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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5217_Библиотеки_им_академика_М_И_Перельмана.pdf
• The most suitable enzyme/enzymes for the target compounds must be used at
optimum concentrations. Enzymes with synergistic effects can be used.
Enzyme Type, Concentration and Composition
• Smaller particles enhance enzyme activity.
The particle size and moisture content of the plant
material.
• The optimal temperature should be selected based on the activity of the enzyme
used.
Reaction Temperature
• The optimal pH should be selected based on the activity of the enzyme used.pH• Enzyme function requires enough time without being overexposed.
Extraction time
• An appropriate ratio should be used to optimize enzyme activity and efficiency.
Solvent-to-solute ratio
Figure 2.15: Key parameters in enzyme-assisted extraction (EAE).
Chapter 2 Methods of obtaining drugs from medicinal and aromatic plants 83
84 İrem Yıldız Özbaş, Severina Pacifico, and Emre Özbaş
compounds from HPLC separations to capillary NMR spectroscopy for structural identification [121].
Solid-phase microextraction (SPME) can be described as a modified version of
SPE and is widely applied across various fields. SPME is a robust solid-based extraction technique developed in the 1990s by Authur and Pawliszyn [122]. SPME differs
from solid-phase extraction in several ways. In SPE, analytes require liquid-phase extraction after sorption. SPE is limited to the extraction of liquid samples, whereas
SPME is a technique that facilitates chromatographic analyses of solutions from challenging matrices in either liquid or gaseous states [123]. SPME is a solvent-free method
aimed at increasing sensitivity by directly injecting all extracted analytes into analytical instruments, in contrast to traditional SPE, which uses larger volumes of extraction phases. An important advantage of the SPME method is its ability to avoid macromolecules and particulate organic materials that obstruct SPE columns [124].
The target analytes, which can be gas, liquid, or solid, are desorbent and analyzed
when a sample is frequently exposed to trace amounts of an extractant immobilized
on a solid substrate for a predetermined amount of time in SPME. To increase the extraction efficiency and selectivity of microextraction, the choice of adsorption materials is important [125]. The amounts and speeds of adsorption are significantly influenced by the analytes’ interactions with the adsorbent surface, which can occur
through hydrogen bonding, π-π, dipole-dipole, electrostatic, or hydrophobic/hydrophilic interactions [126].
In this technique, the substances to be extracted are adsorbed onto high boiling
point polymers coated on the surface of a silica fiber as the stationary phase. The substances absorbed or adsorbed by the fiber coatings are thermally desorbed in a chromatography injection port after the extraction device is exposed to the head space of
the sample or sample solution. Because it only involves a few steps and small sample
sizes, the fiber SPME method is simple to use and offers superior cleanup. The partition equilibrium of analytes between the extraction phase and the sample matrix is
the foundation of SPME, which produces quantitative or semiquantitative results. In
recent years, different analytical tools have been combined with alternative microextraction devices to develop various SPME-related technologies [100].
The integration of SPME with chromatography methods consists of two main
steps. The first involves the absorption of dissolved substances from the sample matrix onto the adsorbent, while the second step transfers the absorbed analytes to a
chromatography inlet system through either thermal or liquid desorption. SPME is
gaining increasing attention as a green and versatile sample preparation technique.
Combining SPME with an automated sampler significantly enhances the speed and efficiency of the process [127].
Chapter 2 Methods of obtaining drugs from medicinal and aromatic plants 85
2.3.3.16 Bioassay-guided fractionation of plant extracts
Bioassay-guided fractionation involves testing an extract for activity, separating it
chemically, and then testing the resulting fractions for activity. This procedure is iterative; until one or more active molecules are isolated, the most active fraction can be
separated and its fractions examined again. With bioassay-guided fractionation, drug
interaction analysis is done in reverse; rather than making and testing a mixture, a
naturally occurring mixture is separated and tested in order to identify any interactions that may be present. Data gathered from bioassay-guided fractionation can be
utilized to measure synergy in bioactive extracts without the need for further experiments and help guide go/no-go choices by treating natural substance extracts as combinations of their fractions [128]. The general steps for bioassay-guided fractionation
and the identification of bioactive chemicals are shown in Figure 2.16 [129, 130].
When biologically active molecules are isolated using chromatographic separation techniques combined with bioassay-guided fractionation of plant extracts, the
fact that subfractions obtained through fractionation exhibit more drug-like properties compared to crude extracts can lead to a focus on more promising compounds for
drug discovery. Performing chemical analysis after the active fraction is isolated also
accelerates the process. Fractionation strategies focused on biological activity instead
of a specific group of components have gained importance in drug development processes involving medicinal and aromatic plants [131].
Common challenges encountered in fractionation under bioanalysis guidance include the potential loss of bioactive biological activity during the fractionation process
or failure resulting from the procedure. Key reasons for this include the degradation
of bioactive components during the process, the presence of components at very low
concentrations, and the bioactivity being due to the synergistic effects of multiple
components. To avoid these issues, it is crucial to identify the target bioactive compounds early in the purification procedure. However, in recent years, the re-isolation
of previously identified plant molecules as a result of fractionation under biological
assay guidance has been encountered. To prevent this, a pre-evaluation step known
as “dereplication” is used [132].
2.3.4 Isolation and purification
Modern research requires the isolation of individual components from plant extracts
and their evaluation as potential drugs, as opposed to the traditional medicine, which
uses whole plant extracts for treatment. Both approaches – using whole extracts and
purifying individual components – have their own advantages and disadvantages. In
some cases, it has been observed that the herbal extract obtained from the whole
plan tor specific parts of the plant, or a mixture of different plant extracts without
isolating the herbal components provides better therapeutic efficacy. In many cases,
• Plant Material
Natural
Source
• Extraction
Crude Extract
• In vitro
bioassay
Fractionation
• In vitro
boassay
Bioactive
fraction
• A single entity
made up of
the bioactive
fraction
Pure
compound
• Bioactive
molecule
Structure
elucidation
Figure 2.16: The general steps for bioassay-guided fractionation of natural sources.
86 İrem Yıldız Özbaş, Severina Pacifico, and Emre Özbaş
Chapter 2 Methods of obtaining drugs from medicinal and aromatic plants 87
the isolation of the “active compound” has resulted in the compound becoming
completely inactive. The generally better therapeutic effects observed when using
whole extracts instead of isolated compounds without any purification steps can be
attributed to the potential synergistic effects of the active components in plants or the
specific properties of the matrix in which they naturally occur in the plant [57].
A lead compound (bioactive pure compound), if found within a mixture of other
compounds derived from a plant source and is intended to be developed as a standalone drug, must be isolated and purified. The process of isolating and purifying bioactive compounds from plants is a highly challenging and complex endeavor. The
physical or chemical differences between each particular natural product determine
the separation. The ease of isolation and purification is tightly associated with the
structure stability and concentration of the compound within the material [133].
The separation and isolation of components from a plant extract is typically performed using chromatographic techniques, followed by the characterization of the
isolated compounds, which is generally carried out using spectroscopic techniques
[134]. Within this framework, chromatographic separation methods are used iteratively to produce fractions enriched with specific components or groups of components, ultimately leading to the isolation of single compounds. Spectroscopic methods
allow the analysis of extracts, fractions, or single compounds and provide insights
into the chemical character and structural properties of the compounds [135].
In recent years, significant advancements have been made in the field of natural
compound isolation. New methods combining chromatographic and spectroscopic or
spectrometric techniques aim to elucidate the structures of known or novel compounds without requiring isolation. There has been an increasing trend toward isolation techniques based on pharmacological or biological activity. Bioassay-guided isolation strategies enable the correlation of chemical profiles of extracts and fractions
with activity data from micro-scale in vitro biological assays. This approach has significantly shortened the time required to identify bioactive compounds [136].
Chromatographic techniques are among the most important bioanalytical methods used in the analysis of natural product sources. By utilizing these methods, different and complex components in a complex plant extract can be separated, identified,
and purified both qualitatively and quantitatively [137]. This section will focus on
chromatographic techniques commonly used in isolation and purification.
2.3.4.1 Thin-layer chromatography (TLC) and high-performance thin-layer
chromatography (HPTLC)
Thin-layer chromatography (TLC) is a chromatographic technique that is simple to
prepare and apply, versatile, sensitive, and highly efficient. It is generally used to separate nonvolatile compounds from plant materials [138].
88 İrem Yıldız Özbaş, Severina Pacifico, and Emre Özbaş
TLC is a chromatographic technique that utilizes the adsorption mechanism to
separate a component from a mixture. Separation, as in all chromatographic methods,
relies on the interaction between the compounds in the mixture and the stationary
phase. The basic principle of the method involves a multistep distribution process
that includes the target compounds, solvents, or mixtures of solvents (mobile phase or
eluent), and the stationary phase (adsorbent). It is applicable for the separation of low
molecular weight compounds. The stationary phase typically consists of materials
such as silica gel, sephadex, aluminum oxides, or cellulose, cast at an appropriate
thickness onto glass, plastic, or aluminum plates. The compounds in the extract migrate on the plate based on their solubility in the mobile phase. Each separated compound is identified by calculating the retention factor (Rf), which is the ratio of the
distance traveled by the compound to the distance traveled by the mobile phase, and
is then compared with known compounds. The method’s key advantages include its
time efficiency and stability against acidic solvents [70].
(TLC is among the earliest developed chromatographic techniques. However, with
the development of devices, automation, and the advancement of new adsorbents and
supports, it is still widely used today. HPTLC is an advanced form of TLC that uses
higher-performance adsorbents. The HPTLC technique is a standardized method that
can be used for the qualitative and quantitative analysis of components in plant samples. At the same time, HPTLC is a technique recognized by the European Pharmacopoeia and widely used for quality control and analysis of herbal medicines and their
preparations in compliance with GMP standards. The TLC method is a versatile, sensitive, and high-efficiency technique with simple sample preparation and application.
With TLC/HPTLC, chromatographic fingerprint analyses can be performed by quickly
comparing a range of plant components with references [46]. Compared to TLC, it offers higher accuracy and reproducibility due to improved separation efficiency and
detection limits. The use of high-resolution sorbents with specific particle sizes and
chemically modified plates, combined with compatibility with various instruments
and detectors, enables rapid quantification of phytochemicals and chromatographic
fingerprint analyses [138–140].
2.3.4.2 High-performance liquid chromatography (HPLC) and ultra-performance
liquid chromatography (UHPLC)
Liquid chromatography is widely regarded as the most popular method for herbal fingerprinting due to its numerous advantages, including broad applicability, high resolution, excellent selectivity, sensitivity, reproducibility, and the capability for full automation [46].
High-performance liquid chromatography or high-pressure liquid chromatography (HPLC) is a modern, powerful, and versatile chromatographic separation technique routinely used to separate, identify, and quantify components from complex
Chapter 2 Methods of obtaining drugs from medicinal and aromatic plants 89
mixtures. HPLC analysis serves as a cornerstone of phytochemical studies, especially
in the characterization and isolation of components from crude plant extracts and in
obtaining their chemical profiles or fingerprints [141].
HPLC operates based on chromatographic separation principles, where analytes
are separated according to their differential interactions with the stationary phase
and the mobile phase. The separation mechanism in HPLC relies on the differences in
the affinities of the compounds to the stationary and mobile phases. The analytes eluting from the HPLC column are detected by various detectors, and their signals are
recorded by a data system [142]. Typically, the stationary phase inside the column engages with the molecules of interest through mechanisms dependent on the type of
separation method employed. A liquid solvent or a mixture of solvents serves as the
mobile phase, facilitating the movement of analytes through the column. The differences in interactions between analytes and the stationary phase result in varying retention times, enabling the separation of components in the mixture. HPLC is particularly suitable for the analysis of nonvolatile and thermally unstable plant metabolites.
Parameters such as the type and properties of the solvent, column temperature, and
flow rate significantly influence the separation efficiency of HPLC. By controlling
these parameters, it is possible to achieve high-resolution and efficient separation of
complex extracts [143].
Ultra-high-performance liquid chromatography (UHPLC) systems are advanced
techniques that operate at significantly higher pressures compared to HPLC and utilize packing columns with particles smaller than 2 μm. This method is widely applied
in various areas of plant analysis, including the chemical profiling of plant components, fingerprinting, dereplication, and metabolomics. Beyond identifying compounds, one of the fundamental applications of both HPLC and UHPLC is the dereplication process, which involves recognizing known metabolites in extracts and is
conducted during the early stages of the fractionation process to expedite analysis
[144]. Compared to HPLC, UHPLC offers numerous advantages, including operating at
high flow rates, significantly reducing analysis time, providing highly efficient separation with excellent reproducibility, enhanced sensitivity, and lower solvent consumption than other analytical methods. This method provides fast and sophisticated chromatographic separation with reduced analysis time, while also ensuring exceptional
precision and selectivity, which helps in the precise and dependable identification of
compound structures across various samples [145].
2.3.4.3 Gas chromatography (GC)
Gas chromatography (GC) is a method capable of performing both qualitative and
quantitative determination of target analytes. It is a chromatographic technique in
which the mobile phase is a gas and the stationary phase is a liquid. In this method,
the chromatography column contains a liquid stationary phase adsorbed onto the sur-
90 İrem Yıldız Özbaş, Severina Pacifico, and Emre Özbaş
face of an inert solid. The migration rate of the compounds to be analyzed relies on
their distribution within the gas phase. In GC, volatile or volatilizable compounds are
vaporized and injected into the chromatographic column, where they are carried
along the column by the flow of the gaseous mobile phase and detected using various
detectors [146].
Gas chromatography/mass spectrometry (GC/MS) is considered the gold standard
for comprehensive qualitative and quantitative analysis of volatile organic compounds found in natural products [147]. In GC-MS analysis, compounds are first injected into the gas chromatograph, where they are separated based on their volatility.
The separated compounds then enter the mass spectrometer, where they are bombarded with electrons and fragmented into ions. These ions are detected by the system’s detectors, allowing for the analysis to be performed [148]. Given that substances
suitable for evaluation by GC – characterized by low molecular weight, medium or
low polarity, and concentrations in the ppb-ppm range – also meet the requirements
for mass spectrometry (MS), the combination of GC and MS forms a highly advantageous and synergistic method. Furthermore, both analytical processes occur in the
same aggregation state, which is the vapor phase [149].
2.3.4.4 Column chromatography (CC)
Column chromatography (CC) is a technique used for the isolation of bioactive compounds identified in plants and for the separation of metabolites in various plant extracts. Additionally, it is a preferred method for the removal of impurities and purification of biological mixtures. This method can be utilized to separate and purify both
solid and liquid samples. The basic principle of CC is based on the separation of compounds by adsorption onto a stationary phase placed inside a narrow column, with
the help of a liquid mobile phase. Compounds are adsorbed by the stationary phase to
varying degrees depending on their chemical structural properties, and elution occurs
in this manner [150].
CC, based on the principle of adsorption, is commonly used in the initial separation stage of plant extracts. The main reasons for its widespread use include the simplicity of the technique, the high capacity of the process, and the low cost of adsorbents like silica gel and macroporous resins. Since the separation process primarily
depends on the adsorption affinities of natural compounds to the surface of the adsorbents, it is crucial to carefully select the adsorbent (stationary phase) and the mobile phase to ensure efficient separation, high recovery of target compounds, and to
prevent irreversible adsorption of target compounds onto the adsorbents [151]. The
use of silica gel as an adsorbent is suitable for the separation of most phytochemical
compounds. Alumina can be used for alkaloids, steroids, and terpenoids, which are
alkaline or neutral lipophilic components. Activated carbon is suitable for hydrophilic
components such as amino acids, carbohydrates, and some glycosides. Polyamide,
Chapter 2 Methods of obtaining drugs from medicinal and aromatic plants 91
based on the formation of various hydrogen bonds, can primarily be used for the separation of phenols, quinones, flavonoids, anthraquinones, tannins, and others [152].
2.3.4.5 Ion exchange chromatography (IEC)
Ion exchange chromatography (IEC) is a widely used fractionation method that enables the separation of ions and ionizable molecules based on differences in their electrostatic properties [153]. Among all LC techniques, it is one of the most widely used
and versatile due to its large sample-handling capacity, broad applicability (especially
to proteins and enzymes), moderate cost, powerful resolving ability, ability to perform
simultaneous quantification, and ease of scalability and automation [154, 155].
IEC can be applied in both solid-gas and solid-liquid systems. Ion exchangers are
used as the stationary phase in ion chromatography. Different types of ion exchangers
are utilized based on their polarity, chemical and physical resistance, particle size distribution, internal and specific surface area, density, porosity, and pore radius distribution [156]. Ion-exchange resin might capture and release the charged molecules by
altering the mobile phase’s ionic strength (e.g., changing pH or salt concentration)
[157]. The type of stationary phase, detection method, and eluent type are the most
important factors affecting the separation quality [158].
2.3.5 Elucidation of the chemical structure
The process of identifying and characterizing phytochemicals is still greatly challenged by the fact that plant extracts typically consist of a mixture of several bioactive
compounds with varying polarities [159]. The complex chemical composition of herbal
samples is represented by distinctive profiles and patterns, called fingerprints, which
can be developed through multiple approaches, such as chromatographic and spectroscopic techniques [46].
The structure of a purified active compound obtained through extraction and isolation methods can be determined using various spectroscopic techniques. Nuclear
magnetic resonance (NMR) spectroscopy is frequently employed for the structural determination of natural products, especially for unknown compounds, as it offers significant advantages. Materials analyzed by NMR can be recovered after analysis.
Other commonly used structural elucidation methods include mass spectrometry (MS)
for determining molecular weight and infrared (IR) spectroscopy for identifying functional groups [160].
Combined instrumental analysis methods are used to profile the structural composition of the numerous and complex secondary metabolites found in plants. In the
study of the effects of phytochemical compounds and the quality control of herbal
medicines, “hyphenated techniques,” which integrate sensitive and rapid analytical
92 İrem Yıldız Özbaş, Severina Pacifico, and Emre Özbaş
methods with online spectroscopic techniques to simultaneously provide both structural and activity information, are widely used. These methods are highly successful
in the rapid online identification of known components, preventing dereplication,
and ensuring the standardization or quality control of a complex extract [161].
2.3.5.1 Nuclear magnetic resonance (NMR)
NMR spectroscopy is a technique applicable for identifying target bioactive metabolites from complex plant extracts. The primary advantages of NMR spectrometers include their ability to perform measurements without requiring any prior sample
preparation or preprocessing. Moreover, its noninvasive nature, rapid operation, and
high sensitivity make it a highly preferred method. However, due to the significant
equipment costs, NMR is predominantly used for the structural elucidation of previously uncharacterized compounds rather than known ones [162].
The sample preparation for NMR is quite simple, as it does not require detailed
pretreatment or fractionation. It is also a highly reproducible method. NMR provides
quantitative and detailed information about the structure of metabolites. However, its
main disadvantages are its low sensitivity and the fact that it generally profiles only
the major components. Additionally, NMR is not very useful for detecting trace components, as it can only detect compounds at concentrations as low as 0.1% [163].
The NMR technique has never lost its importance as it is used not only for elucidating chemical structures but also for structural studies of biomolecules in three dimensions, identifying reaction mechanisms, and ligand binding screening in drug discovery [164].
2.3.5.2 Mass spectrometry (MS) and high-resolution mass spectrometry (HRMS)
Mass spectrometry (MS) is a spectroscopic technique that generates ions from atoms
or molecules in the gas phase and measures their mass-to-charge (m/z) ratios. Mass
spectrometers differentiate ions with different mass-to-charge ratios using static,
pulsed, or periodically changing electric and/or magnetic fields. The main applications
of mass spectrometers include determining molecular mass, elemental and isotopic
compositions, structural elucidation, and quantification [165].
The most prominent separation techniques commonly combined with mass spectrometry (MS) are HPLC, GC, and capillary electrophoresis (CE). GC-MS is the most frequently employed technique among these for the separation and analysis of mixtures
that contain volatile organic compounds or those that can be made volatile, along
with thermally stable components. GC-MS is an inexpensive and highly sensitive
method. However, its applicability is limited compared to other methods, as it is only
suitable for the analysis of volatile compounds and relatively lower molecular weight