Introduction to herbal technology

Medicine is a substance that has nutritive, curative, or preventive properties,

while the term “herbal” refers to a botanical or plant-based preparation. Hence,
the term “herbal medicine” is used for plantbased substances that consist of
nutritive, curative, or preventive properties. Herbal medicine is an
interdisciplinary branch between herbal medicine and Ayurveda as it covers all
fields of herbal medicine related to botany, medicinal plant research,
pharmacognosy, phytochemistry, phytotherapy, botanical medicines, Ayurveda,
natural chemistry, agriculture science, Unani medicine, biotechnology, and
biochemistry. A person who deals with herbs, especially medicinal herbs, is
known as an herbalist. Herbal journals deal with the use of plants in the treatment
of diseases.
Herbal technology Circumscribes all the advancing technical frontiers (except
genes) meant to Tap myriads of modes of Manipulating plants around America.
An oversized range of Technologies have Been developed to reap the Bountiful
merchandise that the plants manufacture, as well as natural Dyes, biofertilizers,
biopesticides And biofuel. Flavourer Technology was the primary step in
Codifying Principles and process scientific ways of this New conception of
fruitfully Managing the plants Around America . For over twenty years Herb
Technology has been on the Cutting edge of botanical medicine development.
Our team of Ayurvedic, Chinese and Western Doctors has formed the traditional
art of flavourer Formulation. Incorporating fashionable scientific Discoveries
with lore, Herb Technology skilled formulas have set the Standard for the clinical
follow of botanical medicine The flavourer trade offers a singular and Strategic
Investment chance that resulted in its rise worldwide.Herbal drugs is associate
knowledge base branch between flavourer drugs And Ayurveda because it Covers
all fields of flavourer drugs associated with biology, medicative plant analysis,
Pharmacognosy, Phytochemistry, botanical medicine, biological science
medicines, Ayurveda, natural Chemistry, agriculture science, Unani drugs,
Biotechnology, and organic chemistry. A person Who deals with herbs,
particularly medicative herbs, is understood as associate healer. Flavourer
journals Deal with the utilization of plants within the treatment of diseasesThe
major downside of recent medicines is their facet effects which can cause life
Threatening of patients. Flavourer medicines even have their list of facet effects
like every alternative Synthetic drug. So it’s essential to guage their clinical
safety and effectuality. Current focus On chemotype-driven finger printing and
connected Techniques needs integration with Genotype-driven molecular
Techniques so associate best characterization of biological science Materials is
feasible. The role of natural merchandise, flavourer drugs, social group and
ancient Medicines is being more and more Appreciated in recent years for the
hindrance and cure of Human components. Being with chemicals numerous
they’re ready to modulate many targets Simultaneously in a very advanced
system.
Different methods of Identification of plants
1)Expert Determination:
The best method of identification is expert determination in terms of reliability or
accuracy. In general the experts have prepared treatments (monographs,
revisions, synopses) of the group in question, and it is probable that the more
recent floras or manuals include the expert’s concepts of taxa. Experts are
typically found in botanical gardens, herbaria, museums, colleges, universities,
etc. However, although of great reliability, this method presents problems of
requiring the valuable time of experts and creating delays for identification
2)Recognition:
It approaches expert determination in reliability. This is based on extensive, past
experience of the identifier with the plant group in .in some groups this is
virtually impossible. Comparison- A third method is by comparison of an
unknown with named specimens, photographs, illustrations or descriptions.
Although this is a reliable method, it may be very time consuming or virtually
impossible due to the lack of suitable materials for comparison. The Use of Keys
and Similar Devices Synopses, Outlines, etc. This is by far the most widely used
method and does not require the time, materials, or experience involved in
comparison and recognition.
3)Comparison:
A third method is by comparison of an unknown with named specimens,
photographs, illustrations or descriptions. Although this is a reliable method, it
may be very time consuming or virtually impossible due to the lack of suitable
materials for comparison.
4)The Use of Keys and Similar Devices (Synopses, Outlines, etc.):
This is by far the most widely used method and does not require the time,
materials, or experience involved in comparison and recognition.
Authentication of plant
Herbal authentication is a quality assurance process that ensures the correct
herbal species and plant parts are used as raw materials for herbal products
Besides, correct identification of herbal plants forming the herbal products is a
prerequisite and fundamental to the whole realm of medicine and science
Moreover, identifying herbal plants as the raw materials should be performed to
ensure that the raw materials used in finished products are suitable for their
intended use In addition, it is crucial to utilize a precise authentication method to
ensure that the herbs used as materials are correct and authentic prior to any
processes in order to ensure the quality and the safety of finished herbal products
Thus, authentication of herbal plants taken up for research or medicinal use is a
necessity to achieve satisfactory results and also to maintain the efficacy and
therapeutic property of the preparations in which these plants are used
Authentication tests usually involve applicable analytical techniques in dealing
with particular sample This is useful in the cases of finished herbal products that
are frequently substituted or adulterated with other morphologically and
chemically indistinguishable varieties Commonly, the methods used to assess the
authenticity of herbs depend on morphological and analogical analysis,
organoleptic characters, deoxyribonucleic acid (DNA)-based, chemical
fingerprinting and many others
1)Macroscopy: Macroscopy involves checking external look or sensory
characters Like color odor, taste, size, shape, fracture etc. botanic identification
of herb is usually done By trained person like biologist. For proper botanic
identification, entire plant at the side of root And flower is required. Botanic
identification is predicated on morphology that involves checking Various
elements of herbs like leaves, flower, root et al.. Leaves and flowers virtually
vital Parts that facilitate in identification of plant. Herb will be ascertained for
color size, shape and Arrangement of leaves and flower. Arrangement of leaves
on stem AN branching is termed Phyllotaxy. Differing types of arrangement of
leaves like alternate distichous, opposite, Decussate, whorled varieties of leaves
arrangement will be useful to spot herb properly. Different types of shapes of
leaves like oval, oblong, obovate, spherical linear, lanceolate, Elliptical,
spatulate, cordate, unsubdividedar one amongst the best tool to spot plants. Even
margins Of leaves will be ascertained to spot herbs. Margins like entire, serrate
rough, sinuate, ciliate, Spinose facilitate in identification of herb. In some cases,
completely different species of plants will be known Only once flowering.
2) Phytochemistry:- once macro and research, preliminary phytochemical
analysis helps To identify plant. Preliminary phytochemical analysis helps to
reveal chemical constituents. Also analytical techniques ar wont to establish
marker compound that is {particular} to particular Herb. UV, MASS, NMR,
HPLC, HPTLC ar habitually employed in trade for identification of Herbs.

3) DNA Fingerprinting:-Use of biotechnological tools for correct identification of

herbs is the upcoming latest technology. Molecular markers like RAPD, 1SSR,
RFLP uses DNA fingerprinting for identification of herb at molecular level.
Molecular markers are nothing but sequence of DNA which is unique to each
plant. First, plant DNA is isolated and then amplified with the help of PCR and
then screened for similar and different patterns. Plants also have DNA patterns
similar to human. Pattern of this DNA can be identified in the for barcoding or
DNA fingerprinting. DNA barcoding identify plant as specie subspecies level or
molecular level, even when small part of plant without flower More recent
technology like genomics, proteomics, transcriptomics and metabolomics
technique can identify plant at genetic level

Different extraction methods including advanced extraction techniques like

supercritical fluid extraction, microwave-assisted extraction, ultrasound assisted
extraction, solid-phase and microwave assisted extraction etc.

2 Extraction
Extraction, as the term is used pharmaceutically, involves the separation of
medicinally active portions of plant or animal tissues from the inactive or inert
components by using selective solvents in standard extraction procedures. The
products so obtained from plants are relatively impure liquids, semisolids or
powders intended only for oral or external use. These include classes of
preparations known as decoctions, infusions, fluid extracts, tinctures, pilular
(semisolid) extracts and powdered extracts. Such preparations popularly have
been called galenicals, named after Galen, the second century Greek physician.
The purposes of standardized extraction procedures for crude drugs are to attain
the therapeutically desired portion and to eliminate the inert material by
treatment with a selective solvent known as menstruum. The extract thus
obtained may be ready for use as a medicinal agent in the form of tinctures and
fluid extracts, it may be further processed to be incorporated in any dosage form
such as tablets or capsules, or it may be fractionated to isolate individual
chemical entities such as ajmalicine, hyoscine and vincristine, which are modern
drugs. Thus, standardization of extraction procedures contributes significantly to
the final quality of the herbal drug.
Methods of Extraction of Medicinal Plants
1)Maceration In this process, the whole or coarsely powdered crude drug is
placed in a stoppered container with the solvent and allowed to stand at room
temperature for a period of at least 3 days with frequent agitation until the soluble
matter has dissolved. The mixture then is strained, the marc (the damp solid
material) is pressed, and the combined liquids are clarified by filtration or
decantation after standing.
Maceration is a simple extraction method that involves soaking the plant
prepared raw material in a coarse or powder form in a solvent of interest at room
conditions for at least three days with intermittent agitation . After the extraction
is completed, the mixture is strained either through sieves or a net with tiny
holes. Subsequently, the marc is pressed, and the liquid extract is cleaned using
either filtration or decantation after standing. Maceration is preferably carried out
in a stoppered container to minimize solvent loss through evaporation. It is
undesirable to obtain an already concentrated extract through evaporation of the
solvent during the extraction process. The product is concentrated frequently by
the use of vacuum evaporation. It is crucial to select an appropriate solvent in the
maceration as the solvent will delineate the phytochemicals classes salvaged
from the samples. The solvent could also enable the extraction of thermolabile
phytochemicals. The procedure has the underlying disadvantage of low
efficiency and long duration of extraction . However, optimized conditions may
attribute significant efficiency to this technique as high phenolic compounds, and
anthocyanins yield were obtained from chokeberry . On the other hand, a study
on C. cajan leaves to extract flavonoids using microwave-assisted , reflux,
ultrasound-assisted, and maceration extractive methods observed that the
maceration techniques afforded the lowest yield . Flavonoids have also been
extracted using maceration from rhizomes of turmeric using nonionic surfactant
Triton X-100 at a temperature of 35 °Cat neutral pH, from fruits of Arbutus
unedo L. at a raised temperature of 79.6 °C in 3.7% diluted ethanol, and from
leaves of Ficus carica and Euphorbia neriifolia using 75% concentrated ethanol at
room temperature . In these studies, the type of solvent used was based on the
polarity of the phytochemicals extracted. For instance, Triton X-100, which is
nonionic but has a hydrophilic side chain, was used to extract less polar
flavonoids, whereas the more polar ethanol, or a mixture of ethanol and water
was used to extract more polar flavonoids Further, Polyphenols, such as
anthocyanins have been extracted from dried fruits of chokeberry using 50%
ethanol as solvent
2)Infusion:- Fresh infusions are prepared by macerating the crude drug for a short
period of time with cold or boiling water. These are dilute solutions of the readily
soluble constituents of crude drugs.
Infusions are dilute solutions containing the readily-soluble constituents of crude
drugs. ¾ Formerly, fresh infusions, prepared by macerating the drug for a short
period in cold water or boiling water were used. ¾ Now, infusions are usually
prepared by diluting one volume of a concentrated infusion to ten volumes with
water. ¾ Concentrated infusions are prepared by modified percolation or
maceration process, which after dilution with water, resemble in potency and
aroma the corresponding fresh infusion. ¾ Infusions are liable to fungus and
bacterial growth, and it is necessary to dispense them within twelve hours of their
preparation.
Infusion is described as a dilute solution of easily soluble constituents of the
plant material. It is an extraction technique in which the plant material is
immersed in boiling solvent, particularly water, and left to stand in a stoppered
container for about 15 min, after which time the extract (tea) is poured off and
separated from the marc using a filter . Tea may be considered as the best
example of an infusion. For example, Caffeine has been extracted from dried
crushed leaves of tea brands alokozay, lipton, tapal, and tetley at brewing times
ranging from 2 to 30 min within the temperature range of 30 to 90 °C . Further,
phenolic compounds were extracted from fruits of Tilia cordata at an optimal
temperature of 95 °C . It has to be noted that some infusions are prescribed to
treat health issues such as diarrhea, bronchitis, and asthma. For instance,
antioxidants; phenols, and flavonoids have been extracted from rhizomes of
various gingers in boiling water for 10 min . Another method of interest similar
to infusion but more efficient than maceration is percolation

Digestion
This is a form of maceration in which gentle heat is used during the process of
extraction. It is used when moderately elevated temperature is not objectionable.
The solvent efficiency of the menstruum is thereby increased.

Digestion is an extractive method similar to maceration and uses slight warming

in the extraction process . Care is, however, exercised to avoid the temperature
altering the bioactive phytochemicals of given plant material. Therefore, there is
increased efficiency in using the extraction solvent due to warming. Mostly
temperatures are kept in the range of 35 to 40 °C but may be increased to a
maximum of 50 °C for tougher plant materials such as barks and materials
containing dismally soluble phytochemicals. During extraction, desired plant
parts are introduced in a container with the appropriate solvent pre-heated to the
indicated temperatures. The optimum temperature is maintained for a period that
may range from half an hour to 24 h with shaking the container at regular
intervals

Percolation:-
This is the procedure used most frequently to extract active ingredients in the
preparation of tinctures and fluid extracts. A percolator (a narrow, cone-shaped
vessel open at both ends) is generally used (Figure 1). The solid ingredients are
moistened with an appropriate amount of the specifi ed menstruum and allowed
to stand for approximately 4 h in a wellclosed container, after which the mass is
packed and the top of the percolator is closed. Additional menstruum is added to
form a shallow layer above the mass, and the mixture is allowed to macerate in
the closed percolator for 24 h. The outlet of the percolator then is opened and the
liquid contained therein is allowed to drip slowly. Additional menstruum is added
as required, until the percolate measures about three-quarters of the required
volume of the fi nished product. The marc is then pressed and the expressed
liquid is added to the percolate. Suffi cient menstruum is added to produce the
required volume, and the mixed liquid is clarifi ed by fi ltration or by standing
followed by decanting.

PERCOLATOR

Hot Continuous Extraction (Soxhlet)

In this method, the fi nely ground crude drug is placed in a porous bag or
“thimble” made of strong fi lter paper, which is placed in chamber E of the
Soxhlet apparatus. The extracting solvent in fl ask A is heated, and its vapors
condense in condenser D. The condensed extractant drips into the thimble
containing the crude drug, and extracts it by contact. When the level of liquid in
chamber E rises to the top of siphon tube C, the liquid contents of chamber E
siphon into fl ask A. This process is continuous and is carried out until a drop of
solvent from the siphon tube does not leave residue when evaporated. The
advantage of this method, compared to previously described methods, is that
large amounts of drug can be extracted with a much smaller quantity of solvent.
This effects tremendous economy in terms of time, energy and consequently fi
nancial inputs. At small scale, it is employed as a batch process only, but it
becomes much more economical and viable when converted into a continuous
extraction procedure on medium or large scale

Aqueous Alcoholic Extraction by

Fermentation :-
Some medicinal preparations of Ayurveda (like asava and arista) adopt the
technique of fermentation for extracting the active principles. The extraction
procedure involves soaking the crude drug, in the form of either a powder or a
decoction (kasaya), for a specifi ed period of time, during which it undergoes
fermentation and generates alcohol in situ; this facilitates the extraction of the
active constituents contained in the plant material. The alcohol thus generated
also serves as a preservative. If the fermentation is to be carried out in an earthen
vessel, it should not be new: water should fi rst be boiled in the vessel. In large-
scale manufacture, wooden vats, porcelain jars or metal vessels are used in place
of earthen vessels. Some examples of such preparations are karpurasava,
kanakasava, dasmularista. In Ayurveda, this method is not yet standardized but,
with the extraordinarily high degree of advancement in fermentation technology,
it should not be diffi cult to standardize this technique of extraction for the
production of herbal drug extracts.
Counter-current Extraction
In counter-current extraction (CCE), wet raw material is pulverized using toothed
disc disintegrators to produce a fi ne slurry. In this process, the material to be
extracted is moved in one direction (generally in the form of a fi ne slurry) within
a cylindrical extractor where it comes in contact with extraction solvent. The
further the starting material moves, the more concentrated the extract becomes.
Complete extraction is thus possible when the quantities of solvent and material
and their flow rates are optimized. The process is highly efficient, requiring little
time and posing no risk from high temperature. Finally, sufficiently concentrated
extract comes out at one end of the extractor while the marc (practically free of
visible solvent) falls out from the other end.
This extraction process has significant advantages:
i) A unit quantity of the plant material can be extracted with much smaller
volume of solvent as compared to other methods like maceration,
decoction, percolation.
ii) CCE is commonly done at room temperature, which spares the
thermolabile constituents from exposure to heat which is employed in most
other techniques.
iii) As the pulverization of the drug is done under wet conditions, the heat
generated during comminution is neutralized by water. This again spares
the thermolabile constituents from exposure to heat.
iv) The extraction procedure has been rated to be more effi - cient and
effective than continuous hot extraction.
ADAVANCE EXTRACTION PROCESS:-
Herbal medicines have been evolving side by side of human culture. Plants are
considered as natural factories for production of various phytochemicals. A large
number of secondary metabolites like alkaloids, phenolics and flavonoids are
synthesized by plants in addition to compounds that are needed for the
reproduction and growth of plants. Advancements in natural sciences led
researchers towards identification and isolation of different bioactive
phytochemicals . Depending on physical nature and the properties of
phytoconstituents, various methods are in use to obtain the crude extract. Among
these various conventional extraction methods including infusion, digestion,
decoction, percolation and maceration are commonly practiced in herbal industry
for crude extraction . Most Pharmacopeias mentioned the use of percolation and
maceration to obtain plants’ crude extractions. However traditional methods of
extraction are time taking, like, 2-7 hours at least are required for maceration, and
this process also requires a large amount of solvents . In case of Soxhlet
extraction the targeted molecules might be decomposed due to high
temperatures . Therefore there was space for invention of new techniques. Hence
alternative extraction techniques like, Supercritical Fluid Extraction (SCF),
Accelerated Solvent Extraction (ASE) and Ultrasonic Assisted Extraction (UAE)
are invented to compensate the increasing demand of natural products. These
techniques reduced the consumption of organic solvent, enabled automation and
shortened extraction time.
Supercritical Fluid Extraction
Supercritical fluid extraction (SFE) is an alternative sample preparation method
with general goals of reduced use of organic solvents and increased sample
throughput. The factors to consider include temperature, pressure, sample
volume, analyte collection, modifi er (cosolvent) addition,flow and pressure
control, and restrictors. Generally, cylindrical extraction vessels are used for SFE
and their performance is good beyond any doubt. The collection of the extracted
analyte following SFE is another important step: signifi cant analyte loss can
occur during this step, leading the analyst to believe that the actual effi ciency
was poor. There are many advantages to the use of CO2 as the extracting fl uid.
In addition to its favorable physical properties, carbon dioxide is inexpensive,
safe and abundant. But while carbon dioxide is the preferred fl uid for SFE, it
possesses several polarity limitations. Solvent polarity is important when
extracting polar solutes and when strong analyte-matrix interactions are present.
Organic solvents are frequently added to the carbon dioxide extracting fl uid to
alleviate the polarity limitations. Of late, instead of carbon dioxide, argon is
being used because it is inexpensive and more inert. The component recovery
rates generally increase with increasing pressure or temperature: the highest
recovery rates in case of argon are obtained at 500 atm and 150° C.
The extraction procedure possesses distinct advantages:
i) The extraction of constituents at low temperature, which strictly avoids
damage from heat and some organic solvents.
ii) No solvent residues.
iii) Environmentally friendly extraction procedure. The largest area of growth
in the development of SFE has been the rapid expansion of its applications.
SFE fi nds extensive application in the extraction of pesticides,
environmental samples, foods and fragrances, essential oils, polymers and
natural products. The major deterrent in the commercial application of the
extraction process is its prohibitive capital investment.

Microwave assisted extraction

Microwave assisted extraction is one of the advanced techniques under thought
now a days. In MAE, microwave vitality is utilized to concentrate plant
metabolites with the solvents. This system has demonstrated its wellbeing for the
vast majority of the specimens because of the ease to handle and to undeerstand
steadiness. Exploration is continuing for functional use of microwaves for
business creation of phyto-constituents, yet at the same time in early stages.
In microwave assisted extraction, heating of matrix is done by the means of
microwaves. Microwaves possess electric field and magnetic fields which are
mutually perpendicular. Heating is brought about by electric field by means of
ionic conduction and dipole rotation. Components absorb microwave energy
depending upon their dielectric properties.

Principle of microwave assisted extraction

Microwaves are part of electromagnetic spectrum of light with a range of 300
MHz to 300 GHz and wavelengths of theses waves range from 1cm to 1m
(Mandal et al., 2007). These waves are made up of two perpendicular oscillating
fields which are used as energy and information carriers. First application of
microwaves includes its interaction with the specific materials which can absorb
a part of its electromagnetic energy and can convert it into heat. Commercial
microwaves use 2450 MHz of energy for this purpose which is almost equivalent
to 600-700W . Practically, microwaves induce dipole rotation in organic
molecules along with heating which causes the destruction of hydrogen bonding.
This causes the traffic of ions which results in a heating effect due to increased
kinetic energies of ions as well as friction between ions due to their continuous
movements and change in directions. Destruction of hydrogen bonding also
increases the penetrating efficiency of the solvents into the plant matrix
Properties of Microwaves
1) Microwaves lie between infrared waves and radio waves in electromagnetic
spectrum.
2) These have wavelengths between 0.01 m to 1 m
3) These can be operated in a frequency range between 300 MHz to 300 GHz.
4) Energy in a microwave photon is 0.037 kcal/mol which is relatively low
compared to that required to break a bond hence the changes brought about by
microwave heating are purely kinetic .
5) Microwaves transfer energy in seconds way faster than the time required by a
molecule to relax
Mechanism of MAE
Heating of solid matrix is brought about by dual mechanisms of dipolar rotation
and ionic conduction. Heating may be due to either one of them or both the
mechanisms taking place simultaneously. In ionic conduction, ions try to migrate
in accordance with changing electric field. In doing so they suffer collisions with
each other and solvent molecules. Furthermore, if solvent molecules resist the
motion of ions, friction is generated eventually leading to liberation of heat. In
dipolar rotation, polar molecules try to align themselves in phase with the
changing electromagnetic field. However due to inertia and inter-molecular
forces of attraction, some resistance is offered which leads to generation of heat.

Ultrasonic-assisted extraction
Ultrasound assisted extraction (USAE) has been used extensively in the last two
decades as an efficient extraction method both in the food and pharmaceutical
industries. The number of papers published in the last three decades related with
this topic has suffered an exponential increase (Figure 1). According to the
applied power, different effects may be sought. At low intensities, the external
and, possibly, the internal mass transfer resistances are affected. Nevertheless, the
product structure remains mainly unaffected. Intermediate intensities may affect
the product structure thus increasing the effects on the internal mass transfer
resistance. If ultrasonic power is further increased, cell disintegration can take
place.
Ultrasonic-assisted Extraction (UAE) is a rapid and effective extraction
technique that uses ultrasound to generate rapid movement of solvents, resulting
in a higher mass transfer speed as well as acceleration of extraction. Compared to
other advanced extraction techniques, UAE is more economic, eco-friendly, and
convenient . it is reported that UAE is a simple and fast technique, which
consumes less energy, time and materials, thus producing more pure products at
higher yields . UAE can be performed under low operation temperature where it
can prevent thermal damage to the extracts and preserves the bioactive
compounds’ properties in term of its structure and molecule, thus UAE is seen as
an ideal option for the edible oil industry . UAE also no prevent the thermal
damage on bioactive compounds but it also avoid the damage in plant materials
UAE can be applied in two different techniques, either by using an ultrasonic
bath or an ultrasonic horn transducer. Ultrasonic bath which was equipped with a
temperature-controlled device which helped the recovery of thermo-sensitive
compounds including essential oils
There are two ways of ultrasonic irradiation which are direct or indirect
contact with sample. Indirect contact means the contact happens through the
walls of the sample such as ultrasonic bath system. Direct contact system is more
effective in extraction process as it can power up to 100 times better than indirect
contact or ultrasonic bath
Recently, there have been many reports on the application of UAE in extraction
of trace organic compounds from soil, animal and plant tissues . Besides, UAE is
also well recognized to exhibit great effects on the rate of various extraction
processes in food, pharmaceutical and cosmetic industries
The advantageous of using UAE are it is easy to handle, safe in term of
atmospheric pressure and ambient temperature, moderate use of solvent and it
also reproducible . It could also be operated at moderate temperature which is
suitable for heat-sensitive compounds . In contrast, the disadvantages of UAE are
it requires filtration step and possible degradation of compounds at high
frequencies.
Ultrasonic-assisted Extraction Process.
UAE is based on the principle of acoustic cavitation ultrasound that induced via
a series of compression and rarefaction waves in the molecules of the medium
through which it is capable of damaging the cell walls of plant matrix as well as
favouring the release of bioactive compounds . During sonication, acoustic
cavitation produces cavitation bubbles which causes the breaking of the plant cell
wall and eventually allows easy percolation of solvent into the extractable
material . In details, the use of ultrasound or sonication is to break the cell
membranes that has the advantage of reducing considerably the extraction time
and increasing the yield extracted. The application of ultrasound ruptures the cell
wall structure and increases the diffusion through membranes; thus, the cell lyses
and hence facilitates the release of cell contents . The cavitation bubbles will
form at sufficient high power which the rarefaction cycle may exceed the
attractive forces of the molecules of the liquid. Such bubbles grow under a
process known as rectified diffusion i.e. small amounts of vapour (or gas) from
the medium enters the bubble during its expansion phase and during
compression, it is not fully expelled. It is the fate of these bubbles when they
collapse in succeeding compression cycles which generates energy. The
symmetric bubbles are created in a homogenous liquid and their collapse in the
bulk liquid are also symmetric leading to localized hot spots (~5000 K and ~2000
atm) and another one is an asymmetric bubbles which cavitation in a
heterogeneous extraction mixture is that the collapse of the bubbles is
asymmetric resulting extremely high speed jets of solvent targeting the vegetal
material and this what makes UAE so effective. As soon as a bubble collapses
near a surface where this could be vessel wall, herbal particles or any suspended
material in the liquid, that bubble deforms taking a doughnut- shape i.e. the
collapse is asymmetrical and forming a high velocity jet, impacting the wall with
the potential to sweep particles away from the surface or indeed cause actual
damage. This is one of the main reasons why ultrasound is very effective for
surface cleaning. During the sonication of a vegetal material in a solvent, the
suspended solids promote asymmetric bubble collapse which generates jets of
solvent towards herbal particles which boost more efficient extraction from them.
Asymmetric bubble collapse is the key parameter of ultrasonically assisted
extraction and as in many ultrasonic processes the acoustic power applied must
be large enough to overcome the cavitation threshold
 Furthermore, ultrasounds also use a mechanical effect which promotes
better penetration of solvent into the sample matrix thus allowing higher
diffusion rates across the cell wall

Solid Phase Extraction (SPE)

Solid phase extraction (SPE) is a common sample pre-treatment technique. It has

been developed from solid-liquid extraction and chromatography methods and is
often used for sample fractionation, purification, enrichment, and concentration.
Similar to liquid chromatography (LC), in the SPE process, interfering
components are separated from analytes due to differences in affinity and
adsorption.
SPE is one of the most common sample pre-treatment techniques used before
HPLC, LC, and GC-MS analysis due to its ease of operation, ability to simplify
analysis, and increase sensitivity. The major advantages of using SPE include:
1. Make the sample simpler and cleaner. Samples often have a
complex composition. Interferents can reduce sensitivity and also
shorten the lifespan of columns and instruments. SPE treatment can
provide clean extraction of analytes and yield more accurate results.
2. Concentration and trace enrichment. SPE is advantageous when
researchers aim for low detection levels, such as parts per billion
(ppb, 10-9) or parts per trillion (ppt, 10-12), as the SPE process
facilitates sample enrichment and purification with ease.
3. Compound fractionation. SPE can effectively separate multiple
compounds from samples based on specific characteristics, such as
polarity or non-polarity.

Solid Phase Extraction Applications

1) Pharmaceutical industry, such as components extraction and research of

metabolite.
2) Environmental Analysis, such as extraction of metals in water.
3) Food testing, such as pesticide and veterinary drug residues.
4) Switch sample matrices to be more compatible with the target
chromatographic method.
5) Concentrate analytes (trace enrichment) for increased sensitivity.
6) Remove interferences that cause high background, misleading peaks, and/or
poor sensitivity during chromatographic analysis.
7) Protect the analytical column from contaminants. Automate the extraction
process.

Steps of Solid Phase Extraction Process

There are four major steps in the solid phase extraction process:
1) Conditioning: This involves priming the cartridge to prepare it for
accepting the target analyte.
2) Sample loading: The sample is loaded onto the cartridge at a suitable flow
rate, allowing the target analytes to be adsorbed or collected by the
adsorbent.
3) Washing: The cartridge is rinsed with a solvent or 100% H2O to remove
interferents that were not retained by the adsorbent. Additional rinses
 can be performed using solvents of increasing polarity (e.g., 5%, 10%,
20%, 100%) to remove compounds based on decreasing polarity.
4) Elution: This step involves releasing the analytes from the adsorbent,
typically using an elution solvent or a combination of solvents.

SPE、LLE、SLE Comparison
Statistically, sample preparation and pre-treatment account for 80% of the time
and costs involved in modern scientific analysis. Adopting simpler and faster
pre-treatment methodologies can provide significant benefits for all scientists.
In addition to SPE, there are several other commonly used sample pre-treatment
methods, such as Liquid-Liquid Extraction (LLE), Supported Liquid Extraction
(SLE), and Protein Precipitation (PPT)
 MODULE 2 :- Isolation And purification techniques
. General Isolation Techniques
1. Extraction strategies
2. Plant material extraction may be a crucial method within the isolation of
natural plant compounds and their Purification.
3. Plant matrices naturally area unit advanced, containing a large vary of
compounds that have varied Physical and
4. Chemical properties . It’s thus imperative to fastidiously, isolate from the
remainder of the plant, Matrices and
5. Make pure, compounds of interest in plants for his or her characterization.
There area unit many ways in which Extraction strategies
6. Can be categorised . During this chapter, they need been categorised
supported the temperatures They work below.
7. Low or temperature strategies
8. Cold extraction methodology
9. The methodology has been represented in literature . Briefly, dried plant
components samples (Cut, Crushed or processed
Chromatographic Technique
Introduction
People on all continents have used tons of to thousands of autochthonous plants
for treatment of Ailments Since prehistoric times. Several plants synthesize
substances that square measure helpful to the Maintenance of health in Humans
and alternative animals. These embody aromatic substances, most Of that square
measure phenols or their oxygen-Substituted derivatives like tannins . Sick
animal Tend to forage plants made in secondary metabolites, like tannins and
alkaloids. Since these Phytochemicals typically have antiviral, antibacterial drug,
antifungal and Anthelminthic properties, a Plausible case are often created for
self-medication by animals within the wild . In line with associate degree
Estimate of the planet Health Organization (WHO), regarding eightieth of the
planet population still Uses herbs and alternative ancient medicines for his or her
primary health care desires. Flavoring medication Products square measure
Dietary supplements that folks fancy improve their health and square measure
oversubscribed as Tablets, capsules, powders, teas, Extracts and recent or dried
plants. Herbals square measure historically Considered harmless and more and
more being Consumed by individuals while not prescription
II. CHROMATOGRAPHIC TECHNIQUES IN HERBAL DRUG
ANALYSIS
Chromatography represents the foremost versatile separation technique and
pronto obtainable. Chromatography Is outlined as technique of isolation and
identification of parts or Compounds or mixture of it’s into Individual parts by
exploitation stationary part and mobile Phase. Plant materials ar separated and
sublimate by exploitation varied chromatographical techniques. Herbal
medication may be a sophisticated system of mixtures. Thus, the strategies of
alternative for Identification of‘ biology drug’ ar principally meant to get a
Characteristic fingerprint of A specific plant that Represent the presence of a
selected quality process Chemical Constituents. Chemical fingerprints obtained
by chromatographical technique and particularly by Hyphenated action, ar
powerfully suggested for the aim of internal control of Herbal Medicines, since
they may represent fitly the “chemical integrities” of the Herbal medicines and
thus be used for authentication and Identification of the seasoner Products.
Skinny layer action (TLC) and High Performance skinny Layer Chromatography
(HPTLC)
Thin Layer Chromatography
Thin layer chromatography is simply known as TLC. It is one of the most
popular and simple chromatographic Technique used of separation of
compounds. In the phytochemical evaluation of herbal drugs
Thin layer action is solely called aid. It’s one amongst the foremost widespread
and easyChromatographic Technique used of separation of compounds. Within
the phytochemical analysis Of flavorer medication, aid is being used extensively
for the subsequent reasons:
1. It permits speedy analysis of flavorer extracts with minimum sample cleanup
demand,
2. It provides qualitative and semi quantitative info of the resolved compounds.
3. It permits the quantification of chemical constituents. Procedure victimisation
HPLC and GLC is additionally allotted in Specific cases.
In aid procedure, the info that may be recorded employing a high
performance aid (HPTLC) Scanner includes the recording, retardation issue (Rf)
values, the colour of the separated Bands, their absorption spectra, λ Georgia
home boy and Shoulder inflection/s of all the resolved bands. All Of these, at the
side of the profiles on derivatization with completely different reagents, represent
the aid Fingerprint profile Of the sample. The data therefore generated
incorporates a potential application in The identification of AN authentic drug, in
Excluding the adulterants and in maintaining the Quality and consistency of the
drug. Aid was the common methodology of alternative for flavorer analysis
Before instrumental action ways like Gc And HPLC were established. Even
Nowadays, aid continues to be oftentimes used for the analysis of flavorer
medicines since numerous Pharmacopoeias like yank flavorer assemblage (AHP)
, Chinese drug monographs And analysis, assemblage of the People’s Republic of
China etc. still use aid to produce First characteristic fingerprints of herbs .
Rather, aid is employed as a better methodology of initial Screening with a semi
quantitative analysis at the side of different chromatographical techniques.
In TLC fingerprinting, the data that can be recorded using a high
performance TLC (HPTLC) scanner includes the Chromatogram, retardation
factor (Rf) values, the color of the separated bands, their absorption spectra, λ
max and Shoulder inflection/s of all the resolved bands. All of these, together
with the profiles on derivatization with different reagents, represent the TLC
fingerprint profile Of the sample. The information so generated has a potential
application in the identification of an authentic drug, in Excluding the adulterants
and in maintaining the quality and consistency of the drug. TLC was the common
method of choice for herbal analysis before instrumental chromatography
methods like GC And HPLC were established. Even nowadays, TLC is still
frequently used for the analysis of herbal medicines since various
pharmacopoeias such As American Herbal Pharmacopoeia (AHP) , Chinese drug
monographs and analysis, Pharmacopoeia of the People’s Republic of China etc.
still use TLC to provide first characteristic fingerprints of herbs . Rather, TLC is
used as an easier method of initial screening with a semi quantitative evaluation
together with other
Advantages: -
 Simple method and cost of the equipment is low.
 Rapid technique.
 Detection is easy and not tedious. Needs less solvent, stationary phase and
time for every separation when compared to column chromatography.
Disadvantages: -
 It is time consuming process for the separation of compounds.
 It is expensive as higher quantities of solvents are required.
 The automated process becomes complicated and therefore costly.
 It has a low separation power.
Application: -
 Separation of mixtures of drugs of chemical or biological origin, plant extracts,
etc.
 Separation of carbohydrates, vitamins, antibiotics, proteins, alkaloids,
glycosides, etc
 Identification of related compounds in drugs.
 To detect the presence of foreign substances in drugs.
Column chromatography
It is a type of absorptions chromatography in which column is used for the
separation of components from mixture.
Column chromatography in chemistry is a chromatography method used to
isolate a single chemical compound from a mixture. Chromatography is able to
separate substances based on differential adsorption of compounds to the
adsorbent; compounds move through the column at different rates, allowing them
to be separated into fractions. The technique is widely applicable, as many
different adsorbents (normal phase, reversed phase, or otherwise) can be used
with a wide range of solvents. The technique can be used on scales from
micrograms up to kilograms. The main advantage of column chromatography is
the relatively low cost and disposability of the stationary phase used in the
process. The latter prevents crosscontamination and stationary phase degradation
due to recycling. Column chromatography can be done using gravity to move the
solvent, or using compressed gas to push the solvent through the column.
Principle: -
Column chromatography is based on the principle of separation by adsorption.It
is a preparative technique used to purify compounds depending upon their
polarity or hydrophobicity. In this technique a mixture of molecules is separated
based on their differential partitioning between a mobile phase and stationary
phase. Column chromatography is more advanced form of separation science
than paper and thin layer chromatography

Requirements:
Stationary Phase: Solid (Silica gel), 80-100 mesh or 100-200 mesh which has a
particle size of 60-200μ.
Mobile Phase: liquid (petroleum ether, Acetone, Ether, Toluene, Esters,
Chloroform, etc.

Advantages of Column chromatography: -

 Any type of mixture can be separated.
 Any quantity of the mixture can be separated (μg to mg of substance).
 Wider choice of mobile phase.
 Automation is possible.
 In preparative type, the sample can be separated and reused

Disadvantages of Column Chromatography: -

 Time consuming method.
 More amount of solvent is required.
 Automation makes the technique more complicated and Expensive.
Application: -
 Separation of mixture of compounds.
 Isolation of active constituents.
 Isolation of metabolites from biological fluids.
 Removal of impurities or purification process
 Estimation of drugs in formulation or crude extracts.

High Performance Thin Layer Chromatography

HPTLC is a modified and an advanced version of TLC technique (Daniel, 1991).
HPTLC- High Performance Thin Layer Chromatography is a sophisticated, a
powerful, reliable, efficient and automated form of TLC having the latest
technical developments for quality assessment and evaluation of botanical
materials.
The advancements include:
❖ Enhancement to the basic method of TLC to automate the different steps
(Automation in HPTLC is useful to avoid uncertainty in application size and
position, when the sample is applied manually to theTLC plate)
❖ Increase the resolution
❖ High sample throughput together with better analytical precision
❖ Accurate quantitative measurements with reduced consumption of mobile
phase per sample
VALUE OF HPTLC
HPTLC today is more than just plates and instruments. It is also a concept,
including a scientific basis, standardized methodology, and validated methods.
In HPTLC separations, normal phase silica gel is most frequently employed,
using adsorption chromatography, which benefits from the ability to separate
substances according to the type, number and position of functional groups.
There are also other modern stationary phases include reversed, amino-, diol-,
and cyano-bonded phases available for HPTLC.This makes setting up two
orthogonal separations possible. In HPTLC, the plates are precoated with a
stationary phase with a typical mean particle size of 5 μm. The plates give better
separations and reproducibility than normal precoated TLC plates (mean particle
size 12 μm) and they also allow more sensitive detection. Shorter developing
distances are required. The number of theoretical plates is in the 5000 range,
while for HPLC the range is 6–10000

USE OF PLANAR CHROMATOGRAPHY IN PLANT DRUG

STANDARDIZATION
 Here we have used High-Performance Thin-Layer Chromatography
coupled with computerized densitometry detection of plant constituents
separated on to thin layer.
 Planar chromatographic applications enabled us to identify in terms of
color and intensity of separated band
 A unique identity of zone can be established by scanning spectrum. This
spectrum acts as a unique identity of particular zone.
 Further a zone purity analysis can be performed indicating purity of
separated zone on to HPTLC.
 Using planar chromatography a unique Chemical Fingerprints / Profiles
may be generated and can also be documents to be used as future reference
in the performance of Quality Control Studies (QCS) or Quality Monitoring
Studies (QMS) of medicinal plants

Different steps of HPTLC fingerprinting

A. Chamber saturation:
Chromatographic chamber is filled with the solvent system 30
minutes prior to development of plate, to get uniform distribution of
solvent vapors in the chamber.
 B. Application of sample and standard:
Sample is spotted on the TLC plate with automatic applicator Linomat IV
attached with the compressed nitrogen gas cylinder and operated with
software winCATS.
➢ To take full advantage of the separation power and reproducibility of
HPTLC, precise automated positioning and volume dosage is mandatory.
➢ At the high end, autosamplers/applicators are available, e.g., from
CAMAG, that require no operator presence and can apply sample as spots
by contact transfer or as a rectangular band by a spray-on technique,
essentially using technology similar to that used in ink-jet printers.
➢ The spray-on technique permits sample application as bands or
rectangles with volumes as little as 0.5 μL to >50 μL. Prior to
chromatography, these rectangles are focused into narrow bands with a
solvent of high elution strength.

C. Selection of mobile phase:

➢ The solvent system is selected considering the nature of the components
to be separated like polar or non-polar and also solubility, affinity and
resolution, as the compound will follow the rule of ‘like dissolves like’.
➢ The desired mobile phase would provide the greatest solubility, while
providing affinity for the sample on the stationary phase.
➢ Highly polar solvents are water, methanol, ethanol, acetone, diethyl
ether, ethyl acetate, etc. while non-polar solvents are dichloromethane,
toluene, chloroform, cyclohexane, petroleum ether, hexane etc. ➢ The
developing solvent must be of high purity.
➢ The presence of small amounts of water or other impurities can produce
irreproducible chromatograms.

D. Chromatographic development and drying:

Sorbent layer thickness of TLC plate is 100µm and due to this smaller
particle size, separations are achieved at low distance route viz. at 3 to 5
cm. Because of this shorter migration distance, less amount of mobile phase
is required and the analysis time is greatly reduced. After development,
remove the plate and Dry in vacuum desiccator.
ADVANTAGES OF HPTLC
❖ Relatively inexpensive (equipment & materials needed).
❖ Efficiency of time (multiple samples in single run).
❖ Small amounts of solvents used.
❖ Provides characteristic fingerprint of the plant with or with out reference
standards.
❖ Appropriate for small or large companies.
❖ Multiple applications (verification of species, identification of
adulterants, basic qualitative assessment, detection of adulterants, product
characterization, quantitative evaluation, raw material or end product
screening).
❖ Minimum amount of training needed to use as an effective qualitative
assessment tool.

High-Performance Liquid Chromatography: -

It is advance technique of column chromatography. It is also known as
high pressure liquid chromatography (HPLC). It is as analytical technique
used for separation, identification and quantification of each component in
a mixture.
Principle: -
The main principle of separation is adsorption. The mixture of component
is mixed in the liquid solvent which administered into column under high
pressure up to 400 atmospheres.
 When a mixture of components is introduced into the column. Various
chemical and / or physical interactions take place between its sample
molecules and the particles of the column packing.
 The components which have more affinity towards the adsorbent travels
slower.
 The component which has less affinity toward the stationary phase travel
faster.
 Hence the component of mixture moves according to their affinities
towards the adsorbent.

Types of HPLC: -
Normal phase HPLC: - Stationary phase is polar in nature (silica gel),
while mobile phase is non-polar in nature (diethyl ether, chloroform). The
non-polar sample will elute out first and polar sample are retained on
column.
Reverse phase HPLC: -Stationary phase is non-polar, while mobile phase
polar in nature. Hence, the polar sample elute out more.
Size – exclusion HPLC: - The column is filled with precisely controlled
substrate. Based upon the difference in molecular sizes the separation of
components of mixture occurs.
Ion – exchange HPLC: - The surface of the stationary phase is ionically
charged which is opposite to the charge of the sample. The mobile phase
used is aqueous buffer which will control ionic strength and ph.

Instrumentation of HPLC: -
[1] HPLC consist of
1. solvent reservoirs
2. solvent degasser
3. gradient valve
4. mixing vessel
5. high pressure pump
6. sample injection loop
7. guard column
8. analytical column
9. detectors
10.waste collector.
Applications: -

In pharmaceutical application: -
 Identify active constituents in dosage forms.
 Evaluate of pharmaceutical product shelf- life.
 Dopamine in levodopa.

In Environment application: -
 Identify diphenhydramine in deposited sample.
 Pollutant bio monitoring.

In Clinical application: -
 Analysis of antibiotics.
 Detection of endogenous neuropeptides in brain extracellular fluids
 In Food and Flavour: -
 Ensuring soft drink consistency and quality.
Analysis of natural contamination e.g., Mercury & phenol in Sea water.
Control the drug stability and quality control