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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5401_Библиотеки_им_академика_М_И_Перельмана.pdf
44 Herbal Pharmacopeia
of low pressure, small vacuum bubbles are formed in the liquid. These bubbles grow over successive
cycles until they reach a critical size. At that time, during a high- pressure cycle, the bubbles burst
viciously. And this produces strong energy. This leads to:
3.5.1.1 Cell Disruption
The collapse of cavitation bubbles disrupts the plant cell walls, thereby increasing the contact area
between the applied solvent and the plant material.
3.5.1.2 Increased Mass Transport
Due to the burst of cavitation bubble, the micro- jets and shock waves are created which increases the
penetration of applied solvent into the plant matrix and bioactive compounds ooze out.
3.5.1.3 Enhanced Solvent Effectiveness
The localized exerted pressure and temperature can increase the solubility of required compounds
in the solvent.
3.5.2 Parts of the UltrasoUnd- assisted extraction systeM
1. Ultrasonic Generator: It converts the electrical energy into high- frequency ultrasonic
waves.
2. Transducer: The electrical ultrasonic waves produced by the generator is then converted
by the transducer into mechanical vibration
3. Sonotrode (Probe): This probe is immersed in the extraction solvent. It transmits the gen-
erated ultrasonic energy into the liquid.
4. Extraction Vessel: This vessel holds the solvent and plant material. It could be a simple
vessel or more complex setup with temperature and pressure controls.
5. Cooling System: This is needed in keeping the temperature low in the extraction system to
prevent the thermal degradation of temperature- sensitive compounds.
6. Power Supply: This provides energy to the ultrasonic generator and transducer.
3.5.3 Method of extraction froM herBs
1. Preparation of Plant Material: Drying and grinding of the herbs is carried out. The plant
material is reduced to particle size to provide a greater surface area for extraction.
2. Selection of Solvent: Suitable solvent is chosen depending on its polarity and solubility of
target compounds. This might include, for example, methanol, water, ethanol etc.
3. Loading of the Extraction Vessel: The powdered herb material and the selected solvent
are loaded into the extraction vessel.
4. Ultrasonication: The ultrasonic probe is submerged into the solvent. The ultrasonic gen-
erator is switched on to generate the ultrasonic waves, which produce cavitation in the
solvent.
5. Extraction: Extraction time, temperature required and herb sample are optimized on the
basis of the specications of the extraction process. This process may take either a few
minutes or several hours.
6. Filtration and Separation: The mixture obtained after extraction is ltered to remove all
solid remains; the ltrate contains the required bioactive compounds.
7. Concentration and Purication: Concentration or purication can be achieved by the use
of various techniques such as evaporation, chromatography, or distillation (Figure 3.3).
Modern Techniques in Herbal Extraction and Analysis 45
FIGURE 3.3 How the plant cell bursts by ultrasonic waves.
3.6 PRESSURIZED LIQUID EXTRACTION (PLE)
3.6.1 definition
Pressurized Liquid Extraction (PLE), also known as “Accelerated Solvent Extraction,” is the process
of extracting required bioactive compounds from solid and/or semi- solid samples by using solvents
at high temperature and/or high pressure. The efciency and speed of the extraction process are
greatly boosted by the technique, which is driven by enhanced solubility and the mass transfer of
target compounds (Goettel et al., 2013).
3.6.2 Working PrinciPle
PLE operates in conditions in which the solvent is maintained above its boiling point but below its
critical point. The high temperature enhances the capacity of the solvent to dissolve the required
compounds. While, the high pressure prevents the boiling off of the solvent and keep it in the liquid
state. This combination of temperature and pressure improves extraction efciency in:
1. Improving Solubility: The solubility of the required phyto- compounds in the solvent is
increased by higher temperatures.
2. Improvement in Mass Transfer: Increased temperature and pressure reduces the surface
tension and viscosity of the extraction solvent. It improves the diffusion of the extraction
solvent in the plant matrix and the release of required compound.
3. Reducing Extraction Time: The improved mass transfer and solubility result in a faster
extraction rate, which reduces the overall extraction time.
3.6.3 Parts of the Ple systeM
1. Solvent Reservoir: It holds the extraction solvent used for extraction.
2. Pump: This is also known as delivery system. It adds the solvent to the SPE cartridge at a
controlled ow rate and pressure.
3. Extraction Cell: This is used to hold the plant sample and the solvent. It is designed to
withstand very high temperatures and pressures.
4. Heater: This heats the extraction solvent to the chosen temperature before it reaches the
extraction cell.
5. Pressure Regulator: This is used to maintain the required pressure within the extraction
system.
46 Herbal Pharmacopeia
6. Collection Vials: These are vessels which collect the extract after it has passed through the
extraction cell.
7. Control Unit: This is used to monitor and control all of the changes which take place together
with the control of pressure, temperature, and ow rate during the extraction process.
3.6.4 Ple extraction Method
1. Sample preparation: The plant sample is dried, ground up, and then kept into the extrac-
tion cell. The cell is then tightly sealed.
2. Solvent selection: Keeping in view the polarity and solubility of required compound, the
solvent is selected.
3. Selection of solvent: A proper selection of the solvent based on the polarity and solubility
of the analytes of interest is made.
4. System setup: It involves setting up the system with the extraction cell loaded with samples.
A heater, collection vials, and lling of the solvent reservoir are the parts of this system.
Heating and Pressurization: The required temperature for the heating of extraction solvent is
maintained. It is then pumped at high pressure into the extraction cell.
Extraction: In the extraction cell, the extraction solvent streams through the sample and dis-
solves the required compounds. The increased temperature and pressure boost the extraction efciency.
Collection: The solvent with the extracted required compounds leaves the extraction cell and
is collected in collection vials.
Repeat Cycles: This process is repeated a number of times with a fresh extraction solvent to
conrm the complete extraction of required compounds.
Post- Extraction Processing: The collected extract of required compounds may then be concentrated or further puried by using any techniques such as evaporation, chromatography, or distillation (Figure 3.4).
FIGURE 3.4 Process diagram of Subcritical Water Extraction (SWE).
Modern Techniques in Herbal Extraction and Analysis 47
3.7 SUBCRITICAL WATER EXTRACTION (SWE)
3.7.1 sUPercritical flUids
The supercritical state is a state of any matter that occurs when any substance or matter is brought to
the zone of its “critical point,” at which it experiences high temperature and pressure. At this stage
the substance shows intermediate characteristics of both liquid and gas and acts both like a “reactant” and like a “catalyst.” This state of substance is described as a supercritical uid.
3.7.2 sUPercritical flUid extraction (sfe)
This is a sample extraction method which is used to extract the required compound from the sample by using the special characteristics of supercritical uids. Supercritical uids become diffused
quickly like gases and can be dissolved as easily as liquids.
Subcritical Water Extraction (SWE): This is one of the most advanced techniques, and
is used for the extraction of required compounds from herbs. In this technique, water is
used as a supercritical uid (at between 100°C and 374°C and high pressure) to extract the
required compounds from herbs. This method takes advantage of the distinctive solvent
properties of supercritical water.
3.7.3 Working PrinciPle of sUBcritical Water extraction (sWe)
The working principle of SWE is based on the fact that the behavior of water in the subcritical
phase is unique. When temperature of water is increased to between 100°C and 374°C and pressure
is increased (to keep the water in liquid state) its “dielectric constant” is decreased, resulting in a
decrease in polarity. This subcritical water dissolves more compounds than is the case when cold
water is used.
3.7.4 Parts of the sUBcritical Water extraction systeM
1. Extraction Vessel: Herb material is placed in the extraction vessel. This vessel should be
made in a manner to withstand high temperature and pressure.
2. Heater: The heater is used to heat the water to acquire the subcritical temperature.
3. Pump: The pump maintains the higher pressure to ensure that the water doesn’t boil and
that it remains in the supercritical phase during extraction.
4. Cooling System: After extraction, this is used to separate the extracted compounds from
water. The solution is cooled within this system.
5. Separator: The “separator” separates the extracted compounds from the water.
6. Control System: This system controls the temperature, pressure, and ow rate and main-
tain them to ensure have the best conditions of extraction (Figure 3.5).
3.7.5 Process of sUBcritical Water extraction
1. Preparation of Plant Material: The sorting, cleaning, and drying of herb material is car-
ried out. This plant material is then ground into ne powder so that the surface area could
be increased for extraction.
2. Loading the Extraction Vessel: The sample is then loaded into the extraction vessel and
water is added into it.
3. Heating: The water is heated to its supercritical stage; it then acts as a supercritical uid.
The system heats the water to the subcritical range. The water acts as a solvent in its subcritical state, effectively dissolving the bioactive compounds.
48 Herbal Pharmacopeia
FIGURE 3.5 Flow chart diagram of Pressurized Liquid Extraction (PLE).
4. Extraction: The water, which is hot and pressurized, is passed through herbal material.
The heated, pressurized water passes through the plant material, extracting the desired
compounds.
5. Separation and Cooling: The extracted material is cooled down, and bioactive compounds
are precipitated or separated out from the water.
6. Collection: The extract is cooled down; this process precipitates or separates the bioactive
compounds from the water.
Analytical Techniques for Herbal Analysis
• High- Performance Liquid Chromatography (HPLC)
• Gas Chromatography- Mass Spectrometry (GC- MS)
• Liquid Chromatography- Mass Spectrometry (LC- MS)
• Fourier Transform Infrared Spectroscopy (FTIR)
• Nuclear Magnetic Resonance (NMR) Spectroscopy
3.8 HIGH- PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)
The use of herbs in medicine is as ancient as humankind. Medicinal herbs have vast usage, not
only used as just medicine, but also in the cooking and perfumery purposes. Herbs generally are
medicinal, culinary, and aromatic plants rich in bioactive compounds and phytochemicals that form
the development row of therapeutic effects. The identication and valuation of these compounds set
the core of health benets as well as quality control for herbal products. High- performance liquid
Modern Techniques in Herbal Extraction and Analysis 49
chromatography (HPLC) is the very efcient analytical technique used in the separation, identication, and quantitation of bioactive compounds in complex herbal matrices. The principles of HPLC
and updates in its application to the separation of bioactive compounds in herbs are outlined in this
paper.
3.8.1 PrinciPles of hPlc
Basically, HPLC is based on liquid chromatography, where the mobile phase—liquid—is carried in
a column that has been lled with a stationary phase to carry out a sample. It is a separation- based
technique in which the interaction of the sample with the stationary phase allows the separation of
compounds based on different afnities. Key components making up an HPLC system include a
solvent reservoir, a pump, an injector, a column, a detector, and a data acquisition system. Selection
of the stationary phase is silica- based for the most part, and the mobile phase would be a mixture
of solvents, relying upon the nature of compounds to be analyzed. RP- HPLC is a mode most commonly used to analyze the bioactive compounds in herbs: nonpolar stationary phases and polar
mobile phases are utilized. Detection varies, but often includes UV- Vis detectors, photodiode array
detectors, and mass spectrometry due to their high sensitivity and specicity.
Sample preparation is an essential part of HPLC analysis to achieve accurate and reproducible
results. Herbals generally need to be dried and powdered into a ne powder prior to extraction. The
preparation of solutions begins with the selection of the extraction solvent, taking into account the
polarity of the target compounds. Generally utilized solvents fall in the class of water, methanol,
ethanol, and mixtures of the above. Available methodologies for extraction are maceration, reux,
ultrasound- assisted extraction, microwave- assisted extraction for best results .
These extracted samples, however, block require further purication to remove interferences
from the substances. Techniques such as SPE and LLE can be used to concentrate and purify the
target analytes. The puried extract that remains is ltered, and is injected into the HPLC system for
analysis.
3.8.2 Bioactive coMPoUnds analysis
3.8.2.1 Phenolic Compounds
Among the wide variety of compounds, great attention has been paid to phenolic compounds such
as avonoids, phenolic acids, and tannins in herbs since these are bioactive compounds. HPLC has
been applied to a wide range of phenolic compound analyses. In general, RP- HPLC separation
employs a C18 column with a mobile phase consisting of water and acetonitrile or methanol, often
modied with an acid like formic acid or acetic acid to improve peak shape and resolution.
3.8.2.2 Alkaloids
Alkaloids are nitrogen- containing compounds that have very important pharmacological activities.
When it comes to the analysis of alkaloids, generally HPLC methods use mobile phases with ionpairing reagents so that such basic compounds are strongly retained and separated. Detecting these
compounds will basically use UV- Vis and MS detectors, hence providing contributions to qualitative and quantitative analysis.
3.8.2.3 Terpenoids
Terpenoids make up a class of bioactive compounds with vast diversity in biological activities,
and they include monoterpenes, sesquiterpenes, diterpenes, and others. Terpenoids are analyzed
by HPLC on practically nonpolar phases with gradient elution for good separation. Additionally,
since a good number of them have volatility, it is very advantageous to analyze them in HPLC
coupled to MS.
50 Herbal Pharmacopeia
Being one of the most common constituents in herbs, glycosides carry another non- sugar moiety
with a sugar moiety. For the most part, glycosides exhibit very varied therapeutic effects. The usual
methodology used to decipher the glycosides is RP- HPLC with gradient elution. The enzymatic
hydrolysis is performed before the analysis to raise the sensitivity of the detection based on the
release of an aglycone.
3.8.3 recent advances in hPlc techniqUes
3.8.3.1 Ultra- High- Performance Liquid Chromatography
Ultra- high- performance liquid chromatography (UHPLC) provides a higher resolution compared
with traditional HPLC. Besides, it reduces analysis time, as it is also quite sensitive. It is comprised
mainly of a column with smaller particles, about sub- 2 μm in diameter, used at higher running pressures, thereby providing better separation efciency. Nowadays, this technique has gained popularity for the analysis of complex herbal extracts.
3.8.3.2 HPLC- MS
Thus, the coupling of HPLC with MS could help not only as a multiple separation tool but also as
an atomic level structural elucidator. It would make it possible to identify those compounds not only
with their mass- to- charge ratio (m/z) but also by the fragmentation patterns. In essence, HPLC- MS
is a very powerful tool for the analysis of a group of compounds which are devoid of chromophores
and therefore cannot be detected with UV- Vis detectors.
The major advantage of PDA detectors is that they record multiple wavelengths simultaneously,
give additional spectral detail on the identity of the analyte. This is highly useful for the identication of co- eluting compounds and to calculate the purity of peaks. HPLC- PDA is utilized for both
qualitative and quantitative analysis of phenolic compounds and other phytochemicals in herbs.
3.8.3.3 Chiral HPLC
Sometimes enantiomers are compounds related to chiral experimentations in HPLC. This kind of
analysis, in analyzing bioactive compounds with a chiral center, is quite signicant, because mostly
the enantiomers can have very divergent biological activities. They use chiral stationary phases or
chiral additives in the mobile phase to gain enantiore solution.
3.8.4 aPPlications of herBal Medicine
3.8.4.1 Quality Control
The relation of the drug from the pharmacokinetic data, or the relation of the data, is often difcult
to interpret to the pharmacologist; hence, HPLC plays an important role in the quality assurance of
herbal products. It lls the gap in ensuring that consistency in herbal preparations is maintained as
over- the- counter drugs, thus conrming the amount of bioactive compound present and their exact
concentration for safety. It is also a very common practice in setting a benchmark in the standardization of herbal extracts toward common reference compounds to conrm quality assurance of the
herbal products.
3.8.4.2 Pharmacokinetic
The pharmacokinetic study describes the absorption, distribution, metabolism, and secretion of bioactive compounds from herbal remedies into blood circulation. HPLC will also characterize their
interaction with biological systems. Metabolomics will, thus, provide a holistic description of the
bioavailability and therapeutic efcacy of herbal drugs in human health.
Metabolomics is a denition of the analytical activity of a biological system within a biological
system. In metabolomic investigations, this is one of the principal tools combined with HPLC and
Modern Techniques in Herbal Extraction and Analysis 51
either MS or NMR. It allows the proling of phytochemicals in herbs and also nds biomarkers that
are related to therapeutic action/toxicity.
HPLC, therefore, is very necessary in the process of the isolation and identication of potential
candidates for new drugs. The high- throughput methods of HPLC are feasible for the quick screening of a large number of samples so that the speed of discovering new compounds can be greatly
improved.
3.8.4.3 Challenges and Prospects for Further Study
Though HPLC is a powerful technique in terms of the analysis of bioactive compounds from herbs,
it does suffer from some challenges. This might cause complications in herbal matrices in terms of
the separation and identication of compounds. In addition, matrix effects are also well known to
affect the quantication in terms of accuracy. In addition, high volumes of organic solvents are used
during sample preparation, which brings an environmental impact into play and raises questions
about the sustainability of the technique.
Future trends in research on herbs with HPLC embrace the design of more selective and sensitive
detectors, the application of various green chemistry principles to bring reduction in the overall
environmental footprint, and the incorporation of advanced chemometrics for the interpretation of
complex data sets. HPLC, coupled with other techniques such as supercritical uid chromatography
and capillary electrophoresis, offers advanced resolution and speed for the analysis. HPLC analysis
of bioactive compounds and phytochemicals from herbs provides one of the cornerstones in this
context, given the techniques’ versatility, precision, and complexity of its matrices. Therefore, this
is a very important tool for research and quality control purposes in herbal medicine. Further
improvements in the technology of HPLC, combined with a large number of new developed analytical techniques in which it is implicated, make it an increasingly powerful tool by which to bring out
the therapeutic potential of the herbal compounds. This means that HPLC has been so important for
the safety, efcacy, and consistency in herbal products with increased demands for the products.
3.9 GAS CHROMATOGRAPHY- MASS SPECTROMETRY (GC- MS)
Herbs have been used since ancient times not only as plants for diet, but also to improve human
health and nutrition. They are an immense source of bioactive compounds and phytochemicals
responsible for the therapeutic activity of herbs, and they possess a wide array of bioactive constituents, which include alkaloids, avonoids, terpenoids, and phenolic compounds coupled with
essential oils. The analysis and characterization of these compounds are important to understand
their role in the promotion of health and the prevention of diseases. Amongst the various analytical
techniques, GC- MS has been reported to be a promising tool for the qualitative and quantitative
analysis of bioactive compounds in herbs. This chapter focuses on describing the principles, methodology, and applications of GC- MS analysis of phytochemicals from herbs.
3.9.1 PrinciPles of gc- Ms
GC- MS is an analytical technique which combines the features of Gas Chromatography (GC) with
those of Mass Spectrometry (MS). A sample is vaporized and passed through a column that is coated
with a stationary phase. As components will interact with the stationary phase differently, they will
be separated based on their varying volatility and afnity.
The chromatographically separated compounds enter the mass spectrometer, in which they
become ionized, usually by electron impact ionization. The ions are then detected after being separated based on their mass- to- charge ratio (m/z). The mass spectrometer produces a spectrum that
shows the relative abundance of ions at each m/z value. It then yields a molecular ngerprint that can
be used in identifying the compound .
52 Herbal Pharmacopeia
3.9.2 saMPle PreParation
Proper sample preparation is essential for accurate GC- MS analysis. Most of the herbs contain a
matrix of compounds: both volatile and non- volatile. These should be isolated and concentrated
before instrument injection. Common sample preparation techniques include:
1. Solvent Extraction: This process uses solvents like methanol, ethanol, or hexane to take
out bioactive components from the herb matrix. The choice of the solvent used depends on
the polarity of the target compounds.
2. Steam Distillation: The process that distills the herb with water to separate the volatile
compounds is used to extract essential oils.
3. Solid- Phase Micro Extraction: Solid- phase micro extraction (SPME) is a solvent- free
technique where volatile compounds from the herb matrix are adsorbed onto a ber coated
with a specic extracting phase.
4. Supercritical Fluid Extraction: SFE employs supercritical CO₂ to efcaciously lift out
bioactive compounds. It’s also employed widely in thermolabile substances.
5. Instrumental Setups: The general conguration of GC- MS comprises an injection port, a
GC column, a mass spectrometer, and a data processing system. The injection port used is
heated to a very high temperature for the purpose of the vaporization of the sample. The GC
column is, in most cases, a fused silica capillary column and it has to be selected depending
on the nature of the compounds to be analyzed. Such a column is kept in a programmable
temperature oven that facilitates the separation of compounds.
The GC- MS system has an ion source, a mass analyzer, and a detector. The ion source ionizes
molecules whereas the mass analyzer resolves such ions according to their m/z ratio, which the
detector records in terms of ion abundance. Most modern instruments of GC- MS are coupled
with large libraries of mass spectra that help in the identication of compounds through spectral
matching.
3.9.3 aPPlications of gc- Ms in herBal analysis
GC- MS has been excellently applied in the analysis of several bioactive compounds in herbs. Some
of the prime applications are discussed below:
3.9.4 endoWed oil analysis
These oils, as well as their therapeutic functions, are essentially composed of volatile aromatic
compounds that can be classied into four classes: terpene, alcohol, ester, and phenolic. The sensitivity and resolution needed for the analysis of essential oils usually make GC- MS a technique of
choice. For instance, analysis of the essential oil from Lavandula angustifolia, or lavender, shows
that important constituents responsible for the sedative activity of lavender oil include linalool, linalyl acetate, and camphor.
3.9.5 alkaloids and Phenolic coMPoUnds
Alkaloids represent nitrogen- containing compounds of important pharmacological activity, for
example, morphine from the opium poppy (Papaver somniferum) and caffeine from the coffee plant
(Coffea arabica). GC- MS will be able to quantify and identify alkaloids. Similarly, phenolic com-
pounds such as avonoids and tannins having strong free- radical scavenging activity will lend them
very well to analysis by the GC- MS method.
Modern Techniques in Herbal Extraction and Analysis 53
3.9.6 terPenoids
Terpenoids are large and diverse classes of naturally occurring organic chemicals formed by vecarbon isoprene units. They give the aroma to many herbs and avorings. The GC- MS analysis
of terpenoids from herbs like rosemary (Rosmarinus ofcinalis) and thyme (Thymus vulgaris) is
aimed at furnishing information regarding the chemistry of their composition and associated health
benets.
3.9.7 qUantitative analysis
Quantitative analysis using GC- MS is performed by determining the concentration levels of some
compounds in an herb sample. The method therefore includes the plotting of calibration curves
using known concentrations of standard compounds. Because the peak area in the chromatogram is
directly proportional to the concentration of the compound, the quantitation is quite accurate.
3.9.8 data analysis and interPretation
GC- MS data are analyzed by matching the recorded mass spectra with the reference spectra in the
libraries such as NIST and Wiley. The retention time, along with the mass spectrum, helps in the
proper identication of the compounds. The software tools of different levels do facilitate easier
automatic matching and peak selection, thus enabling faster data analysis.
3.10 LIQUID CHROMATOGRAPHY- MASS SPECTROMETRY (LC- MS)
Herbal bioactive compounds and phytochemicals occupy a great deal of interest in research with
regard to their therapeutic and nutritional applications, as the popularity of natural products is
increasing. Within this context, LC- MS has proven to be very powerful; the major advantages of
this technique over the others considered in this chapter are very high sensitivity and specicity,
and the fact that complex mixture analysis can be performed. In essence, this chapter outlines the
identication and quantication of bioactive compounds and phytochemicals from herbs using
LC- MS.
In LC- MS, liquid chromatography provides physical separation, while mass spectrometry supplies the mass analysis, thereby allowing a combination to both identify and quantify mixtures of
compounds based on a mass- to- charge ratio. The scope covers a broad array of bioactive compounds
and phytochemicals, such as alkaloids, avonoids, terpenoids, and phenolic acids.
3.10.1 PrinciPles of liqUid chroMatograPhy- Mass sPectroMetry
Liquid chromatography (LC) is a general technique, mainly based on the principle that different
compounds have differential interactions between a stationary and a mobile phase. In most cases,
the stationary phase refers only to a column packed with some solid particles, while the mobile
phase refers to a liquid solvent owing through the column. These will allow other compounds
contained in a sample the possibility to interact to varying degrees with the stationary and mobile
phase, permitting them to elute at different times—retention times. Separation is the real essence of
analyzing complex mixtures, such as herbal extracts.
Mass spectrometry is the technique used to quantify the mass- to- charge ratio of ions. In support
of LC- MS, the compounds separated by LC are ionized and introduced into the mass spectrometer.
There is a different way of carrying out the process of ionization, including electrospray ionization
and atmospheric- pressure chemical ionization. Once these compounds are ionized, their m/z ratios
are detected to obtain both qualitative and quantitative information.