Chemical Composition and Biological Activities of* Apis mellifera *Bee…*

thermodynamic conditions of the fluid that will pass through the material, thus

*Apparatus of supercritical fluid extraction of essential oils [40] (1) CO2 tank; (2) cooling bath; (3) pump; (4) oven; (5) extraction container; (6) vial; (7) air compressor; (8) flow meter; V1-V5. Flux control valves.*

In supercritical fluid extraction, there are two periods: the first is called static, at which the valve V4 is closed for about 30 minutes; then, the dynamic conditions are adjusted and V4 is opened, thus initiating the dynamic period and the extraction itself, because at this point, the essential oil begins to be poured into the

Essential oils biosynthesized by aromatic plants can be directly influenced by multiple factors such as genetic, anthropic action, environmental conditions, geographical origin, circadian regime, seasonality, stage of development, and others

Variability in content and composition of the EOs and other secondary metabolites is a way that the plant finds to better adapt to the exposed conditions, since the metabolic activity of the plants is a chemical interconnection between the plant and

The composition of EOs in plants of the same species that are living in the same place, but that have different chemical profiles, may be influenced by different genetic factors [51]. Other factors that significantly influence both quantitative and qualitative chemical variability of EOs are seasonal and circadian variations, which are related, respectively, to different periods of the year, and to day and night variations [52]. The chemical composition of the EO constituents obtained from the same plant organ may vary according to the species and extraction method used.

*Apis mellifera* bees produce propolis by chewing resins collected from trees by adding salivary enzymes to them. The wax produced is used to cover hive failures, besides having antibacterial, antioxidant, antifungal, and antiviral activities, thus

Due to these properties, the extraction of propolis essential oil has gained prominence in the research field, being reported the presence of compounds such as terpenoids, alcohols, aldehydes, hydrocarbons, and aliphatic ketones in its chemical composition [53, 54]. And due to geographic factors, bee types, and

**3. Chemical composition of** *Apis mellifera* **essential oil**

*DOI: http://dx.doi.org/10.5772/intechopen.92130*

extracting the essential oil.

**2.4 Factors that influence EO composition**

the environment it's inserted [47, 50].

helping to protect the bees themselves.

collecting vial [35].

[42–49].

**Figure 3.**

*Volatile Compounds, Chemical Composition and Biological Activities of* Apis mellifera *Bee… DOI: http://dx.doi.org/10.5772/intechopen.92130*

**Figure 3.**

*Essential Oils - Bioactive Compounds, New Perspectives and Applications*

extraction methods [32, 33]. Supercritical fluid extraction arose from the need of new techniques that could minimize chemical changes and optimize extraction time [34]. This technique is considered an innovative "green" separation process to obtain natural products, such as EOs, and presents a prominent role in food and pharmaceutical industries [35, 36]. Among many possible supercritical fluids, CO2 is the most widely used. Its critical point is reached at pressure of 72.9 atm and temperature of 31.2°C, which makes it not harmful to EO thermolabile molecules, thus preventing the chemical changes that occur in classic extraction processes [20, 36]. In addition, CO2 is an inert gas, which means it is not reactive and can be eliminated simply by pressure decrease at the extractor

*Representative schemes of direct (wet) steam distillation (A) and indirect steam distillation (B) [20].*

Carbon dioxide has characteristics that justify its use as supercritical fluid, such as low viscosity, high diffusivity, and density close to that of liquids [20]. Other factors related to CO2 also help to understand the importance of using this gas as a supercritical fluid: non-toxicity, non-flammability, insipidity [20, 37], noncorrosivity, non-explosivity [35], great availability [36], and selectiveness [38]. It is also noteworthy that supercritical fluid extraction provides the purest EOs, as no trace of solvent remains after the end of the process, and no external substances are

In addition to providing a purer product, extraction using supercritical CO2 is also more advantageous in relation to extraction time, as it is faster than conventional methods [35]. The low viscosity and high diffusivity of the supercritical fluid increase

Despite being a very sophisticated, advantageous, and efficient method for the production of EOs, supercritical fluid extraction has some disadvantages regarding installation costs and equipment maintenance [20], besides high energy consump-

The supercritical fluid extraction system (**Figure 3**) is basically constituted by a carbon dioxide cylinder, cooling bath, high-pressure pump, oven, extraction container, vial, air compressor, flow meter, and flux control valves [35, 40, 41]. The process begins when the CO2 contained in the cylinder is pumped into the cooling bath, in which it is liquefied and then pressurized by the high-pressure pump. The compressed CO2 is then transferred to the main extraction cell, maintaining the required process temperature. These processes guarantee the ideal

its penetration power based on the high mass transfer rate of solutes, allowing efficient extraction of compounds in the plant material. In addition, low viscosity contributes to lower fluid transport costs [36]. The efficiency of supercritical carbon dioxide extraction is due to the fact that it is a nonpolar solvent, similar to EO's

**126**

outlet [20].

**Figure 2.**

constituents [35].

present in the extracted material [39].

tion to set pressure and temperature [26].

*Apparatus of supercritical fluid extraction of essential oils [40] (1) CO2 tank; (2) cooling bath; (3) pump; (4) oven; (5) extraction container; (6) vial; (7) air compressor; (8) flow meter; V1-V5. Flux control valves.*

thermodynamic conditions of the fluid that will pass through the material, thus extracting the essential oil.

In supercritical fluid extraction, there are two periods: the first is called static, at which the valve V4 is closed for about 30 minutes; then, the dynamic conditions are adjusted and V4 is opened, thus initiating the dynamic period and the extraction itself, because at this point, the essential oil begins to be poured into the collecting vial [35].

#### **2.4 Factors that influence EO composition**

Essential oils biosynthesized by aromatic plants can be directly influenced by multiple factors such as genetic, anthropic action, environmental conditions, geographical origin, circadian regime, seasonality, stage of development, and others [42–49].

Variability in content and composition of the EOs and other secondary metabolites is a way that the plant finds to better adapt to the exposed conditions, since the metabolic activity of the plants is a chemical interconnection between the plant and the environment it's inserted [47, 50].

The composition of EOs in plants of the same species that are living in the same place, but that have different chemical profiles, may be influenced by different genetic factors [51]. Other factors that significantly influence both quantitative and qualitative chemical variability of EOs are seasonal and circadian variations, which are related, respectively, to different periods of the year, and to day and night variations [52]. The chemical composition of the EO constituents obtained from the same plant organ may vary according to the species and extraction method used.
