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Chapter 2 Methods of obtaining drugs from medicinal and aromatic plants 73
2.3.3.6 Hydrodistillation and steam distillation
These two methods are commonly used for extracting essential oils from plant materials, based on the principle of separating components according to differences in their
physical properties. In hydrodistillation, plant samples are placed in a closed container with an appropriate amount of water. The mixture is then boiled or subjected
to direct steam. The steam carries the extracted essential oil to a condenser, where it
cools down and forms a liquid mixture. This process also results in the creation of a
by-product called “hydrosol,” a water component containing part of the plant’s essence [83].
Steam distillation is a suitable extraction technique for temperature-sensitive materials such as oils, resins, hydrocarbons, and other compounds that are waterinsoluble and can be separated at their respective boiling points. It has been used for
many years for the extraction of essential oils from plants. The process involves distilling a component or mixture of components at temperatures significantly lower
than their specific boiling points. Fresh or dried plant material is placed in the steel
chamber of the apparatus, and the generated steam passes through the plant material,
penetrating its cells, softening them, and facilitating the volatilization of the essential
oil. Once released, small droplets of oil are formed and mix with the steam (carrier),
passing into a cooling system. The mixture condenses there, forming a liquid mixture
where the oil phase is typically at the top. The less dense oil is easily separated from
the water [84].
Hydrodistillation and steam distillation are traditional extraction methods for isolating essential oils. The primary advantage of these methods is their low cost. However, they also have disadvantages, including low extraction yield, partial loss of volatile components, lengthy processing times, and the potential degradation of some
components. Despite these drawbacks, these two methods remain the most commonly
preferred techniques for essential oil isolation [85].
2.3.3.7 Soxhlet extraction
The Soxhlet extractor, developed by German chemist Franz Ritter Von Soxhlet in 1879,
has remained a popular apparatus for many years and is widely used today for the
extraction of natural source compounds. It has also served as a reference model for
newly developed extraction techniques [59].
For the extraction process, the finely ground dry sample is first placed into the
extraction chamber of the Soxhlet apparatus. This chamber typically consists of a porous bag or “thimble” made of filter paper or cellulose. The extraction solvent is then
heated above its boiling point in the distillation flask. Vapors from the boiling solvent
move into the condenser, where they condense and drip back onto the sample. Once
the solvent reaches the siphon level, the siphon empties the solution back into the dis-
74 İrem Yıldız Özbaş, Severina Pacifico, and Emre Özbaş
tillation flask. The solutes dissolved in the solvent are transferred to the bulk liquid in
the flask. As the solvent flows back into the solid plant material bed, the dissolved
metabolites are retained in the distillation flask. This allows the hot solvent to circulate through the material multiple times. Since only pure solvent is vaporized, fresh
solvent is used in every cycle, while the extracted metabolites remain in the solvent
flask. This process is repeated until the extraction is complete [47, 59]. Therefore, it is
a time-consuming process that requires multiple cycles to complete the extraction.
Prolonged exposure of bioactive compounds to high temperatures can result in the
degradation of thermolabile components, reducing their quality. The efficiency of
Soxhlet extraction depends on various factors such as the average particle size of the
material, extraction time, and the choice of solvents, whether polar or nonpolar [86].
Soxhlet extraction is a straightforward and practical technique that allows for an
endless cycle of extraction using a new solvent until all of the solute in the raw material has been extracted [58]. In fact, the primary characteristic of Soxhlet system is the
gradual recycling of the extracting solvent, which prevents the solvent from potentially settling during the maceration step, which is traditionally seen upon simple contact between the solvent and the sample matrix, and displaces transfer equilibrium to
ensure a high extraction yield [56].
Soxhlet extraction can be applied to solid and semisolid plant materials, but it is
primarily used for extracting components from solid samples. A dry, finely divided
solid is the acceptable sample for Soxhlet extraction. A number of variables, including
temperature, solvent-sample ratio, and agitation speed, must be taken into account.
The extraction solvents are usually pure organic solvents or their mixtures, and high
purity is required for these solvents. However, this increases exposure to toxic organic solvents and their environmental impact [47].
Although the Soxhlet extraction method has drawbacks, such as prolonged processing times and significant solvent usage, it is still frequently utilized for plant material extraction because of its simplicity [56]. In recent years modern versions of Soxhlet extractors, including pressurized, automated, ultrasound-assisted, and microwaveassisted versions, have also been developed [47].
2.3.3.8 Advanced extraction techniques
There are several methods available for extracting plant materials. Due to the long
processing times, high solvent consumption, and low yields associated with traditional
extraction techniques, the use of modern extraction methods has increased in recent
years. Innovative and more environmentally friendly advanced extraction techniques, which minimize the use of synthetic and organic chemicals, have been developed to replace traditional methods. Most of these techniques use mechanisms such
as heating and ultrasonic vibrations to break down cell walls more rapidly, enhancing
the solubility of desired active compounds and improving extraction efficiency [87].
Chapter 2 Methods of obtaining drugs from medicinal and aromatic plants 75
Modern methods are highly automated, allowing for the simultaneous control of
multiple parameters. By selecting the most suitable technique, both sample and solvent consumption can be reduced. These methods enable efficient extraction in a
shorter time, often providing extracts with higher yield and quality compared to conventional methods. One common feature of these techniques is their ability to operate
at high temperatures and pressures. Many modern methods are much more suitable
for the extraction of heat-sensitive and volatile compounds compared to traditional
methods [88].
The most prominent modern extraction methods include microwave-assisted extraction, pressurized liquid extraction, supercritical fluid extraction, and ultrasonicassisted extraction. These techniques are suitable for industrial-scale extraction of active compounds from plants. Additionally, the use of green technology, which combines
the use of green solvents such as deep eutectic solvents and ionic liquids, offers a good
alternative for the extraction of natural compounds, achieving higher yields with less
solvent and energy consumption [89]. A brief comparison of the advantages and disadvantages of advanced extraction techniques is presented in Table 2.3.
Table 2.3: The advantages and disadvantages of advanced extraction techniques.
Method Advantages Disadvantages References
Ultrasoundassisted
extraction
Pulsed-electric
field extraction
Microwaveassisted
extraction
Reduced reaction/preparation times
Minimal material consumption
Effective and economical solvent use
Increased sample throughput
Short extraction time
High efficiency
Short extraction time
High efficiency
Low energy
Less solvent
Low extraction temperature
Short extraction time
Ease of use
High efficiency
Less amount of solvent
Automation of the instrument
Easily coupled with other analytical
methods
Low energy consumption
Decline of extraction of power
with time
High cost
Nonselective
Heat can damage thermal
labile compound
Free radical formation
Expensive equipment
Efficiency dependent on the
conductivity of the
environment
It can damage heat-sensitive
compounds
Limited penetration depth
Uneven heating in complex
matrices
Equipment and maintenance
cost
[88, 90]
[91, 92]
[8, 93]
76 İrem Yıldız Özbaş, Severina Pacifico, and Emre Özbaş
Table 2.3 (continued)
Method Advantages Disadvantages References
Supercritical
extraction
Pressurized liquid
extraction
Enzyme-assisted
Extraction
Solid-phase
microextraction
Short extraction time
High selectivity
Suitable for thermally unstable
compounds
Better diffusivity
Requires less sample and solvent
Environmentally friendly
Ability to operate at low temperatures
Minimal waste production
Ease of automation
Possibility of on-line coupling with
separation and detection techniques
High efficiency and extraction yield
Green technology
Automation
Reducing time and solvent consumption
Protection sensitive compounds
Selectivity
Sustainable and eco-friendly
High efficiency
Low energy consumption
Simple recovery with reduced solvent
usage
Ease of use
Low cost
High efficiency
Rapidity
Being solvent-free
A lack of requirement for special
equipment
Improved sensitivity
Automation, miniaturization
High-throughput performance
Online coupling with various analytical
instruments
Limited to low-polarity
compounds
Low extraction yields
Expensive
High instrument cost [96, 97]
Slow process
Difficulty in achieving optimal
conditions
Expensive
Fiber breakage
Sample carry-over problems
pH instability
[94, 95]
[76, 98, 99]
[100, 101]
2.3.3.9 Ultrasound-assisted extraction
Ultrasound-assisted Extraction (UAE) is a technique that utilizes high-frequency
sound waves for the extraction of target compounds. UAE is considered an environmentally friendly technology due to its ability to reduce the need for organic solvents.
Chapter 2 Methods of obtaining drugs from medicinal and aromatic plants 77
The significant increase in the production of targeted plant-based molecules makes
UAE a highly efficient method [102].
In ultrasound-assisted extraction, also known as sound wave-assisted liquid extraction, sound waves are emitted into the medium through devices that generate ultrasonic sound, enabling the extraction process. This method typically relies on sound
waves produced by devices operating at frequencies ranging from 20–50 kHz, which
disrupt the structure of the cell walls in the sample, thereby facilitating the penetration of the solvent into the plant material [69].
Several parameters influence the extraction efficiency in UAE. These include the
frequency and intensity of ultrasonic waves, the type of solvent, extraction time, and
temperature. These factors can be optimized based on the characteristics of the material and components to be extracted, ensuring that target compounds are not degraded or subjected to thermal damage, thereby maximizing extraction efficiency.
UAE can be performed in two ways: bath extraction and probe (horn) extraction. In
bath extraction, the sample container holding the plant material is immersed in a liquid medium (usually water) or placed in a bath directly exposed to ultrasonic waves.
In probe extraction, ultrasonic horns are applied directly to the sample. Both methods
generate cavitation through vibrations, breaking down cellular barriers and facilitating the extraction of desired compounds [90].
In UAE, solvents such as ethylene glycol, water, ionic liquids, and its oligomers,
glycerol, or other solvents derived from biomass can be used [103]. This method enables the extraction process to be carried out with lower energy consumption, shorter
durations, and at lower temperatures. Additionally, it requires fewer instruments and
smaller solvent volumes, making it an environmentally friendly approach [104].
2.3.3.10 Pulsed-electric field extraction
In extraction processes, nonthermal technological methods are gaining increasing importance as alternatives to thermal treatments. Pulsed electric field (PEF) is a technique that uses moderate to high electric fields to reduce the damage caused by traditional heating methods to plant materials. PEF extraction enhances mass transfer by
disrupting the matrix in which the components are embedded within the plant material. PEF technology is a promising alternative to many other extraction methods, as it
allows the extraction of plant components without affecting their activities. The use of
PEF for extraction has increased extraction yield, shortened processing time, prevented the decomposition of temperature-sensitive materials due to the absence of
thermal treatment, reduced energy costs, and eliminated negative environmental impacts. Recently, this technology has also been employed to stimulate the biosynthesis
of metabolites in plants beyond its application in extraction [91, 105].
The fundamental principle of PEF-assisted extraction is based on placing plant
material between two metal electrodes and exposing it to repetitive short pulses of
PEF
Transfer of large and small molecules into the
intracellular region
Transfer of proteins into the cell
membrane
Cytoplasmic fusion
Cell disruption
Figure 2.13: Effects of PEF on cells.
78 İrem Yıldız Özbaş, Severina Pacifico, and Emre Özbaş
moderate electric fields and low energy input. This process induces permeabilization
of plant cell membranes through pore formation, a phenomenon known as electroporation or electropermeabilization. This technique shows great potential for the selective recovery of target intracellular compounds. One of the primary reasons why
purer extracts can be obtained with PEF is its selective effect on the cytoplasmic membrane, allowing the targeted discharge of intracellular components without disrupting
the overall cell structure. As a result, the need for additional purification steps is reduced. For this reason, PEF is applied to plant tissues as a pretreatment that facilitates
extraction [106].
PEF technology is a nonthermal, minimally invasive, and environmentally friendly
technique. Due to its ability to enhance mass transfer of intracellular components
through electroporation, PEF has found its place in plant extraction processes. The effects of PEF on cells are illustrated in Figure 2.13. The electric field applied disrupts the
cell’s lipid bilayer membrane, alters its permeability, and facilitates contact between
the solvent and target compounds. This results in an increase in extraction efficiency.
Consequently, it reduces the solvent temperature and concentration required for extraction. Lower temperatures help preserve the structural and bioactive properties of
heat-sensitive compounds during extraction [92].
The efficiency of electroporation is generally improved by increasing crucial factors
like the strength of the electric field, treatment duration, and application temperature.
Additionally, the ease, speed, and scalability of adapting PEF to industrial tools make
it a versatile technology for integration with other methods. However, PEF parameters must be tailored to each species, considering their structures, sizes, and other factors that influence extraction efficiency [107].
Chapter 2 Methods of obtaining drugs from medicinal and aromatic plants 79
2.3.3.11 Microwave-assisted extraction
Microwave-assisted extraction is a technique in which the sample is extracted by applying microwave energy in an appropriate solvent. In this method, high-frequency
microwave energy as a type of electromagnetic waves with wavelengths ranging from
1 mm to 30 cm is used. Microwave energy heats the sample from the inside out, providing simultaneous and homogeneous heating. Since microwave energy accelerates
heating, it also speeds up the extraction process and allows the use of less solvent.
This type of extraction enables rapid and effective extraction of the materials. Key
parameters impacting extraction efficiency include the choice of solvent, operating
temperature, microwave power, exposure time, as well as the properties of the plant
material, its matrix, and particle size [108].
The basic principle of the microwave-assisted extraction method involves heating
intracellular water, leading to the breakdown of plant cells and allowing the solvent
to penetrate the plant matrix, resulting in the transfer of components into the solvent.
Microwaves disrupt hydrogen bonding in organic molecules and induce dipole rotation. This causes ions with increased kinetic energy to continuously move and change
direction. The disruption of hydrogen bonds also enhances the ability of solvents to
penetrate the plant matrix [109].
Microwave radiation’s most significant characteristic is its interaction exclusively
with the dipoles of polar or polarizable substances (solvents and samples). The heat
generated through microwaves is transferred via conduction on the surface of these
materials. Since energy transfer occurs solely through dielectric absorption, nonpolar
liquids exhibit very weak heating. Microwave-assisted extraction (MAE) is selectively
applied using solvents with high dielectric constants and polar substances. MAE is
suitable for certain secondary metabolites, such as phenolic acids and some flavonoids, but not for those sensitive to thermal degradation, such as anthocyanins and
certain tannins [110]. In a closed MAE system, it is possible to reach temperatures 2–3
times the boiling points of certain solvents (such as acetone, acetone-hexane, dichloromethane-acetone). This significantly increases the extraction efficiency of components
from the plant matrix. Solvents like water, methanol, and ethanol have high microwave absorption capacities and can rapidly increase in temperature, thereby reducing the processing time [93].
2.3.3.12 Supercritical extraction
Supercritical fluid extraction (SFE) is an environmentally friendly extraction technique. Its key feature is the use of supercritical fluids as solvents. These fluids operate
above their critical temperature and pressure, showing physicochemical features that
exhibit a balance between gas-like and liquid-like behaviors (Figure 2.14) [111]. The
low viscosity and high diffusivity of supercritical fluids allow the solvent to penetrate
Figure 2.14: The supercritical fluid region.
80 İrem Yıldız Özbaş, Severina Pacifico, and Emre Özbaş
the material more effectively, significantly reducing extraction time. Among supercritical fluids, carbon dioxide (CO
native to organic solvents. CO
) is the most widely used solvent as an excellent alter-
2
is an efficient solvent for an extensive variety of com-
2
ponents, and its inert, nontoxic, safe, and recyclable nature makes it ideal for SFE.
Moreover, its low cost and easy availability further enhance its appeal as a solvent for
this method. SFE can be performed at low temperatures, preserving the biological activity of heat-sensitive compounds. The ability to reuse the solvent minimizes waste
generation, contributing to economic and environmental sustainability [94]. Some of
the solvents that can be utilized as supercritical fluids, apart from carbon dioxide, include hydrocarbons such as pentane, butane, and hexane; aromatic solvents like benzene and toluene; alcohols (methanol, ethanol, isopropanol, n-butyl alcohol); and
gases such as ethylene and propane and water [112].
The main drawback of supercritical CO
polar or moderately polar substances are easily dissolved by CO
is its low polarity, which limits its use. Non-
2
, a nonpolar mole-
2
cule. As a result, SFE is primarily used for the extraction of nonpolar or moderately
polar compounds such as lipids, essential oils, and carotenoids. To overcome this limitation, CO
can be combined with polar organic solvents (modifiers) for the extraction
2
of polar compounds. Small amounts of cosolvents such as ethanol or water can be
used to enhance the solubility of polar compounds. These cosolvents, being more
polar than CO
, increase the polarity of the supercritical mixture, significantly im-
2
proving extraction efficiency [113].
There are three approaches to the implementation of supercritical extraction:
static, dynamic, and a combination of both modes. In the static mode, the supercritical
solvent is allowed to contact the plant matrix for a specific period. In the dynamic SFE
mode, fresh supercritical solvent is continuously introduced over the plant material.
Consequently, the flow rate of the supercritical fluid is directly proportional to extraction efficiency. When both modes are combined, the process begins with static extrac-
Chapter 2 Methods of obtaining drugs from medicinal and aromatic plants 81
tion for certain duration, followed by a transition to the dynamic mode. This combination positively influences extraction efficiency [95].
SFE allows for shorter operation times, versatile applications, and safer, greener
experiments through the use of a low-viscosity fluid for the extraction of plant-based
components. Another key advantage of SFE is its ability to establish a direct online
connection with a chromatographic method (e.g., GC, HPLC), enabling the immediate
measurement of components after extraction [114].
2.3.3.13 Pressurized liquid extraction
Pressurized liquid extraction (PLE), first introduced in 1995 as accelerated solvent extraction (ASE), is also referred to by several other names, including pressurized solvent extraction, enhanced solvent extraction, and superheated liquid extraction
(SHLE). When water is used as the extraction solvent, the technique is known as pressurized hot water extraction (PHWE) [97].
PLE is an automated and rapid extraction method that utilizes liquid solvents at
high temperatures and pressures, enabling more efficient extraction of components
from solid and semisolid plant matrices. Increasing temperature and pressure significantly enhances the extraction performance compared to traditional methods [115]. In
this technique, solvents are maintained in a liquid state above their boiling points
under high pressure. Using solvents at temperatures above their atmospheric boiling
points provides several advantages, including improved solubility, enhanced diffusion,
and better mass transfer mechanisms, which facilitate the extraction of target plant
components. Additionally, under high temperatures and pressures, the viscosity and
surface tension of solvents are reduced, allowing for deeper penetration into the solid
matrix. This accelerates solvent penetration and the overall extraction process, thereby
increasing the efficiency of bioactive compound extraction from plant materials [116].
PLE can generally be performed in static mode, dynamic mode, or a combination
of both modes. In the dynamic mode of the extraction procedure, fresh solvent is continuously pumped through the sample, resulting in a constant shift in equilibrium
and an increase in the mass transfer rate. However, its main disadvantages include
the requirement for larger solvent volumes and the necessity of a concentration step
to dilute the components in the extract before chromatographic analysis of the target
compounds. Extraction efficiency in dynamic mode is equal to or higher than in static
mode, and the extraction time is generally similar in both modes. However, incorporating a pre-extraction step in static mode before transitioning to dynamic mode can
reduce the overall extraction time [117].
Widely used for years, PLE provides numerous benefits compared to traditional
extraction methods, such as faster extraction times, lower solvent usage, reduced
costs, and the ability to scale the extraction process easily to industrial levels [118].
82 İrem Yıldız Özbaş, Severina Pacifico, and Emre Özbaş
2.3.3.14 Enzyme-assisted extraction
The fundamental principle of enzyme-assisted extraction is the catalytic action of enzymes to hydrolyze and break down plant cell walls. This process enables the intracellular components to be released under optimal experimental conditions. Initially, the
plant cell wall binds to the active site of the enzyme. The substrate-enzyme interaction between the cell wall and the enzyme induces a conformational change in the
enzyme upon binding. This structural alteration in the enzyme leads to the breakdown of the bonds within the cell wall, releasing the active components from the
plant cells. This method significantly preserves the bioactive potential of the extracted
compounds [98].
Enzyme-assisted extraction (EAE) is particularly necessary for phytochemicals
bound within the lignin-polysaccharide network of certain plants. These phytochemicals are stabilized by hydrophobic interactions, such as hydrogen bonds and van der
Waals forces, making their separation highly challenging. In such cases, phytochemicals are often dispersed in the cytoplasm and cannot be extracted through standard
solvent-based methods. To address this issue, specific enzymes are employed. These
enzymes hydrolyze structures like cellulose and lipids, facilitating the release of
bound phytochemicals. Enzyme treatment is employed as a pretreatment to degrade
cell walls, enhancing the extraction efficiency of phytochemicals [76].
EAE is performed using two primary approaches: enzyme-assisted aqueous extraction (EAAE) and enzyme-assisted cold pressing (EACP). EAAE is typically applied for the
extraction of oils and other lipophilic components from seeds, while EACP is commonly
used to hydrolyze seed cell walls with enzymes, thereby enhancing extraction efficiency
[99]. The parameters affecting the EAE procedure are listed in Figure 2.15 [99, 119].
2.3.3.15 Solid-phase microextraction
Solid-phase extraction (SPE) operates on a principle similar to liquid-liquid extraction
(LLE), involving the partitioning of dissolved substances between two phases. However, in SPE, one phase is a liquid while the other is a solid (sorbent). The stationary
phases used in solid-phase extractions are the same type as those used in liquid chromatography columns. The stationary phase is housed in a glass or plastic column.
Commercial SPE cartridges are designed in the form of injectors, are single-use, and
have a capacity of approximately 1–10 mL. Solid-phase extraction is often used as a
sample preparation step to clean the sample before performing chromatographic or
other analytical methods for determining the quantities of components in the sample
[120]. Solid-phase extraction was initially employed as a purification method before
HPLC or GC analysis. However, its application has expanded and it is now commonly
used for the rapid fractionation of crude plant extracts or for transferring purified