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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_6035_Библиотеки_им_академика_М_И_Перельмана.pdf
TABLE 8.3
Name of the
Authentic
Drug Source
Maidenhair tree Leaflet Alzheimer’s disease and
(Continued)
Type of
Plant Part
Used
Used for Medicinal Purpose Presence of Any Kind
Parkinson’s disease
Marker Used Application References
of Adulterants or
Substituent
Not present DNA-
barcoding
technique
Authentication of herbal
dietary supplements using
DNA-barcoding technique
163
Little (2014)
Wan Chak
Motluk
Indian Rhubarb Roots Laxative, reduces
Rhizome Reduces inflammation. Hidden Ginger AFLP marker Identification and
inflammation, antibacterial,
purging heat, curing kidney
disorders, and anticancerous
R. emod, R. hotaoense,
R. undulatum, and R.
compactum
morphological characterization
of phytoestrogens-producing
plant using AFLP markers
ISSR marker Authenticating the defined
species of Indian Rhubarb
Keeratinijakal
(2010)
Wang (2011)
et al.
164
FIGURE 8.2 Methodology using pictures for DNA-based markers.
⏎
165
FIGURE 8.3 A description of the Parallel Approach for biological activity-guided fractionation of different
plant extracts.
⏎
In the first phase, at least three fractions of extracts—such as 100% aqueous, 100% etha-
nolic, and water–ethanol extracts (50:50 v/v)—are gathered and examined in the primary
screening for target biological activity.
In the first phase, at least three fractions of extracts—such as 100% aqueous, 100% etha-
nolic, and water–ethanol extracts (50:50 v/v)—are gathered and examined in the primary
screening for target biological activity.
To extract the desired chemicals, chromatographic separation is applied to the highest
active subfraction(s) produced at Step 2. Prior to being tested for the intended biological
166
activity, each drug is purified using the appropriate purification techniques, such as
column chromatography, preparative HPLC. The chemical structures of the compounds
exhibiting the best biological activity are clarified using contemporary techniques such
as mass spectrometry (MS), nuclear magnetic resonance (NMR) spectroscopy, and liquid
chromatography–mass spectrometry (LC–MS).
8.5.2 SEQUENTIAL APPROACH
This method is mostly applied to plants chosen using a random selection strategy when it is
unknown what their biological activity is. Figure 8.4 provides a summary of the biological
screening, isolation, and extraction/fractionation procedures used in this strategy. The
experiment can be broken down into the following two stages.
FIGURE 8.4 An overview of the steps in the sequential process for fractionating plant extract based on
biological activity.
⏎
167
The extraction of the plant material and the fractionation of the extracts take place concurrently at this stage. After performing extractions in solvent systems with increasing polarity ,
fractions are sequentially collected, for example, using chloroform, ethanol, petroleum
ether, ethyl acetate, and water. Target biological activity is tested for in all fractions.
The stage one fraction experiment with the highest levels of biological activity is chosen,
and the chemicals responsible for a specific level of biological activity are isolated using
the techniques outlined in the preceding plan. The isolated compounds are analyzed using
cutting-edge analytical techniques such as MS, LC–MS, NMR spectroscopy, and Fourier
transform infrared spectroscopy (FTIR) to ascertain their structural makeup. The first step
involves the primary screening (Figure 8.4) to determine efficacy, whereas secondary
screening involves identifying the mechanism of action with in vitro molecular screening.
In both methods, a variety of polar and nonpolar solvents are used to extract the plant
material. Yet, the way the material was extracted and divided remained basically constant.
It is typically easy to predict the chemical classes of chemicals present in fractions or
different forms of extract based on the polarity of the solvent. Examples of lipophilic
molecules (low-polarity contents) include oils, fatty acids, steroids, hydrocarbons, and
low-polarity terpenoids. These substances are extracted using nonpolar solvents such as
n-hexane and ether. In contrast, ethyl acetate and chloroform extracts frequently contain
medium polarity substances such as phenolics and alkaloids. Highly polar and oxygenated
compounds such as minute carboxylic acids, sugars, glycosidic alkaloids, and avonoids
are often produced by aqueous or methanol/ethanol extracts.
8.6 STRUCTURE ELUCIDATION OF ISOLATED COMPOUNDS
The structure of particular molecules is ascertained using information from a range of
various spectroscopic techniques, such as infrared (IR), ultraviolet–visible (UV–V is), mass
spectroscopy , and NMR. Exposing an organic molecule to electromagnetic radiation, some
of which it absorbs but not all, is the basic concept of spectroscopy . By keeping track of the
amount of electromagnetic radiation absorbed, a spectrum can be produced. Each bond in
a molecule has a unique spectrum. The structure of the organic molecule can be ascertained
from these spectra. Most often, spectra from UV, visible, and IR region of electromagnetic
spectrum are used by scientists to clarify structural details.
• UV Spectroscopy: It can be used for the qualitative analysis and identification of
particular type of extract in both biological and pure form of compounds. In order
to do quantitative research, UV–Vis spectroscopy is used since aromatic chemicals
168
are potent UV chromophores. Natural chemicals can be identified via UV–Vis
spectroscopy. It has been discovered that phenolic compounds, such as tannins,
anthocyanins, phenols, polymer dyes, form a complex with iron using UV–Vis
spectroscopy . Furthermore, it was demonstrated that spectroscopic UV–V is methods
give details on the composition of the overall polyphenol content while being less
selective (Wang et al., 2016a). Total phenolic acids (360 nm), anthokyanids (520
nm), flavones (320 nm), and phenolic extract (280 nm) were all measured using
UV–Vis spectroscopy (520 nm). This procedure is less expensive and takes less
time than previous approaches.
• IR Spectroscopy: Some IR light frequencies will be absorbed as they pass through
an organic molecule sample, while other frequencies will pass through the sample
undetected. A molecule will experience vibrational alterations as a result of being
exposed to IR light, which is connected to IR absorption. IR spectroscopy can therefore be considered a form of vibrational spectroscopy. The different bonds (C–C,
C=C, CC, C–O, C=O, O–H, and N–H) have different vibrational frequencies. It
is possible to detect whether an organic molecule possesses these kinds of bonds
by investigating the absorption pattern in a particular IR spectrum. To identify the
chemical components and define the structural constituents, FTIR, a high-resolution
analytical technique, is applied. Herbal extracts or powders can be quickly and
nondestructively fingerprinted using FTIR.
• NMR Spectroscopy: NMR is primarily concerned with the magnetic properties
of numerous atomic nuclei, including those of the carbon, proton, and hydrogen
an isotope of carbon. It has allowed multiple researchers to study molecules by
capturing the differences between the distinct magnetic nuclei and offering a precise
representation of their locations inside the molecule (Tu et al., 2019). Additionally,
it will display which atoms are present in close-by groupings. It is possible to find
out the number of atoms each habitat possesses.
• Identification of Chemical Substances Using MS:
When organic molecules are subjected to laser or electron irradiation during MS,
they become charged ions with a high energy. Plotting a fragmented ion’s rela-
tive abundance against its mass/charge ratio yields a mass spectrum. A precise
molecular formula can be determined using knowledge of the areas where the
molecule has been broken and relative molecular weight can be calculated using
MS. Prior studies have used HPLC, column chromatography, and bioactivityguided solvent extraction to separate and purify bioactive chemicals from pith.
While the method of tandem MS is applied, MS yields an abundance of informa-
tion for the elucidation of the structures of the molecules. Thus, even in the lack
of a pure standard, the combined use of HPLC and MS enables quick and precise
detection of important compounds in medicinal plants. LC–MS has recently been
used extensively for phenolic compound analysis. Electrospray ionization (ESI)
is a favored source as it has high ionization efficiency for various important
phenolic extracts. Figure 8.5 summarizes the structural elucidation process of
several pure bioactive extracts from significant naturally occurring sources.
169
FIGURE 8.5 A framework for the structural clarification of novel phytochemicals.
⏎
8.7 BIOLOGICAL SCREENING OF EXTRACTS/FRACTION/ISOLATES
The animal models are utilized for biological screening of pure isolated extracts and natural
sources, but there are some drawbacks to this method, including the need for huge sample
volumes, drawn-out experimental procedures, a lack of sensitivity , and ethical concerns. It
is very difficult to obtain bioactive pure chemicals in sufficient quantities for animal testing
because the yield of these compounds from natural sources is so low (Liska, 1998). On the
other hand, toxic effects observed in cell type screening based on toxic effects seen in cellbased screening, which may have shown good safety profiles due to liver detoxification in
the animal body, prospective successes could be harmful.
Numerous mobile and molecular bioassay techniques have improved as a result of the
development of studies in the eld of life sciences, which have revealed various pathophysiological approaches and medication action mechanisms. HTS approaches can be used
to some extent with those bioassays (Kell, 1999). The HTS procedures should signicantly reduce the sample amounts needed for screening, which are wanted in micrograms,
170
enabling the assessment of natural chemicals extracted in incredibly minute amounts.
Additionally, the expansion of the identity of bioactive chemicals (hits) as prospective
“ lead” molecules as indicators has been made possible by advancements in automation
technology, computer software, and microuid regulator, making it possible to conduct
bioassay for a large number of samples quickly (Sittampalam et al., 1997).
When evaluating medications physiologically, it is important to consider the impact of
the solvent used on the drug’s dissociation and molecular or conformational organization.
Dimethyl sulfoxide (DMSO) can dissolve a variety of polar and nonpolar compounds,
and is one of the most often used solvents for this. The solubility of nonpolar molecules is
reduced by absorbed water due to hygroscopic property of DMSO. Due to harmful effects
on the cells or test organisms and low miscibility with the assay media, other organic
solvents are normally not advised; nevertheless, the toxic effects are always neutralized by
the solvent controls (Gray et al., 2012). A positive in vivo activity of the test compound is
usually anticipated when it displays positive in vitro results, although the in vitr o screening
outcomes not always result into in vivo activity due to a number of variables, namely,
membrane permeability, biodegradation, dose, and solubility in an organic medium.
However, rather than relying just on one in vivo screening, several are needed to estab-
lish the efcacy of a true drug candidate’s biological prole.
The screening procedure would be substantially improved, and it would be essential for
the pharmacological evaluation and quality control of NPs to use a thorough HTS technique
that enables the identication of potentially active components in NPs. The process could
be accelerated by selecting compounds with medicinal value among the hundreds of
chemicals found in NP extracts using a bioassay-guided isolation and screening approach
(Butler et al., 2014). Following the isolation of the active compounds from the targets,
the structures and activities of the active compounds can be studied using bioinformatics
approaches and chromatographic procedures combined with MS or other detectors. This
not only makes it possible to identify substances that might be bioactive but also offers
crucial details on their molecular and cellular mechanisms of action (Muhammad et al.,
2017). Importantly, ligand’s interactions with targets at the cellular or molecular level can
be investigated using the biological screening assay . Most research on biological screening
methods to date have concentrated on the use of biological chromatography, including
molecular biochromatography and biomembrane chromatography , and magnetic separation
techniques to screen target components from NPs (Ciesla and Moaddel, 2016).
8.7.1 CELL CULTURE-BASED ASSAY
Cell-based screening methods are becoming increasingly and more crucial for locating
the active ingredients in NP extracts. Since whole living cells are employed, all of the
cell’s receptors, channels, enzymes, and other components are accessible for the screening
of test compounds (Liu et al., 2014). The discovery of bioactive substances in NPs has
already been effectively accomplished using a variety of cell-based screening techniques.
In cell culture systems, cells are combined with NP extracts for these screening techniques
before being rinsed with buffer to get rid of any unattached compounds. A range of
171
chromatographic analysis techniques are used to analyze the chemical components after
the cells and the components that have been precisely bound have been digested. A
conventional or hollow fiber (HF) cell culture system can be used to screen for target
cell-based bioactive components, depending on the culture technique (Hong et al., 2011).
The active ingredients in NPs have been screened using the traditional cell culture-based
fishing approach. Briefly, NP extracts are treated with cells grown on a culture bottle or
board before the unbound components are removed by washing. The cells are subsequently
treated with a hydrochloric acid phosphate buffered saline solution (pH 4.0) to denature
them and release the cells’ associated components, which are then investigated in further
detail (Sun et al., 2015).
The HF cell culture-based screening technology has been widely used for the extraction
and identication of active compounds due to its simplicity, speed, high enrichment, little
solvent consumption, and low cost (Zhang et al., 2014). Using this technique, a preset
number of living cells were introduced into the ber lumen using a syringe in order to
screen and sh for active chemicals. Following a U-shaped bend, the HF was added to the
NP extract. The NP extract was stirred for 3 h at 37°C. As soon as the HF was removed
from the NP extract solution, the seals at both ends of the ber were shattered. The HF
containing the cell target analyte was separated from it using methanol centrifugation, and
the supernatant was subsequently analyzed using HPLC (Wu et al., 2017).
Cell culture-based screening techniques have been shown to be useful for identifying
and analyzing bioactive candidates from NPs, but they still require a long incubation time,
making them difcult to use for HTS.
8.7.2 DIALYSIS
Semipermeable dialysis membranes that are impermeable to target molecule-active
chemical complexes have been used in equilibrium dialysis to discriminate between bound
and unbound NP constituents (Qi
et al., 2006). When NP extracts and target molecules
are mixed in a dialysis bag, the unattached compounds easily pass through the membrane
and are eliminated, but the potential bioactive compounds connected to the targets cannot
because the dialysis membranes have a certain molecular weight cut-off. Today, the main
application of equilibrium dialysis is to isolate active compounds by coupling equilibrium
dialysis with HPLC or ultrahigh-performance liquid chromatography with MS or other
detectors to profile NP extracts before and after interaction with targets (Hou et al., 2013).
8.7.3 MICRODIALYSIS
As a more advanced form of equilibrium dialysis, microdialysis has been widely used in
pharmacological and physiological research and has attracted significant interest in the
fields of analytical chemistry and pharmaceuticals. The majority of microdialysis systems
consist of a microdialysis pump and cellulose membrane-coated probes. The extracts and
target proteins are initially incubated in this screening method before the probe is introduced
172
to the mixture for microdialysis. The microdialysate and a control microdialysate are
then gathered for HPLC analysis after equilibrium has been reached. In comparison to
equilibrium dialysis, more targets (such as enzymes, serum albumins, and DNA) have
been integrated with microdialysis and HPLC–MS to offer a speedier way of NP bioactive
component screening (Wang et al., 2016b). Dialysates are examined for each analyte until
a steady level is attained.
8.7.4 ULTRAFILTRATION
Similar to microdialysis and equilibrium dialysis, ultrafiltration is a technique based
on a semipermeable membrane with a low-molecular-weight cut-off that allows liquids
and solutes below a certain molecular weight to flow through the membrane. Due to its
simplicity and dependability without requiring an equilibrium technique, ultrafiltration has
been shown to be a quicker screening method than equilibrium dialysis or microdialysis
(Liu et al., 2013). In an ultrafiltration system, NP components that are specifically bound
to high-molecular-weight target molecules pass through the membrane while unbound
mixture components are left behind. Additionally, it has been demonstrated that using a
centrifuge to separate a liquid from a solid lowers the possibility of bacterial development. The unbound substances were removed from the chamber solution containing the
ligand–receptor mixture by centrifugal force or pulse pressure in these models, whereas
the ligand–receptor complexes were retained after the NP extracts were incubated with
the targets (Li et al., 2014). The ligands were then released from the receptor by washing
the membrane with the proper eluent, such as methanol. Finally, the active compounds
were found and assessed using chromatography. One of the limitations of ultrafiltration is
that the unbound compounds cannot be completely removed from the system. Therefore,
consistent washing of semipermeable membranes is essential in this screening paradigm to
avoid nonspecific adsorption of ultrafiltration membrane-ligand.
8.7.5 CHROMATOGRAPHY
Bioaffinity chromatography, a major and well-liked method predicated on the interactions
between bioactive compounds and immobilized targets, has already been used to successfully screen bioactive components from NPs (DeMoraes et al., 2016). To preserve only the
putatively active compounds that precisely bind to the targets (cell membrane, protein, or
liposome) in the screening model, the targets are used as a stationary phase coupled with
a carrier (such as gel or silica particles). The NP extract is initially injected into an affinity
column loaded with target-covered carriers in order to enable robust binding of interest
components with the immobilized targets. The targets are then exposed to detector analysis
after the bound compounds are separated from them using an elution buffer.
Using an online size exclusion chromatography (SEC) column in connection with
a column-switching HPLC–DAD (Diode Array Detector) or HPLC–MS, a system for
chromatography by size exclusion (SEC) instantly lters a mixture of biological molecules