**3. Techniques for extraction of bioactive compounds from aromatic plants**

When considering quality and composition of an EO, the method of extraction plays a crucial role. EO are extracted from different part of the plants – flowers, buds, seeds, leaves, bark, herbs, wood, fruits, and roots – and different extraction techniques should be used in different conditions for understanding the extraction selectivity.

Furthermore, separation, identification, and characterization of bioactive compounds are only possible by an appropriate extraction process of plant matrix.

Different techniques could be used to extract bioactive compounds; many of them remain almost same through hundreds of years. All these techniques have some common objectives: (a) to extract targeted bioactive compounds from complex plant sample, (b) to increase selectivity of analytical methods, (c) to increase sensitivity of bioassay by increasing the concentration of targeted compounds, (d) to convert the bioactive compounds into a more suitable form for detection and separation, and (e) to provide a strong and reproducible method that is independent of variations in the sample matrix [66].

#### **3.1. Conventional extraction techniques**

Bioactive compounds from plant materials can be extracted by various classical extraction techniques. Most of these techniques are based on the extracting power of different solvents in use and the application of heat and/or mixing. To obtain bioactive compounds from plants, the existing classical techniques are maceration [19, 67], expression [25], solvent extraction [19], and hydrodistillation.

**Hydrodistillation** is a traditional method for extraction of bioactive compounds and EOs from plants. Organic solvents are not involved, and it can be performed before dehydration of plant materials. There are three types of hydrodistillation: water distillation, water and steam distillation, and direct steam distillation [68]. In hydrodistillation, first, the plant materials are packed in a still compartment; second, water is added in sufficient amount and then brought to boil. Alternatively, direct steam is injected into the plant sample. Constituents that are insoluble in the water but volatile enough to be driven off by the steam come over and are cooled, condensed, and collected in the receiving vessel. The condensed mixture flows from the condenser to a separator, where oil and bioactive compounds separate automatically from the water [69]. At a high extraction temperature, some volatile components may be lost. This drawback limits its use for thermolabile compound extraction. EOs extracted by hydrodistil‐ lation need further purification, especially drying, to remove water.

#### **3.2. Nonconventional extraction techniques**

It was found that the major drawbacks of conventional extraction are necessity of costly and high purity solvent, solvent or water removal, low extraction selectivity, thermal decomposi‐ tion of thermolabile compounds and long extraction time [70]. In recent years, to overcome these limitations, so-called nonconventional extraction techniques are presented: ultrasoundassisted extraction [71], pulsed electric field–assisted extraction [19, 72], enzyme-assisted extraction [19, 73], microwave-assisted extraction [19, 74], and supercritical fluid extraction (SFE).

SFE was systematically investigated over the past decades [48]. SFE is performed by using fluids in supercritical state at temperature higher than their critical temperature and under the pressure higher than their critical pressure. Supercritical fluid possesses gas-like properties of diffusion, viscosity, and surface tension and liquid-like density and solvation power [7]. These characteristics enable easy penetration of the fluid in supercritical state into herbal material and the extraction of secondary herbal metabolites.

For several practical reasons, more than 90% of all analytical SFE is performed with carbon dioxide (CO2). Apart from having relatively low critical pressure (74 bar) and temperature (32°C), CO2 is relatively nontoxic, nonflammable, available in high purity at relatively low cost, and easily removed from the extract. The main drawback of CO2 is its lack of polarity for the extraction of polar compounds [75]. The limitation of low polarity of carbon dioxide has been successfully overcome by the use of chemical modifier [76].

A basic SFE system consists of the following parts: a tank of mobile phase, usually CO2; a pump to pressurize the gas; co-solvent vessel and pump; an oven that contains the extraction vessel; a controller to maintain the high pressure inside the system; and a trapping vessel. Usually, different type of meters such as flow meter and dry/wet gas meters could be attached to the system. The major variables influencing the extraction efficiency are temperature, pressure, particle size, packing density and moisture content of feed material, extraction time, flow rate of CO2, and solvent-to-feed-ratio [9, 77]. By varying the extracting conditions, it is possible to obtain extract with the maximum content of desired active substances. The only disadvantage of industrial application of SFE, as opposed to conventional methods, is the larger investment in equipment due to working in conditions of elevated pressure. The costs of production, however, are significantly reduced; the process is simpler and more cost-efficient; and quality of the final product good [7].

Supercritical carbon dioxide (SCCO2) is known as a good solvent for a wide range of natural bioactive principles. In the last 10 years, studies on the extraction of classical compounds like essential and seed oils from various sources: seeds, fruits, leaves, flowers, rhizomes, etc., with or without the addition of a co-solvent, have been published [78]. Extraction of bioactive compounds from plant material using SCCO2 has been indicated as a favorable technique for producing solvent-free extracts suitable for wide use in pharmaceutical, biomedical, cosmetic, and food industries [79].
