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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_6035_Библиотеки_им_академика_М_И_Перельмана.pdf
TABLE 9.3 (Continued)
Extraction Methods Principle Advantages Disadvantages Reference
High hydrostatic
pressure-assisted
extraction
Enzyme-assisted
extraction
Natural deep
eutectic solvent
extraction
Isostatic and Le Chatelier’s
principle
Biocatalyst for breakdown
of cell membrane
Directly by acting on
hydrogen bonds and
indirectly by cell lysis
Reduced damage to cells or denaturation of
protein, environment-friendly, desirable for
thermolabile compounds
Increased extraction yield by cell lysis Additional long operation in wet
NADES are usable and recyclable, nontoxic,
synthesized with low energy, easily get
decomposed, stability at higher temperatures
High cost of investment, changes
internal structure of fragile
material, batch type process
conditions
NADES possess high viscosity,
thereby affecting hydrogen bond
interactions, reducing the diffusion
coefficients, low mass transfer, and
long extraction time
Xi (2006)
Latif and Anwar
(2009), Jha and Sit
(2022)
Mbous
(2017), Vanda
et al. (2018)
193
et al.
194
diffusion by increasing the surface-to-volume ratio. This technique is generally recommended for the extraction of heat-labile components.
This technique is desirable for the separation of bioactive molecules from harder plants.
In this process, the solid material is simmered in boiling water for 15–60 min or until the
amount of water added turns half. This method is considered suitable for water-soluble
thermostable components. Similar to maceration it also follows the principle of molecular
diffusion (Azwanida, 2015). This procedure is generally used during the preparation of
ayurvedic extracts referred to as “quath” or “kwath” (Hussain et al., 2019).
This process involves the passage of boiled solvent through the plant material at a controlled
rate of 5–7 drops/min until the extraction is completed before evaporation. Its continuous
operation makes this technique more efficient than maceration for extraction. Initially,
during the process, less solvent is required as a wetting agent for dried solid material for
2–4 h within the equipment; later the solid material is submerged completely. At this point
the equipment is sealed from the top and the process of extraction is continued for next 24 h
(Azwanida, 2015). This process is mostly used to isolate bioactive compounds for the
formation of tinctures and extracts in liquid form (Hussain et al., 2019).
This method is usually employed for the partitioning of bioactive components that are volatile
in nature. It is primarily used to remove the essential oil from the solid material of plant parts
or plant waste. Three different hydrodistillation methods are used, that is, water distillation,
water and steam distillation, and direct steam distillation (Vankar, 2004). In this technique,
solid material is boiled in water to obtain essential oil and bioactive components after the
vapor condensation. Heating leads to hydrolysis and decomposition of the solid cell wall
and breaks the bounded form of the target compound from a matrix (Rassem
et al., 2016,
Omeroglu et al., 2019). Direct steam can also be used in the solid matrix.
It is a simple chemical method that is desirable for the extraction of volatile bioactive
components. Its working mechanism is similar to that of maceration. However, this technique requires less time for extraction compared to maceration. In this technique, the solid
material is steeped in boiled or cold water followed by the separation and concentration of
solvent which is typically done under a vacuum (Hussain et al., 2019).
195
It is an automatic continuous process that is followed using a Soxhlet apparatus. In the
year 1879, the scientist, Franz Ritter Von Soxhlet from Germany, invented this apparatus
(Soxhlet, 1879). This technique was basically designed for fats and lipid extracts but was
later also used to extract valuable bioactive components. In this technique, the ground solid
material is wrapped in a porous bag or thimble and placed in the apparatus. In the round
bottom flask, the solvent is heated, vaporized into the sample, and condensed. When the
solution in the thimble reaches the overflow level, it is aspirated by a siphon, which returns
the solution to the distillation flask. The solution that returns contains dissolved components
which then remain in the flask itself. It is a continuous process for 8 h (Azwanida, 2015). It
can be used as a benchmark to compare the working efficiency of novel extraction methods.
This technique periodically in a constant motion brings fresh solvent in direct contact with
solid matrix and maintains a relatively elevated temperature during extraction.
9.4.2 NOVEL EXTRACTION METHODS
It is an advanced form of extraction technique where solvent in the form of fluid is used
under pressure ranging between 200 and 400 bar and temperature between 40 and 60°C. The
major solvents used are carbon dioxide, toluene, ethylene, and ammonia. In this technique,
a solvent at its supercritical condition behaves both as a liquid and a gas, possessing both
the diffusivity of a gas as well as the solvating power of a liquid (Al Khawli et al., 2019).
During extraction, the solvent at its supercritical point is introduced into the substance
containing the bioactive compounds. CO2 is the most commonly used solvent which gets
converted into its supercritical stage when the applied pressure is beyond 7.38 MPa and
temperature is above 31°C. The applied pressure increases the solubility and helps in the
quick penetration of solvent to the cell wall. Hence, the bioactive molecules are solubilized
with the supercritical fluid and come out from the solid material along with the fluid itself
(Torres-Ossandón et al., 2018).
It is a dielectric constant-based heating technique that possesses electromagnetic radiations
working at frequencies starting from 300 MHz to 300 GHz and wavelength between 1 and
100 cm. In this technique, the object absorbs electromagnetic energy and converts it into
heat. During microwave exposure, the electric and magnetic fields flow in a perpendicular
direction, and heating takes place due to ionic conduction and molecular dipole rotation.
Commercially , the microwaves used possess a frequency of 915–2450 MHz (Kaderides et al.,
2019). The extraction process involves the polarized material and dipoles of the polar
solvent interacting with the electromagnetic waves and trying to align themselves with
196
the changing field (Chan et al., 2011). This orientation of polar material with changing
field direction generates heat and alters the cell structure, thereby partitioning the bioactive
components from a solid matrix into the solvent. In addition, the yield of the extract is
improved when the solid matrix is drenched completely with the solvent. The effectiveness
of MAE depends on various factors such as sample material, sample size and moisture
content, components being extracted and their dielectric constant, properties of solvent
used, solid-to-solvent ratio, as well as microwave conditions, that is, extraction time,
temperature, frequency , and microwave power (Chan et al., 201 1). However , the solubility,
dielectric constant, and dissipation factor of the solvent are considered the most crucial
factors during extraction. Polar solvents act as superior for retaining more microwave
energy due to their higher dielectric constant (Wang and Weller, 2006). In most cases, the
extraction time and microwave power required during the operation range from 30 s to 10
min and 25 to 750 W, respectively (Kaufmann and Christen, 2002).
It is a sound wave technique that works at a frequency range beyond normal hearing, that
is, 20 kHz–100 MHz. Ultrasound waves also possess characteristics of sound waves, that
is, reflection, interference, adsorption, and scattering and can transmit through all three
matters. This technique possesses cavitation by creating a region of compression (positive
pressure) with rarefaction (negative pressure) (McClements, 1997). In addition, when
the negative pressure is elevated enough, cavitation takes place leading to the collapse of
formed bubbles. However, cavitation only occurs with liquid or liquid-containing solids.
This technique amplifies the mass transfer rate, allowing faster access of solvent to the cell
component of the solid matrix, and thereafter, diffusion of components from the cellular
membrane into the solvent occurs simultaneously with cell wall rupture (Mason et al., 1996).
It is a safe, sustainable, cost-effective, as well as environment-friendly process. The factors
that influence the extraction efficiency include extraction time and temperature, polarity
and amount of solvent, as well as ultrasound source (frequency, intensity) (Tiwari, 2015).
The cavitation decreases with an increase in ultrasound frequency and intensity, thereby
affecting the extraction yield. However, a material with high viscosity considerably requires
a higher amplitude to achieve the required cavitation point (Capelo-Martnez, 2009).
It is an advanced form of conventional extraction methods. It involves the application
of high pressure that makes extraction easier and more efficient by accelerating the
solubility as well as improving the mass transfer rate of the solid matrix with solvent. The
pressure used helps maintain the liquid state of the solvent even after reaching its regular
boiling point. Hence, this technique is also known as high-pressure or accelerated solvent
extraction (Nieto et al., 2010). For achieving the highest recovery of bioactive ingredients,
polarity as well as volume of extracting solvent, pressure, temperature, sample size, and
197
extraction cycle plays a significant role. It is important that the choice of solvent used for
extraction must depend upon the solid matrix from where the component is extracted. This
technique acts as an effective method for essential oil extraction from plant material. When
compared with the conventional Soxhlet extraction method, PLE is considered a powerful
tool in dramatically decreasing operational time and solvent use (Richter et al., 1996). In
the current scenario, PLE possesses great potential as a substitute for the SCFE method for
the extraction of polar molecules (Kaufmann and Christen, 2002).
It is a nonthermal extraction process that is used to recover bioactive compounds from
a plant matrix at a minimal temperature. In this technique, the plant matrix is exposed
to the electric energy which is responsible for the cause of electroporation. The applied
electric potential transfers the ions and molecules within the cell near the cell membrane
and separates the electric potential depending upon their charge in the membrane. When the
transmembrane potential between two membranes exceeds 1 V, repulsion occurs between
charge-bearing molecules creating nano or microporation in the cell membrane. Hence, it
helps the bioactive components present in the cell’s plasma to come out from the membrane
(Shorstkii et al., 2020). PEF treatment is typically performed using simple circuits; however,
its efficiency is affected by various factors such as specific energy input, field strength, pulse
number, materials being treated, and treatment temperature (Heinz et al., 2003). It has been
reported that the plant material treated with PEF (500–1000 V/cm for 10–2–10–4 s) exhibits
membrane injury without a considerable increase in temperature (Lebovka et al., 2002).
In addition to the PEFE method, HPPAE is also considered a nonthermal technique for
extracting bioactive components. It is a high-pressure processing method the working of
which is based on isostatic and Le Chatelier’s principle for the extraction of bioactive
components. In this technique, pressure ranging between 100 and 600 MPa is applied
uniformly throughout the product. This applied pressure induces the breakdown of
ionic bonds, leading to a decrease in volume due to the electrostriction force acting on
water. Hence, high pressure alters the existing structure of the cell’s molecule (protein
denaturation, breakdown of hydrophobic bonds, and salt bridge) and decreases the mass
transfer resistance in the cell internally (Linton et al., 2001). In addition, the higher-pressure
difference between the cell outside and interior of cell membranes enhances solvent
penetration into the cell for the extraction of bioactive components (Grassino et al., 2017).
According to the Food and Drug Administration, HHPAE is a noninvasive method that
does not cause denaturation or major damage to cells and is considered an environmentally
friendly technology (Xi, 2006). Certain parameters, such as solvent type and pressure,
solvent volume, number of extraction cycles, extraction time, and temperature, all have a
significant impact on extract recovery (Xi
et al., 2011).
198
It is a biological technique usually applied to those components where conventional
techniques are not effective for their extraction as well as when they are tightly bound to
the cell wall. Different enzymes are used in this technique to break down the plant cell
wall, allowing the bioactive active components present inside the cell plasma to ooze out
of the cell (Panja, 2018). The enzyme binds to the active site of the cell wall, which is
composed of a network of polysaccharides and lignin, and breaks the glycoside bond of
the cell wall as well as the proteolytic bond in the middle lamella (Zhang et al., 2019).
Enzymes such as cellulose, hemicellulose, polygalacturonase, xylanase, pectin esterase,
polygalacturonase, amylase, protease, are being used for complete biomass extraction (Liu
et al., 2016; Barbosa et al., 2020). Further, the enzyme composition, concentration, enzyme/
substrate ratio, solvent type, solid-to-solvent ratio, extraction temperature, pH, and time all
have a significant impact on enzyme activity disruption and disintegration of cell wall
structure and release of the target bioactive components. EAE is accomplished using two
methods: enzyme-assisted aqueous extraction (EAAE) and enzyme-assisted cold pressing
(EACP). EAAE is a green process of extraction that combines the usage of an aqueous
method of extraction by using an aqueous solvent with enzymes. Aqueous extraction
ruptures the cellular tissues and heats proteins–polysaccharide colloids, denaturing them,
whereas enzyme accelerates the rate of target component release, enabling the recovery
of extractable components like oils. Despite this, conventional cold pressing led to less
emulsion formation during oil extraction because of the EACP’s use of nonaqueous solvents
and disruption of the cell wall structure due to enzymatic activity (Latif and Anwar, 2009).
In this technique, a natural deep eutectic solvent is used for the extraction of bioactive
components. A deep eutectic solvent is considered a eutectic mixture of two or three organic
components that possess a melting point much lower than either individual component. DES
contains various components including choline, urea, organic acids, sugars, etc. However,
natural deep eutectic solvents (NADES) are a new DES derivative. They are regarded as
“natural” due to the constituents being used are primary metabolite groups, such as organic
acids, sugars, bases, and amino acids (Dai et al., 2013). NADES generally act as a pretreatment solvent that works on the basis of two mechanisms, that is, directly by interacting with
active components through hydrogen bonds or indirectly by destroying the cell wall for the
release of active components from the solid matrix (Kalhor and Ghandi, 2019).
9.5 CONCENTRATION AND PURIFICATION OF BIOACTIVE MOLECULES USING CHROMATOGRAPHIC TECHNIQUES
Various traditional as well as novel techniques can be used for the extraction of bioactive components from various natural sources. During extraction, most of the techniques
199
use liquid solvents of polar or nonpolar nature which help in the extraction of particular
bioactive molecules, but after completion of extraction we need to concentrate the extract
by removing solvents by either simple evaporation or heating at atmospheric pressure or
either by use of rotary vacuum evaporator (Hamid et al., 2020b, 2022). Further, to isolate
the individual bioactive molecule, some purification techniques need to be followed, such
as column chromatography.
Utilizing open-column chromatography, an adsorption–desorption method, and
partitionable solvents, the interfering chemicals from the crude extract are removed
during the purication stage. Solid phase extraction (SPE) cartridges, Sephadex LH-20,
polyamide, Amberlite, styrene-divinylbenzene (XAD16, XAD 4, EXA 118, EXA-90,
SP70), and acrylic resins (EXA-31, XAD-7) are a few examples of materials that are
frequently used to separate phenolics from crude sample extracts (Antolovich et al.,
2000, Silva et al., 2007, Li et al., 2005, Scordino et al., 2004). However, in many studies
for purication and partial concentration, SPE was used prior to partitioning using HPLC
(Castaneda-Ovando et al., 2009, Rostagno et al., 2005, Michalkiewicz et al., 2008). In
addition to the aforementioned processes, the separation and purication processes are
most important to get pure and natural products with active fractions of target molecules.
The separation depends on the unique natural product’s chemical or physical dif ferences.
And this isolation is based on the afnity of isolating compounds with the solvents which
makes the column chromatography technique very complex (Zhang et al., 2018).
9.5.1 SEPARATION BASED ON ADSORPTION PROPERTIES
Due to its ease of use, excellent capacity, and affordable adsorbents like silica gel and
macroporous resins, adsorption column chromatography is commonly employed for the
partitioning of natural products principally in the first separation step. The adsorption
affinities of the natural produce for the surface of the adsorbents differ, which forms the
basis for the separation (Zhang et al., 2018). According to estimates, silica gel served as the
foundation for around 90% of phytochemical separation (on a preparative scale). A polar
absorbent having silanol groups resembles silica gel. The silica gel holds to molecules
via hydrogen bonding and interactions between dipoles. In silica gel columns, polar
molecules are, therefore, kept longer than nonpolar ones. A common method for separating
natural polyphenols, such as anthraquinones, phenolic acids, and flavonoids, is polyamide
column chromatography which involves the mechanism of hydrogen bonds establishment
between the target compounds, the mobile phase, and the polyamide absorbents (Zhang
et al., 2018). Gao et al. (2011) investigated how polyphenols, including flavonoids and
phenolic acids behaved during chromatography on a polyamide column. The quantity of
phenolic hydroxyls with their locations within the molecule had an impact on the capacity
of adsorption and it was discovered that the polyamide served as a hydrogen bond acceptor.
Adsorptive macroporous resins are macroporous polymer adsorbents lacking ion exchange
groups that may specifically adsorb practically any kind of naturally occurring substance.
Electrostatic forces, complex formation, hydrogen bonds, and size-sieving interactions
among the resins along with the natural products in solution are some of the adsorptive
200
processes of adsorptive macroporous resins (Zhang et al., 2018). Meng et al. (2017) used
D101 macroporous resin to extract the total saponins from Panacisjaponici rhizome
(PJRS). They discovered that PJRS included more than 73% of main saponins, chikusetsu
saponins IVa, IV, and V, as well as pseudo ginsenoside RT1.
9.5.2 SEPARATION BASED ON PARTITION COEFFICIENT
The liquid–liquid extraction principle which is the foundation of partition chromatography
(PC) relies on the level of mixing properties in two separate immiscible liquids. As initial
step contains one of the liquid phases coated with solid (cellulose, carbon, silica gel, etc.)
phase used as the stationary phase, and the other liquid phase is used as the mobile phase.
The easily removable stationary solid phase and inconsistent results of this type of PC have
made them less common in use nowadays. As bonded phases are frequently employed to
separate a range of natural products, particularly in the last purification step, commercially
available alkyl (C8, C18, cyano, aryl) and amino-modified silanes are frequently used
(Zhang et al., 2018).
9.5.3 SEPARATION BASED ON THE MOLECULAR SIZE
The molecular sizes of the natural products are used to determine whether to separate
them using gel filtration chromatography (GFC) or membrane filtration (MF). Smaller
molecules can pass across the semipermeable membrane in MF while the bigger molecules
are retained. In accordance with the pore size of the membrane used, MF of raw materials
may be divided into three categories: microfiltration, ultrafiltration, and nanofiltration
(Zhang et al., 2018). Oleuropein the predominant component, was concentrated almost ten
times in the nanofiltration retentate, and the antioxidative and antibacterial polyphenols and
flavonoids were recovered by nanofiltration (Khemakhem et al., 2017). Other names for
gel filtration chromatography include size exclusion chromatography and gel permeation
chromatography . In GFC, tiny molecules are retained for a longer period of time than lar ge
molecules (Zhang et al., 2018). The Sephadex (G-types) were employed for the partitioning
of hydrophilic molecules like peptides and are created by cross-linking dextran (Sila and
Bougatef, 2016). The separation of natural products also made use of cross-linked agarose
(Tan et al., 2010) and polyacrylamide (bio-gel P) (Li et al., 2013).
9.5.4 SEPARATION BASED ON IONIC STRENGTH
Based on variations in molecules’ net surface charges, they can be separated using ionexchange chromatography (IEC). IEC may be used to separate several natural compounds,
including alkaloids and organic acids with ionization-capable functional groups. By altering
the potency of ions of the mobile phase (e.g., by altering pH or salt level), the molecules having
charge could be captured and released by ion-exchange resin. Alkaloids were separated using
201
cation ion-exchange resins, while natural organic acids and phenols were separated using
anion-ion-exchange resins. Using cation ion-exchange resin (Dowex 50WX8), the anthocyanins (positively charged) from the neutral phenolic components in XAD-7-treated kiwifruit
fruit extract were separated (Comeskey et al., 2009).
9.5.5 OTHER MODERN SEPARATION TECHNIQUES
By distilling the high molecular weight compounds under vacuum at a temperature much
lower than their boiling point, molecular distillation isolates the molecules (Zhang et al.,
2018). Borgarello et al. (2015) used molecular distillation with artificial neural networks
to extract a thymol enrichment fraction from the essential oil of oregano. The resulting
fraction could stabilize the sunflower oil and possess antioxidant characteristics.
GC may be the best preparative approach for the separation of volatile chemicals due to
its high separation efficiency, quick separation, and analysis. Due to the unavailability of
commercial Prep-GC, the split device, injection port, column, and GC equipment’s trap
device must be transformed for preparative separation (Wang et al., 2011).
Supercritical fluid is used in SFC as the mobile phase. These supercritical fluids have
qualities of low viscosity, high dissolving capacity, and diffusivity, which allow quick and
efficient separation, SFC combines the benefits of both LC and GC, as a result, SFC may
utilize a longer column and shorter stationary phase particles than HPLC, resulting in a
higher number of theoretical plates and improved separation. According to Zhang et al.
(2018), SFC may be used to separate nonvolatile or thermally sensitive substances for
which GC is inapplicable.
Due to its distinct qualities such as high selectivity, cheap cost, or ease of preparation,
molecular imprinting technology has been a popular separation technique in the past
ten years. When the template molecules are taken out of the MIP (molecular imprinted
polymer), several complementary holes are created that retain the memory of the size,
shape, and functional groups of the template molecules. As a result, the MIP will specifically recognize and bind to the template molecule or its analogs. MIPs have been frequently
202
utilized to separate natural goods or as solid-phase extraction sorbents to enhance
the minor components in samples of herbal materials (Zhang et al., 2018). The three
main curcuminoids, curcumin, dimethoxycurcumin, and bisdemethoxycurcumin, were
extracted from the TCM Curcumalonga rhizome using the thermoresponsive magnetic
MIP (You et al., 2014).
Multiple stationary phases with multiple columns are used in simulated moving bed
(SMB) chromatography. Rotary valves that regularly swap the intake (eluent and feed)
and output (raffinate and extract) replicate the counter-current movement of the bed. The
SMB process is a strong instrument for the large-scale partitioning of natural products
with the benefit of using less solvent in less time. It is a continuous separation method
(Zhang et al., 2018).
From the plant matrix, due to wider availability of bioactive compounds, complex nature,
and different properties, it is very complicated to separate the desired compounds in a pure
state only by using single-column chromatography technique. The effectiveness of partition of multidimensional segregation based on solid phase extraction with the linking of
numerous columns with diverse stationary phases is considerably improved (Zhang et al.,
2018). Natural product separation is becoming more quick, efficient, as well as automated
as more competitive multidimensional separation technology enters the market. A total of
five antioxidants, including two alkaloids (glusodichotomine AK, glusodichotomine B)
and three flavonoids (homoeriodictyol, luteolinandtricin), were isolated through a twodimensional HPLC (RP/HILIC) approach from Arenaria kansuensis on NP-XAmide and
RP-C18HCE preparative columns (Cui et al., 2017).
9.6 IDENTIFICATION AND CHARACTERIZATION OF BIOACTIVE MOLECULES
Bioactive compounds are the phytochemicals found in natural sources, for example, plants,
fruits, and vegetables, and thus exhibit an effect on cells and tissues of living organisms.
Bioactive compounds are among the most commonly contained compounds in botanicals
and herbal formulations used for therapeutic purposes, with approximately 20,000
medicinal plants in 91 nations, as per the WHO (Hafizah et al., 2016).
Generally, the key phases for proper application of bioactive compounds from natural
sources include the processes such as extraction, screening through pharmacological
ways, separation/isolation, characterization of bioactive compounds, toxicological as well
as clinical assessment. Figure 9.1 provides an overview of common approaches used to
extract, isolate, and characterize bioactive compounds from natural sources. Usually , during