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Modern Techniques in Herbal
3
Extraction and Analysis
Sadaf Javaria
Institute of Food science and Nutrition, Gomal University,
D.I. Khan, Pakistan
Muhammad Zareef
School of Food and Biological Engineering, Jiangsu University,
Zhenjiang, China
Muhammad Nadeem
Institute of food science and Nutrition, University of Sargodha,
Sargodha, Pakistan
3.1 INTRODUCTION
Since ancient communities, phytochemicals from plant sources have beneted many communities
around the world. Healthcare systems use metabolites as drugs in the management of various diseases. Additionally, phytochemicals are utilized as lead compounds in the synthesis of medicines.
The chapter gives an overview of updated methodologies for the extraction, isolation, and analysis of
bioactive compounds from herbal sources in relation to the keen interest in therapeutic value concerning naturally occurring phytochemicals of plant origin (Abubakar & Haque 2020). Products of herbal
origin in medical applications have been used since time immemorial; however, modern technologies
drove this very ancient practice into a new era of precision (Dastangoo et al., 2020). It calls, therefore,
for the adoption of more sensitive and advanced extraction methods in the identication and exploitation of this bank of phytochemicals in herbs that can be used as medicine (Abhari & Khaneghah
2020). The chapter thus acts as a single tool for researchers, practitioners, and aspirants in the industry
who mean to direct state- of- the- art methodologies in herbal extraction and analysis toward drug discovery, quality control, and the development of evidence- based herb formulations. It has done so by
pulling together theoretical underpinning, helpful methodologies, and incipient trends.
3.2 HERBAL EXTRACTION
Extraction in herbalism is one of the most important processes in releasing the potent core of botanical blessings. To harness the potential of herbs, one needs to know and work under the rules of
extraction. Here, it is dened what the basic conception of herbal extraction, solvent, variables of
the extraction efciency, and principles are.
3.2.1 P
Essentially, extraction is a procedure of separating the active ingredient from the matrix of the
plant. The technique can be proven to arise out of the basic concepts of physics and chemistry. The
basic premise of extraction is the concept of solubility, which can be described as the capability of
36
rinciPles of extraction
: U
nlocking natUre’s BoUnty
Modern Techniques in Herbal Extraction and Analysis 37
a compound to dissolve well in a solvent. Hence the nature of what is extracted, and the amount
of its extraction from the matrix into the solvent, is based on the extractability of the solute in the
solvent. For example, alkaloids and avonoids are pungent compounds that nd solace in dissolving
in polar solvents such as ethanol or water. On the other hand, more non- polar substances from the
plants, which are essential oils, are effectively extracted by a non- polar solvent like hexane or ether.
Another subsection of the principal part is diffusion, which is the process by which the molecules
go from an area of high concentration to one of lower concentration (Brunner, 2005). It is as if they
are spreading out to achieve a uniform concentration. This helps to ensure that the diffusion process
extracts all of the compounds from the plant material. Among the basic scientic concepts that arise
from this focus on the areas of chemistry and physics are solubility, diffusion, and mass transfer.
These are essentially the principles employed to extract the active compound from herbs. Let us
delve a little further into each of these topics.
1. Solubility: Solubility refers to the ability of a solute to dissolve in a solvent to form a
solution. Each solute and solvent pair has a specic saturation point, beyond which no
further solute can dissolve. During plant material extraction with a solvent, the release
of plant material involves the extraction of various compounds. The quantities of these
extracted compounds depend on their solubility in the chosen solvent. The degree of solubility depends on their composition, with different substances having different solubility.
Meanwhile, nonpolar compounds, such as essential oils, are well extracted in nonpolar solvents such as hexane or ether, whereas polar compounds, such as alkaloids, avonoids, and
sugars, prefer better solubilization in polar solvents like water or ethanol (Li et al., 2012).
Selecting the solvent for best extraction requires knowledge of the nature of solubility of
the target compound.
2. Diffusion: This is the process by which the solute or, in some cases, even the solvent
molecules are pushed into the solution from a high concentration of the solute to a lower
concentration of the conditioning solvent. Importantly, this process works toward equilibrating the solutes and conditioning solvents, hence pushing the solutes in the solvent into
it (Adegbola et al., 2017). This allows complete extraction of plant material in compounds
in herbal extraction. During the extraction process, targeted compounds are dissolved into
the solvent phase from the plant matrix. The rate of diffusion depends on numerous factors,
such as particle size, the characteristics of the solvent, temperature, and agitation (Afroz
et al., 2015). To control and optimize these mentioned factors improves the diffusion and
hence an increase in the extraction efciency.
3. Mass Transfer: This process refers to the transfer of solutes among phases, such as from
solid plant material to a liquid solvent. The mechanism of mass transfer between solid plant
material and the solvent phase, thus controls the dissolution and diffusivity mechanism of
the target chemicals in terms of their extraction from the plant matrix and During desorption, the chemical compounds are released from the plant surface into the solvent For
instance, dissolution is the process that relates to the solubilization of molecules in a solvent; and diffusion is what allows the molecules to move around freely in the solvent (Chen
et al., 2011). In the same article, Chen and his colleagues proceeded to describe desorption
process as chemicals migrating from the plant’s surface and moving into a liquid.
The net effect of this means, therefore, is that efcient mass transfer will proceed when the principles are followed by high extraction yields because a value addition aspect will make extractions
of the targeted species very complete (Dababi, et al., 2020). It may also repay even those herbalists
who want to extract more than they need of some components without contaminating their extracts
with unwanted substances. Indeed, this optimization of the extraction conditions, based on parameters such as solubility by diffusivity and mass transport rate, may enable the attainment of bioactive
chemical- rich extracts that have been shown in the past to produce desirable medicinal effects.
38 Herbal Pharmacopeia
3.2.2 choice: solvent selection of a sUitaBle MediUM
Menstruum is a solvent that is used for the extraction of compounds in the medicinal plant. Selecting
an appropriate solvent is one of the best solutions to proper extraction efciency. The choice of
menstruum depends very much on the type of the herb, on the part of the herb from which extraction
is to be done, and the nature of the biologically active compounds. Generally, polar solvents such
as water, methyl alcohol, or methanol and ethyl alcohol or ethanol are used for the extraction of
polar compounds; for nonpolar compounds, hexane, and dichloromethane are used. In liquid- liquid
extraction, two miscible solvents are taken, for example, water and dichloromethane; water- hexane;
water- ether, etc. In all mixtures, however, water is an indispensable component because of its high
polar power and its miscibility with organic solvents (de Menezes Rodrigues et al., 2017).
Properties of the Solvent
Water: Water is considered a solvent that is universal. In the extraction of polar compounds,
water is considered to be the most popular polar solvent. It is cheap, dissolves a broad
range of solvents, is highly polar, and is non- ammable and non- toxic (Zahari et al., 2020).
However, it also has some disadvantages: it promotes microbial growth, it may cause hydrolysis and a large amount of heat is required in case of concentrating the extract of water.
Alcohol: Alcohol is polar in nature like water, also. The miscibility in water is excellent and
has a property of extracting polar secondary metabolites. When its concentration exceeds
20%, then it acts as a natural self- preservative, but it is nontoxic in lower concentrations.
Unlike water, a little heat is called for in extracting the concentrate but none of the fats,
waxes, and gums are soluble in it. Alcohols are volatile and ammable.
Chloroform: This is non- polar in nature. It is extensively used in the extraction of fats, oils,
terpenoids, and avonoids, and so on. Chloroform is soluble in alcohols, has a sweet smell,
and is colorless. However, used widely and in large quantities, it has a mild sedative effect.
It has recently been reported to have carcinogenic properties (Zhang et al., 2018).
Ether: The solvent is non- polar and has been used in extracting alkaloids, terpenoids couma-
rins and fatty acids. It is water- soluble and tasteless, and has a low boiling point. It is also
a very stable and not reactive compound given that it doesn’t react with acids, bases, and
metals. However, it is very volatile and ammable (Díaz- Batalla et al., 2006).
Extraction efciency: factors affecting maximum yield and quality Solvent is an important
factor inuencing efciency, selectivity, and safety in the extraction process, and thus critically affects overall herbal extraction. In pharmacopeia, the alcohols used as solvents are
specied, but various parameters have to be considered in selecting a solvent appropriate
for extracting with herbs (Diep, et al., 2020). We shall consider them in detail.
Herb extraction is a complex process in which many levels of interactions blend to nally determine
how well one can extract targets out of one’s material. High efciency in extraction is essentially
required to maximize yield, keep optimum product quality, and, above all, to minimize the use of
resources.
The following factors are responsible for this phenomenon:
1. Particle Size: This is the particle size for which the herbal material is composed that domi-
nates the efciency of extraction.
Crushing to a ne powder exposes a large surface area for the permeation of the solvent
into the particle and also increases the contact of the solvent with the target compounds or
solutes. Since divalent ions and carboxylates cannot interdiffuse, a higher surface needs to
be achieved. The ner particle sizes present an increased rate and higher yield of extraction
compared to a larger surface area. For this reason, the herbal materials must be ground or
milled accordingly so as to maximize the efciency of their extraction.
Modern Techniques in Herbal Extraction and Analysis 39
2. Temperature: The temperature is known to signicantly inuence kinetics involved in
extraction by changing solvent solubility and diffusivity, apart from chemical reactions.
Generally, high extraction temperatures enhance the rate of mass transfer by increasing
the solvent’s capacity to dissolve solutes, thereby accelerating the kinetics of the process.
However, if the temperature goes beyond a certain limit, the destruction of the thermolabile
stuff occurs, causing the synthesis of unwanted byproducts (Dymek et al., 2021). Optimal
temperatures have to be selected carefully between target compound stability and its rate
of extraction within a reasonable period.
3. The Extraction Time: This clearly determines the yield and composition of the nal
extract. The extraction times clearly determine the concentration of extracted compounds.
Unwanted compounds may be extracted or degradation of heat- labile components takes
place within an extended extraction time (Fomo et al., 2020). Crude time, such as a long
extraction time in favor of a longer duration, allows for the diffusion and equilibrium of
solutes. Balance of these two assures best possible yield with good quality extracts.
4. Solvent Volume- to- Raw Material Ratio: The ration of the volume of the solvent to the
material plays a factor in terms of how well the compound will be extracted, as this will
determine the velocity and penetration of the solvent into the particles and hence will
simultaneously affect the rate of dissolution of the compound.
There is a general rule of thumb that dictates that the greater this ratio of solvent to
material used, the more efcient the extraction will actually be, because the available quantity of solvent is increased as is the mass transfer rate (Geng et al., 2015). However, this
tends to escalate processing costs and environmental concerns in case too many solvents
are used for that purpose. The optimization of the ratio between the solutes and the liquid
medium is required in order to get the maximum extraction efciencies but also the minimum solvents for the extraction process.
5. pH: The pH of the extraction medium mostly affects the ionization state and the solubility
of some of the compounds amounting to their extraction efciency.
At specic pH levels, some substances, in particular, exhibit high solubilities whereas
others undergo chemical decompositions or precipitation. Modication of the pH of the
extracting solvent to be in line with this range can hence be used to enhance extraction
selectivity as well as efciency. Alkaloids, phenolics, and organic acids are mostly picked
in herbal substances (Zygler et al., 2012).
6. Agitation: In mass transfer, we need agitation to achieve adequate permeation of the sol-
vent through the plant matrix, thereby enabling the effective exposure of the desired compounds during extraction.
This will, however, be greatly enhanced by mechanical stirring, shaking, or agitation
through the use of ultrasound, among others, which increases the rate at which extraction
occurs because of increased surface area available for contact with the solvent and, therefore, the diffusing mass transfer resistance (Omeroglu et al. 2019). The proper observation
of such agitation techniques allows the proper mixture circulation, thereby achieving an
even mixture and maximizing yields from the extractions process.
7. Plant Variability: Different types of plants, variation within plant species, how they are
grown, and how they are harvested all lead to a difference in the composition and concentration of benecial compounds in the herbal materials.
This makes the extraction process complicated; hence, it is just as important to decide
on plants under standardized methods of extraction so that there is always consistency in
quality and effective results. These criteria include the age of the plants, its habitat, and the
way it is to be handled after harvesting (Omeroglu et al. 2019).
8. The Method of Extraction: The extraction method to be used for the extraction of
compounds from your herbal material is of great concern with regard to efciency in
extraction.
40 Herbal Pharmacopeia
There are some commonly available methods, which include maceration, percolation, Soxhlet
extraction, supercritical uid extraction, and ultrasound- assisted extraction. Each method, therefore,
achieves a balance between efciency, selectivity, and processing time (Tsao and Deng Z., 2004).
The selection of the appropriate extraction method in relation to the nature of the herbal material
and the desired product characteristics are thus very essential and play a crucial role in gaining good
results from an extraction process.
1. Modern Extraction Techniques
• Supercritical Fluid Extraction (SFE)
• Microwave- Assisted Extraction (MAE)
• Ultrasound- Assisted Extraction (UAE)
• Pressurized Liquid Extraction (PLE)
• Subcritical Water Extraction (SWE)
3.3 SUPERCRITICAL FLUID EXTRACTION (SFE)
Supercritical Fluid Extraction (SFE) is a technique which is used for the extraction of specic components from the source or material, in which supercritical uids act as the extracting solvent. In
other words, SFE is a process of separating extract from the matrix with the help of supercritical
uids which act as an extracting solvent.
A supercritical uid is an enormously compressed uid that signies the properties of both gases
and liquids. They are created by raising temperature and pressure beyond the critical point of a substance. These have high density and are non- compressible (Shinde et al., 2009).
Supercritical uids (SF) have exclusive characteristics whereby they can diffuse through solids
like a gas and simultaneously be able to dissolve materials like a liquid. The most common supercritical uids in use within modern industry are carbon dioxide and water. Carbon dioxide is the SF
which is most commonly used for extraction purposes, because it has relatively low critical temperature and pressure. It is also non- toxic and readily available.
3.3.1 Working PrinciPle of sfe
The working principle of Supercritical Fluid Extraction relies on the unique characteristics of supercritical uids. When the substance is heated above its critical temperature and critical pressure, it
changes into a supercritical state where it shows the characteristics of both liquids and gases. At this
stage, the uid can penetrate the solid material and can dissolve the desired components effectively.
3.3.2 Parts of the sfe systeM
1. Pump: A pump is used to pressurize the carbon dioxide and convey it to the extraction
vessel.
2. Extractor: The extraction vessel in which the raw materials (the herbs) are kept. Here the
supercritical carbon dioxide interacts with the material for the extraction of the desired
compounds.
3. Heater: The heater maintains the desired temperature at which the carbon dioxide remains
in its supercritical state.
4. Separator: Once the extraction process is completed, the carbon dioxide and the extracted
compounds are transferred to the separator where the pressure is decreased, and the carbon
dioxide is reverted to the gas, leaving the extract to separate out.
5. Condenser: This apparatus condenses the carbon dioxide gas back into its liquid form for
reuse in the system.
6. Flow Meter: This monitors and controls the rate of carbon dioxide.
Modern Techniques in Herbal Extraction and Analysis 41
FIGURE 3.1 Method of extraction from herbs using SFE.
3.3.3 Process of extraction
1. Selection of Herbs: For extraction, the herbs which pose essential oils or active com-
pounds are selected.
2. Preparation: In the SEF of herbs fresh plant material is used commonly. When a fresh
sample is extracted from herbs, it contains a high degree of moisture. This could result in
mechanical difculties, for example, the restrictor may become clogged because of ice formation. To avoid this problem, the solution is mixed with anhydrous Na2SO4 (Figure 3.1).
3. Grinding: The raw material (herbs) is ground into a ne powder to enhance surface area
for extraction.
4. Loading: The powdered herbs are loaded into the extractor vessel through a pump which
exerts high pressure.
5. Pressurization: CO₂ is pumped into the extractor vessel and heated to reach its supercriti-
cal state.
6. Extraction: The supercritical CO₂ penetrates the herb matrix and dissolves the target
compounds.
7. Separation: The mixture of CO₂ and dissolved compounds is transferred to the separator,
where the pressure is lowered. The CO₂ becomes gaseous and separates from the extracted
compounds.
8. Collection: The extract is collected from the separator.
9. Recycling: The gaseous CO₂ is condensed back to a liquid state and recycled back into the
system.
3.3.4 aPPlications
1. It is used for the extraction of compounds used in avours, fragrances, and essential oils.
2. It is used for the extraction of bioactive compounds from herbs, which are used in nutra-
ceuticals and pharmaceutical products.
3. It is also used for the purication of natural compounds and products
42 Herbal Pharmacopeia
3.4 MICROWAVE- ASSISTED EXTRACTION (MAE)
Microwave- Assisted Extraction (MAE) is a method which is used for the extraction of bioactive
compounds from raw materials such as herbs and plants through the use of microwave energy.
Microwave energy has the strong ability to warm up solvents and plant materials in a very short time
for the extraction of required compounds. Heat is generated following ionic conduction and dipole
rotation mechanism through microwaves by interacting with polar compounds for example water
and organic components in the plant medium. In MAF, heat and mass are transferred in the same
path which creates a synergistic effect, this quickens the extraction process, and extraction yield is
also improved. It is also regarded as a “green technology” because in the MAE process the usage of
organic solvent is decreased. There are two types of Microwave- Assisted Extraction method:
1. Solvent- free extraction: usually used for volatile compounds.
2. Solvent extraction: usually used for non- volatile compounds.
3.4.1 Working PrinciPle
The MAE works on the principle that with microwave radiations polarizable materials and dipoles of
polar solvent are changed. Microwaves are a type of electromagnetic radiations which have frequencies ranging from 300 MHz to 300 GHz. The polar molecules and ions in the solvent and plant material are oscillated when microwaves are applied to the plant material and heat is produced through
dipole rotation and ionic conduction. The mass transfer rate, breaking the cell walls of plant material
and releasing the required compounds into the solvent, are enhanced due to this localized heating .
3.4.2 coMPonents of a MicroWave- assisted extraction systeM
1. Microwave Generator: This part of microwave- assisted extraction system generates
microwaves of a specic frequency. A frequency of 2450 MHz is generally used for MAE.
2. Microwave Cavity: It is extraction chamber. The microwave cavity is made to contain and
homogeneous reection of microwaves.
3. Sample Holder: This is a container which holds the plant/herb material and extraction
solvent during the process of extraction.
4. Temperature and Pressure Sensors: The temperature and pressure inside the extraction
chamber can be monitored and controlled to provide and ensure the optimum extraction
conditions.
5. Cooling System: This keeps the temperature within safe limits to avoid the decomposition
or degradation of required compounds.
6. Magnetic Stirrer: This ensures the homogenous mixing of the solvent and sample to
increase extraction efciency.
7. Control Unit: This is the unit that manages temperature, pressure, microwave power and
extraction time settings.
3.4.3 Method of extraction froM herBs By Mae
1. Preparation of Plant Material: Herbs are washed, dried, and ground up to make a ne
powder to increase the surface area for extraction. Dried plant material still contains traces
of moisture, which acts as a target for microscopic heating.
2. Selection of Solvent: A proper and well- suited solvent which must be transparent is chosen
on the basis of polarity of required compounds. The solvent used must be transparent to
microwave radiation to ensure efcient absorption by the sample. Commonly used solvents
include water, ethanol, methanol, or their mixtures.
Modern Techniques in Herbal Extraction and Analysis 43
Flow chart of MAE process
High pressure application on the cell wall of plant material
Oozing out of required components.
Application of microwave radiations
Heating and evaporation of moisture
Swelling and rupturing of cell wall of plant material
FIGURE 3.2 Flow chart diagram of Ultrasound- Assisted Extraction (UAE).
↓
↓
↓
↓
3. Loading the Sample: In the sample holder, a prepared sample of herb is placed along with
the chosen solvent.
4. Microwave Treatment: The cavity sample holder is placed in the microwave. The micro-
wave generator is turned on and the microwave increases the temperature of the sample
quickly by penetrating in it. This results in the dehydration of the cellulose of the plant
material as the temperature increases. This reduces its mechanical strength.
5. Extraction Process: The cell walls of the herb’s material are ruptured due to localized
heating. It helps to release the bioactive compounds into the extraction solvent. The yield
and quality of the extracted compounds could be enhanced by the adjustment of extraction
time and microwave power (Figure 3.2)
6. Cooling and Filtration: The mixture is cooled after extraction is completed. Then
it is ltered to separate the plant residue from the solvent containing the extracted
compounds.
7. After the extraction process, the mixture is allowed to cool. It is then ltered to separate
the solvent containing the extracted compounds from the plant residue. After the extraction
process, the mixture is allowed to cool. It is then ltered to separate the solvent containing
the extracted compounds from the plant residue.
8. Concentration and Purication: The obtained extract could be concentrated by evaporat-
ing the solvent, while the isolation of desired compounds will require additional purication steps.
3.5 ULTRASOUND- ASSISTED EXTRACTION (UAE)
Ultrasound waves: the waves which have exceeded the audible frequency range > 20 kHz are designated as ultrasound waves. When these waves pass and spread through the medium, the compression
and rarefaction of particles of the medium results. UAE technique could be combined either with
temperature called “thermosonication” or pressure “manosonication.”
Ultrasound- assisted extraction is the technique in which the application of ultrasonic waves
improves the extraction of bioactive compounds from a wide range of material herbs. This method
improves efciency and extraction yield by using sound waves of high frequency which create cavitation bubbles in the solvent used for extraction. It then bursts on the surface of plant matrix, a shock
damage to plant call wall increases the mass transfer of required compounds from the call membrane
into the solution.
3.5.1 Working PrinciPle
This phenomenon of UAE works on the principle of cavitation. This phenomenon is created when
ultrasonic waves pass through a liquid; cycles of high and low pressure are developed. In the cycle