Introduction:

The nature has been a source of various therapeutic agents from the thousands of
years. Various modern drugs have been derived from natural sources, which having
an impressive therapeutic activities. Over the last century,a number of top selling
drugs have been developed from natural Products. The natural products includes
the plant-based preparations which is referred as" herbal". These herbal products
get a more acceptability from the people's due to it's more advantages and less side
effects. Recently, the herbal formulations are mostly preferred as compared to
synthetic one. Hence the demand of the herbal formulations in the market get
increased. So fulfill the requirements of the people's there is maintenance of
standard of the formulation is becoming a task during the formulation. Due to the
high demand in the market the various types of adulteration in done in the quality
of the crude drug or herbal formulations. So, to maintain the standard various
advanced herbal technology are derived, that helps in the identification,
authentication, isolation, purification and Standardization of the crude drugor
extract of herb. In all these processes the various conventional and modern
techniques are involved. For the identification of the herb the expert determination,
recognition, comparison, the use of key and similar devices like methods are used.
Recently, polyclave method,peak- a- boo or window card key and computerized
identification are used for the identification. The authentication of herb is a quality
assurance process that ensures the correct herbal species. For the authentication of
the herb or herbal products the macroscopic, microscopical and chromatograqphy
(TLC, HPLC) methods are used. DNA-based method, chemical fingerprinting are
the modern method of Authentication. For the preparation of plant formulations
Extraction is the first step to remove the active constituents from the crude Drugs.
For the extraction Maceration, Percolation, decoction, digestion, Immersion,steam
distillation, supercritical fluid extraction, counter current extraction, soxhlation,
Microwave assisted extraction etc. these are conventional and modern techniques
used for Extraction. After the extraction the isolation and purification process are
done by using various techniques to obtaining the pure compounds from the
extract. Then the standardardization by the various morphological, microscopical,
chemical physical, biological parameters the standard, quality, purity is confirmed.
MODULE 1 : IDENTIFICATION,AUTHENTICATION AND EXTRACTION OF HERB
Introduction to herbal technology
Herb is the plant material which is derived from parts of plant which might be
fragmented,entire or in powered form.[1] Because of their therapeutic importance
considered as a source of nutrition. [2] Herbal drug technology is helps in the
converting botanical materials into medicines in this Standardization and quality
maintenance with modern scientific techniques and traditional knowledge is most
important.[3] In herbal medicines the interest of people's is increased significantly
in all the countries. The demand of the herbal medicines is enhanced, hence there
is need to adoption of proper systemic methods for the identification,
authentication, extraction,isolation and purification.It also includes various
standardization techniques to ensure quality, purity, safety and potency and to
develop modern methods for their quality control in order to get the maximum
benefit from these herbal medicines. [4] As per the world Health Organization
(WHO) Herbal medicines are all those medicines which are obtained from the
plant, parts of plant and herbal preparations .[5]in the last decade there have been
significant advances in herbal medicine technology. The traditional drug system
dating back to ancient civilizations could demonstrate the safety of herbal
medicines. It is time to make decisions about the safety and efficacy of herbal
medicines. The legal status and licensing mechanism for herbal medicinal products
also varies from country to country. The World Health Organization (WHO) has
established specific guidelines for assessing the safety, efficacy and quality of
herbal medicinal products as a prerequisite for global harmonization. The progress
of science and technology penetrates far into phototherapeutics products.[6]
Different methods of identification of plant
Identification of experimental material is one of the most fundamental
requirements of any field in the life sciences. The discovery and naming of living
organisms (any life form including plants, animals or microbes) has attracted much
attention throughout history. Identification is "the process of assigning a specimen
to a (pre-existing) taxon". Plant species have been considered the basic unit of
biodiversity and it is the level at which most evolutionary studies have focused.
The want for plant species identity is each various and widespread, and consists of
programs for plant breeding, agricultural seed industry, meals processing,
conservation biology, forensic evaluation and lots of different components of plant
science. Traditionally, identity of plant species has relied closely on morphological
characters.[7]

❖ Methods of identification of plant:

1. Expert determination
2. Recognition
3. Comparison
4. Use of Keys and Similar Devices (Synopses, Outlines, etc.)
1.Expert Determination
The best method of Identification is expert determination of reliability or accuracy.
In general, experts have produced treatments (monographs, reviews, synopses) of
the group in question, and the most recent floras or handbooks are likely to
contain the expert's taxa concepts. This method requires more time of the expert
for the identification. It causes the delay in identification of plant.
2. Recognition
It approaches skilled determination in reliability. This can be supported extensive,
past expertise of the symbol with the plant cluster in question.
3.Comparison
A third method is to compare an unknown with named specimens, photographs,
illustrations, or descriptions. Although this is a reliable method, it can be time-
consuming or practically impossible due to the lack of suitable reference materials.
4.Use Keys and Similar Devices (Synopses, Outlines, etc.)
This is with the aid of using some distance the Maximum extensively used
technique and does now no longer require the time, materials, or revel in
concerned in Assessment and recognition.[8]
Recently, the polyclave identification, Peck-a-boo or Window Card Key, and
computerized identification are investigated as a modern method of identification.
[9]

 Authentication of plant
Medicinal plants have been used for centuries around the world to maintain
health and treat diseases, especially chronic ones. However, for reasons of safety
and effectiveness,
adulteration and the use of fake materials as substitutes have become a major
concern for users and industry. Therefore, the authentication of medicinal plants is
of paramount importance. Morphological, anatomical, chemical and DNA markers
solve the problem by distinguishing real material from adulterants, substitutes and
counterfeit drugs.[10] Methods of Authentication
1.Macroscopic examination
It involves the comparison of morphological characters which might be visible with
the bare eye or below low magnification with descriptions of the plant or botanical
drug in Floras or Monographs. Characters consisting of size, form and coloration of
leaves (or leaf fragments), flora or Culmination are usually utilized in macroscopic
identification.
2.Microscopic examination
Microscopic examination focuses on anatomical structures in plant material that
are only visible with the help of a microscope. Features such as the shape and
structure of the trichomes (hairs), the arrangement of the stomata in the
epidermis, the presence or absence of compounds such as mucus, starch or lignin,
or the presence of tissues with characteristic cells could be used for microscopic
identification of herbal medicinal products.
3.Chromatography
Chromatography is the separation of chemical compounds in a mixture. There are
several chromatographic techniques, but they are all based on the same basic
principles. Thin layer chromatography (TLC) is commonly used to authenticate
herbs, and most herbal pharmacopoeia monographs include a TLC identification
test. TLC separates mixtures of compounds to leave a "fingerprint" of the
separated compounds on a silica gel-coated plate.This fingerprint may be as
compared with that of an true pattern or natural reference compounds. High-
overall performance liquid chromatography (HPLC) is every other form of
chromatography broadly used in the authentication and evaluation of natural
substances. Yet every other type, Gas chromatography, is used specifically for
crucial oils and fatty acids. [11]
 The following Institutes are involved in the authentication of herbs
Names of institutes
1. Central council for research in Ayurveda and Siddha (CCRAS)
2. Central Council for Research in Unani Medicine (CCRUM)
3. Central Council for Research in Homeopathy (CCRH)
4. Central Council for Research in Yoga and Neuropathy (CCRYN)
5. Central Council for Indian Medicines (CCIM)
6. Central Council for Homeopathy (CCH)

 Laboratories:
1. Pharmacopoeial Laboratory for Indian Medicines (PLIM)
2. Homeopathy Pharmacopia Laboratory (HPL)

 National institutes
1. National Institute of Homeopathy (NIH)
2. National Institute of Ayurveda (N!A)
3. National Institute of Unani Medicine (NIUM)
4. National Institute of Neuropathy(NIN)
5. National Institute of Siddha (NIS)
6. Institute of Post-graduate Training and Research in Ayurveda (IPGTRA)
7. Rashtriya Ayurved Vidyapeeth (RAV)
8. Morarji Desai National Institute of Yoga (MDNIY) [12]
 Different Extraction methods
Extraction, as the term is used in pharmacy, involves the separation of medicinally
active
parts of plant or animal tissues from inactive or inert components through the use
of selective solvents in standard extraction processes. The products thus derived
from plants are relatively impure liquids, semi-solids or powders intended solely
for oral or topical use. These include classes of preparations known as decoctions,
infusions, liquid extracts, tinctures, pillar (semi-solid) extracts, and powdered
extracts. Such preparations were popularly called galenic, in honor of Galen, the
Greek physician of the second century.drugs are to achieve the therapeutically
favoured component and to dispose of the Inert cloth with the aid of using remedy
with a selective solvent referred to as menstruum. The extract therefore received
can be prepared to be used as a medicinal agent in the Shape of tinctures and fluid
extracts, it is able to be similarly processed to be incorporated in any dosage shape
together with pills or capsules, or it is able to be fractionated to isolate man or
woman chemical entities together with ajmalicine, hyoscine And vincristine, that
are modem tablets. Thus, standardization of extraction Methods contributes
significantly to the final first-rate of the natural drug.Extraction is the process in
which from the insoluble substance the seperation of soluble constituents are
occurred.[13]
Factor affecting on
extraction:

Fig 2: Factor affecting on extraction efficiency

 Various methods of extraction:
• Maceration
• Infusion.
• Ultrasonic extraction
 Decoction.
• Digestion
• Percolation
• soxhlation (Hot continuous extraction)
 Supercritical fluid extraction
• Counter current extraction
• Accelerated solvent extraction
 Maceration
In maceration, the powered drug is placed with a solvent in a stoppered container
and allow to stand at room temperature for least 3 days with frequent agitation.
Then, this mixture is strained, Marc is pressed and the liquid is clarified by
filtration.
 Infusion
Fresh infusions are prepared by briefly soaking the raw drug in cold or boiling
water. These are diluted solutions of the easily soluble components of raw drugs
 Digestion
The digestion is like a maceration,only difference is that in digestion gentle heat is
used during the extraction process. Due to the heat solvent efficiency to menstrum
is enhanced.
 Decoction
In this system, the crude drug is boiled in a specified extent of water for a defined
time; it's far then cooled and strained or filtered. This Process is appropriate for
extracting water-soluble, heat-strong constituents. This system is generally utilized
in training of Ayurvedic extracts called "quath" or "kawath". The beginning ratio of
crude drug to water is fixed, e.g.1:four or 1:16; the extent is then delivered right all
the way down to one-fourth its authentic volume with the aid of using boiling all
through the extraction process. Then, the extract is filtered.
 Percolation
Percolation extraction is a traditional extraction technique used withinside the
processing of traditional Chinese medicines. After medicinal cloth powder is
positioned in a percolation tank, the Extraction solvent is constantly added, and
percolation extract is collected Simultaneously. Extraction that includes passing a
liquid (solvent) thru a plant. Percolation is an extractive method this is performed
at room temperature and that actually means "pass A liquid thru a strong cloth
drop via way of means of drop."Coarse plant particles should be loaded into a
percolator and immersed in a suitable solvent for 24 to 48 hours, then collect the
percolates at the bottom of the percolator.

Fig.3: Percolation
During the percolation process, new solvent must constantly be added to the top
of the percolation apparatus. It is more efficient than the immersion method due
to the difference in concentration maintained throughout the process. However,
this procedure is complex and consumes a lot of solvent and Time.
 Hot continues extraction (soxhlation)
The process of transferring the partially soluble components of a solid to the liquid
phase using a Soxhlet extractor. The principle of solvent extraction is extremely
simple. The aim is to dissolve (solvate) a target molecule or group of compounds
(solutes) with a liquid (solvent) and remove it from solid plant material. The
solvent is then separated from the solute to concentrate the solute.A Soxhlet
extractor is a bit of laboratory equipment invented in 1879 with the aid of using
Franz von Soxhlet. It Changed into at the beginning designed for the extraction of a
lipid from a stable material. Typically, Soxhlet Extraction is used whilst the
preferred compound has a restricted solubility in a solvent, and the impurity is
insoluble in that solvent. It permits for unmonitored and unmanaged operation
while efficaciously recycling a small quantity of solvent to dissolve a bigger
quantity of material. This is a continuous process of extraction with a hot organic
solvent. The powdered plant material is taken in a thimble which is placed in the
Soxhlet extractor. The extractor, which has a siphoning system, is fitted on top of a
round bottom flask. A condenser is fitted at the top of the extractor. Enough
quantity of the extracting solvent is poured in to the flask placed on a heating
mantle. On heating the solvent evaporates, rises to the condenser, where
condenses and drains back to the extractor holding the thimble with the plant
material. When the extractor becomes full with the hot solvent. The solvent
siphons down to the flask along with the extracted constituents. The recycling of
the evaporated solvent is allowed to continue until the extraction is complete.
 Fig.4:Soxhlet apparatus
 Counter current extraction
In Counter Current Extraction (CCE), wet raw material is pulverized by toothed disc
disintegrators to create a fine slurry. In this process, the material to be extracted is
moved in one direction (usually in the form of a fine slurry) in a cylindrical
extractor where it comes into contact with the extraction solvent. The more the
source material is agitated, the more concentrated the extract becomes.
Therefore, complete extraction is possible if solvent and material amounts and
their flow rates are optimized. The process is highly efficient, takes little time and
poses no risk of high temperatures. Eventually, a sufficiently concentrated extract
comes out of one end of the extractor, while the pomace falls out of the other end
(virtually free of visible solvent).
 Ultrasonic Extraction
The process entails using ultrasound with frequencies starting from 20 kHz to 2000
kHz; this will increase the permeability of cell partitions and produces cavitation.
Although the method is beneficial in a few cases, like extraction of rauwolfia root,
its large-scale software is restrained due to the better costs. One disadvantage of
the process is the occasional but regarded deleterious impact of ultrasound energy
(greater than 20 kHz) at the active parts of medicinal flora through formation of
unfastened radicals and therefore unwanted modifications within side the drug
molecules.
 Supercritical fluid extraction
In the supercritical state, the supercritical fluid comes into contact with plant
tissue. By controlling different temperatures, pressures, and different types and
levels of entrainers, the supercritical fluid can selectively extract components with
different polarities, boiling points, and molecular weights sequentially. This
process is called the Supercritical Fluid Extraction (SFE) process. The critical point
of a pure substance is defined as the highest temperature and pressure at which
the substance can exist in liquid-vapor equilibrium. At temperatures and pressures
above this point, a single homogeneous fluid known as the supercritical (SF) fluid
forms. SF is heavy like a liquid while having a low viscosity like a gas. SF has a fairly
large diffusion coefficient and could solve many compounds well. Various materials
can be used as the SF,For example- Ammonia, ethane, difluorodichloromethane,
heptane, etc., the most common SF being CO2. The critical temperature of CO2 (Tc
= 31.26 °C) is close to room temperature and the critical pressure (Pc = 7.2 MPa) is
not too high. CO2 also has a number of other benefits, such as: Non-toxic,
odorless, non-flammable, chemical stability and low cost, which has made it the
most commonly used solvent in SFE.Supercritical fluid extraction (SFE) is a way
wherein supercritical fluid as an extraction medium is introduced to materials
containing goal components, and extraction is completed primarily based totally
on variations in solubility. In particular, using supercritical carbon dioxide as an
extraction medium gives
many benefits in a lot of fields, which include brief extraction times, simplified
operation, and advanced extraction performance in comparison with the natural
solvent extraction method. Also, solvent elimination following extraction is lots
easier, permitting extra correct thing concentrations to be determined.[13]
 Microwave-assisted extraction
Microwave Assisted Extraction (MAE) is a process that uses microwave energy to
heat solvent in contact with a sample to break down analytes from the sample
matrix into the solvent. The ability to rapidly heat the sample-solvent mixture is
inherent in MAE and is the main advantage of this technique. By using closed
vessels, the extraction can be performed at elevated temperatures, which
accelerates the mass transfer of target compounds from the sample matrix. A
typical extraction procedure takes 15 to 30 minutes and uses small solvent
volumes in the range of 10 to 30 mL. These volumes are approximately 10 times
smaller than the volumes used in conventional extraction techniques.In addition,
the sample throughput is increased because several samples can be taken at the
same time. In most cases, analyte recovery and reproducibility are improved
compared to traditional techniques, as applications.[16] The high and fast
extraction yield with lower solvent consumption and the protection of the
thermolabile components are some of the attractive properties of this new and
promising microwave-assisted extraction (MAE) technique. A brief theoretical
background of microwave heating and the basic principles of using microwave
energy for extraction have been presented for better understanding.[17]
MODULE 2: ISOLATION AND PURIFICATION TECHNIQUES
 General Isolation techniques
The isolation and purification of bioactive compounds from plants is a technique
that has seen new developments in recent years [18, 19]. This modern technique
offers on the one hand the possibility to keep pace with the development and
availability of many advanced bioassays and on the other hand offers precise
isolation, separation and purification techniques. The aim of the search for
bioactive compounds is to find a suitable method that can detect bioactivity in the
starting material, such as antioxidants, antibacterial agents or cytostatics,
combined with simplicity, specificity and speed. [20] In vitro techniques are
generally greater suited than in vivo assays due to the fact animal experiments are
expensive, take greater time, and are at risk of moral controversies. There are a
few elements that make it not possible to discover very last strategies or protocols
to isolate and symbolize positive bioactive molecules. This might be because of
one of a kind parts (tissues) in a plant, a lot of to be able to produce pretty one of
a kind compounds, further to the various chemical systems and physicochemical
homes of the bioactive phytochemicals.[21]
Both the choice and the gathering of plant substances are considered number one
steps to isolate and signify a bioactive phytochemical. The subsequent step
includes a retrieval of ethno-botanical facts to parent viable bioactive molecules.
Extracts can then be made with diverse solvents to isolate and purify the lively
compounds which can be chargeable for the bioactivity.Column chromatographic
strategies may be used for the isolation and purification of the bioactive
compounds. Developed contraptions which include High Pressure Liquid
Chromatography (HPLC) accelerate the method of purification of the bioactive
molecule. Different sorts of spectroscopic strategies like UV-visible, Infrared (IR),
Nuclear Magnetic Resonance (NMR), and mass spectroscopy can identify the
purified compounds.[22]
 Chromatographic techniques
It is a common practice in isolation of these bioactive compounds that a number
of Different separation techniques such as TLC, column chromatography, flash
chromatography, Sephadex chromatography and HPLC, should be used to obtain
pure compounds.
 Thin layer chromatography (TLC)
TLC is a simple, fast and inexpensive method that gives the researcher a quick
answer as to how many components are in a mixture. TLC is also used to support
the identity of a compound in a mixture when comparing the Rf of a compound to
the Rf of a known compound. Additional tests include spraying phytosanitary
products that cause color changes corresponding to the phytochemicals present in
a plant extract; or viewing the plate under ultraviolet light. This has also been used
to confirm the purity and identity of isolated compounds.[23]
 High Performance Liquid Chromatography (HPLC)
For the isolation of natural compounds the High Performance liquid
chromatography ( HPLC) is a versatile and widely used techniques. [24]At present,
this technique is gaining popularity among various analysis techniques as it is the
first choice for fingerprinting studies for quality control of herbal plant.[25] Natural
products are often isolated after evaluating a relatively crude extract in a biological
assay to fully characterize the active ingredient. The biologically active moiety is
often only present as a minor component in the extract, and the resolution of
HPLC is ideal for the rapid processing of such multicomponent samples on both
analytical and preparative scales. Many benchtop HPLC instruments are now
modular in design and include a solvent supply pump, a sample introduction
device such as an autosampler or manual injection valve, an analytical column, a
guard column, a detector, and a recorder or printer. Chemical separations may be
finished the usage of HPLC via way of means of utilising the truth that sure
compounds have different migration prices given a specific column and cell
segment. The volume or diploma of separation is commonly decided via way of
means of the desire of desk bound segment and cell segment. Generally the
identity and separation of phytochemicals may be finished he usage of isocratic
system (the usage of single unchanging cell segment system). Gradient elution
wherein the share of organic solvent to water is altered with time can be ideal if a
couple of pattern issue is being studied and vary from each other.HPLC purification
of the compound of interest is the process of separating or extracting the target
compound from other (possibly structurally related) compounds or impurities.
Each compound must show a characteristic peak under certain chromatographic
conditions. Depending on what needs to be separated and how closely related the
samples are, the chromatographer can select conditions such as the right mobile
phase, flow rate, detectors and columns to achieve optimal separation.[26]
 Column Chromatography
Column chromatography in chemistry is a chromatography technique used to
isolate a Single chemical compound from a mixture. Chromatography is capable of
separate materials primarily based totally on differential adsorption of compounds
to the adsorbent; compounds pass through the column at exceptional rates, letting
them be separated into fractions. The approach is broadly applicable, as many
exceptional adsorbents (normal phase, reversed phase, or otherwise) may be used
with a extensive variety of solvents. The approach may be used on scales from
micrograms as much as kilograms. The fundamental gain of column
chromatography is the extraordinarily low price and disposability of the stationary
phase used withinside the process. The latter prevents cross contamination and
stationary phase degradation because of recycling. Column chromatography may
be performed the usage of gravity to transport the solvent, or the usage of
compressed fueloline to push the solvent through the column.[27]
 High Performance Thin Layer Chromatography (HPTLC)
High Performance Thin Layer Chromatography (HPTLC) technique is a sophisticated
and automated form of the thin-layer chromatography (TLC) with better and
advanced separation efficiency and detection limits and is often an excellent
alternative to GC and HPLC. Applications of HPTLC include phytochemical and
biomedical analysis, herbal drug quantification, active ingredient quantification,
fingerprinting of formulations, and check for adulterants in the formulations.
HPTLC is useful in detecting chemicals of forensic concern. Various advance
techniques in reference to HPTLC like hyphenations in HPTLC- MS, HPTLC-FTIR and
HPTLC-Scanning Diode Laser have made HPTLC a power analytical tool in the field
of analysis. Experts are of the opinion that HPTLC future to combinatorial
approach and the utilization of instrumental HPTLC toward the analysis of drug
formulations, bulk drugs, and natural products will increase in the future.[28]
 Purification techniques for isolated Phytoconstituents
Phytochemical separation is a process in which plant extract components or active
parts are individually isolated and purified into monomeric compounds by physical
and chemical methods. Classic isolation methods such as solvent extraction,
precipitation, crystallization, fractional distillation, salting and dialysis are still
commonly used today. On the other hand, modern separation technologies such
as column chromatography, high performance liquid chromatography,
ultrafiltration and high performance liquid chromatography, droplet
countercurrent chromatography also play an important role in the separation of
phytochemicals. [29]
MODULE 3: METHODS OF STANDARDIZATION OF HERBAL DRUG
 Standardization:
The standardization of herbal medicines is the process of prescribing a set of
standards or inherent characteristics, constant parameters, final qualitative and
quantitative values that include a guarantee of quality, efficacy, safety and
reproducibility. It is the process of developing arid agreeing on technical standards.
The specific standards are developed through experimentation and observation
that would lead to the prescribing process for a range of properties exhibited by
the particular Drugs. Therefore, standardization is a tool in the quality control
process. [30] American Herbal Product association defines: "Standardization refers
to the body of information and control necessary to product material of
reasonable consistency. This achieved through minimizing the Inherent variation of
natural product composition through quality assurance practices applied to
agricultural and manufacturing processes.[31]
 Need of standardization
In the global perspective, there is a shift towards the use of herbal medicines as
the dangers and shortcomings of modern medicine become more
apparent.Regulatory agencies strictly follow various quality standards prescribed
for raw materials and finished products in pharmacopoeias, formulations and
manufacturing operations through statutory good manufacturing practices to
maintain purity, safety, potency and effectiveness. These procedures would
logically apply to all types of medicines, whether they belong to the modern
medical system or to one of the traditional systems. The popularity of the herbal
products get increased in all over the world, one of the barriers
to their acceptance is the lack of a standard quality control profile. The quality of
the herbal medicinal product, i.e. the profile of the components in the final
product, has an impact on ineffectiveness and safety. However, due to the complex
nature and inherent variability of herbal drug components, it is difficult to
establish quality control parameters, although it is hoped that modern analytical
techniques will help circumvent this problem. In addition, the components
responsible for the claimed therapeutic effects are often unknown or only partially
explained. This is further complicated by using the combination of herbal
ingredients as used in traditional practice. It's common to have up to five different
herbal ingredients in a single product. Therefore, batch-to-batch variation begins
with the collection of the raw material itself, since there is no reference standard
to identify it. These fluctuations multiply during storage and further processing.
Therefore, standardization for medicinal and herbal products should cover the
entire field of study, from medicinal plant cultivation to clinical use.Plant materials
and natural remedies derived from them constitute big portion of world
marketplace and in this repect the world over recognized guidelines for their
quality evaluation and quality control are necessary.[32]
 Standardization of single drug or compound Formulation
The natural formulation in general may be standardized as to formulate the
medicament the use of raw material collected from distinctive localities and a
comparative chemical efficacy of various batches of components are to be
observed. The preparations with higher scientific efficacy are to be selected. All
the ordinary physical, chemical and pharmacological parameters are checked for
all of the batches as a way to pick the very last finished product and to validate the
entire production process. Standardization is an critical thing for preserving and
assessing the fine and protection of the polyherbal components as those are
mixtures of multiple herb to gain the choice healing effect.[33] minimizes batch to
batch variation; assure safety, quality and efficacy of compound Formulation.[34]
Standardization of herbal formulation requires implementation of Good
Manufacturing Practices.In addition,study of various parameters such as
pharmacodynamics, pharmacokinetics, dosage, stability, self-life, toxicity
evaluation, chemical profiling of the herbal formulations is considered essential.
Heavy metals contaminations, Good Agricultural Practices (GAP) in herbal drug
standardization are also equally important.[35]

 WHO guidelines for herbal drug Standardization

 Reference to the identity of the drug. Botanical Evaluation- sensory characters,
foreign organic matter, histochemical evaluation, quantitative measurements
etc.
 Refers to the physicochemical character of the Drug. Physical and chemical
identity, Chromatographic fingerprints, ash values, Extractive values, moisture
content, volatile oil and Alkaloidal assays, quantitative estimation protocols Etc.
 A reference to the pharmacological parameters, Biological activity profiles,
bitterness values, Hemolytic index, astringency, swelling factor, Foaming index
etc.
 Toxicity details- pesticide residues, heavy metals, Microbial contamination like
total viable count, Pathogens like E. coli, Salmonella, P. aeruginosa, S. aureus,
Enterobacteria etc.
 Microbial contamination.
 Radioactive contamination.
 Also
Quality control of crude drugs material, plant preparations and finished products.
Stability assessment and shelf life
Safety assessment; documentation of safety based on experience or toxicological
studies. Assessment of efficacy by ethno- medical information and biological
activity evaluations. [32]
 Critical factor affecting on quality control of herbal drug
 Microscopic evaluation
Quality control of herbal drugs has traditionally been based on the appearance
and today microscopic Evaluation is indispensable in the initial identification of
herbs, as well as, in identifying small fragments of crude or powdered herbs, and
detection of foreign matterand adulterants. A primary visual evaluation, which
seldom needs more than a simple magnifying lens, can be used to ensure that the
plant is of the required species, and that the right part of the plant is being used.
At other times, microscopic analysis is needed to determine the correct species
and/or that the correct part of the species is present. For instance, pollen
morphology may be used in the case of flowers to identify the species, and the
presence of certain microscopic structures such as leaf stomata can be used to
identify the plant part used. Although this may seem obvious, it is of prime
Importance, especially when different parts of the same plant are to be used for
different treatments. Stinging Nettle is a classic example where the aerial Parts are
used to treat rheumatism, while the roots are Applied for benign prostate
hyperplasia.
 Foreign matter
Herbal drugs should be made from the stated part of the Plant and be devoid of
other parts of the same plant or other plants. They should be entirely free from
moulds or Insect including excreta and visible contaminant such as sand stones,
poisonous and harmful foreign matter and chemical residues. Anima! matters such
as insects and "invisible" microbial contaminants, which can produce toxins, are
also among the potential contaminants of herbal medicines .Macroscopic
examination can easily be employed to determine the presence of foreign matter,
Although, microscopy is indispensable in certain special cases (for example, starch
deliberately added to "dilute" the plant material). Furthermore, when foreign
matter consists, for example, of a chemical residue, TLC is often needed to detect
the contaminants.
 Ash content
To determine ash content, the plant material is burnt and the residual ash is
measured as total and acid-insoluble ash. Total ash is the measure of the total
amount of material left after burning and includes ash derived from the part of the
plant itself and acid-insoluble ash. The latter is the residue obtained after boiling
the total ash with dilute hydrochloric acid, and burning the remaining insoluble
matter. The second procedure measures the Amount of silica present, especially in
the form of sand and siliceous earth.
 Heavy metals
Contamination by toxic metals can either be accidental or intentional.
Contamination by heavy metals such as Mercury, lead, copper, cadmium, and
arsenic in herbal remedies can be attributed to many causes, including
environmental pollution, and can pose clinically relevant dangers for the health of
the user and should therefore be limited. The potential intake of the toxic metal
can be estimated on the basis of the level of its presence in the product and the
recommended or estimated dosage of the product. This potential exposure can
then be put into a Toxicological perspective by comparison with the so called
Provisional Tolerable Weekly Intake values (PTWI) For toxic metals, which have
been established by the Food and Agriculture Organization of the World Health
Organization (FAO-WHO) .A simple, straightforward determination of heavy metals
can be found in many pharmacopoeias and is based on colour reactions with
special reagents such as Thioacetamide or diethyldithiocarbamate, and the
amount present is estimated by comparison with a standard. Instrumental
analyses have to be employed when the metals are present in trace quatities, in
admixture, or when the analyses have to be quantitative. Generally, the main
methods commonly used are atomic absorption spectrophotometry (AAS),
Inductively coupled plasma (ICP) and neutron activation analysis.
 Microbial contaminants and aflatoxins
Medicinal plants may be associated with a broad variety of microbial
contaminants, represented by bacteria, fungi, and viruses. Inevitably, this
microbiological background depends on several environmental factors and exerts
an important impact on the overall quality of herbal products and preparations.
Risk assessment of the microbial load of medicinal plants has therefore become an
important subject in the establishment of modern Hazard Analysis and Critical
Control Point (HACCP) schemes. Herbal drugs normally carry a number of bacteria
and Molds, often originating in the soil. Poor methods of harrvesting, cleaning,
drying, handling, and storage may also cause additional contamination, as may be
the case with Escherichia coli or Salmonella spp. While a large range of bacteria
and fungi are from naturally occurring microflora, aerobic spore-forming bacteria
that frequently predominate. Laboratory procedures investigating
microbialcontaminations are laid down in the well-known Pharmacopeias, as well
as, in the WHO guidelines. Limit values can also be found in the sources
mentioned. Generally, a complete procedure consists of determining the total
aerobic microbial count, the total fungal count, and the total Entero-bacteriaceae
count, together with tests for the presence of Escherichia coli, Staphylococcus
aureus, Shigella, and Pseudomonas aeruginosa and Salmonella spp. The European
Pharmacopoeia also specifies that E. coli and Salmonella Spp.Should be absent
from herbal preparations. Materials of vegetable origin tend to show much higher
levels of microbial contamination than synthetic products and the requirements
for microbial contamination in the European Pharmacopoeia allow higher levels of
microbial contamination in herbal remedies than in synthetic parmaceuticals. The
allowed contamination level may also depend on the method of processing of the
drug. For example higher contamination levels are permitted if the final herbal
preparation involves boiling with water. The presence of fungi should be carefully
investigated and/or monitored, since some common species produce toxins,
Especially aflatoxins. Aflatoxins in herbal drugs can be dangerous to health even if
they are absorbed in minute amounts.
 Pesticide residues
Even though there are no serious reports of toxicity due to the presence of
pesticides and fumigants, it is important that herbs and herbal products are free of
these chemicals or at least are controlled for the absence of unsafe levels.
 Radioactive contamination
Dangerous contamination, however, may be the consequence of a nuclear
accident. The WHO, in close cooperation with several other international
organizations, has developed guidelines in the event of a widespread
contamination by radionuclides resulting from major nuclear accidents. These
publications emphasize that the health risk, in general, due to radioactive
contamination from naturally occurring radio nuclides is not a real concern, but
those arising from major nuclear accidents such as the nuclear accident in
Chernobyl and Fukushima may be serious and depend on the specific radionuclide,
the level of contamination, and the quantity of the contaminant consumed.[36]
 Standardization and quality control of herbal drugs : Parameters
According to WHO, standardization and quality control of herbals is the process
involved in the physicochemical evaluation of crude drug covering aspects, such as
selection and handling of crude material, safety, efficacy and stability assessment
of finished product, documentation of safety and risk based on experience,
provision of product information to consumer and product promotion. Attention is
normally paid to such quality indices
such as:
 Morphology and organoleptic evaluation
It includes the morphological characters like colour, odor, taste, shape,size
etc.Detail characteristics includes fractures, texture and venation.
 Microscopic and histological evaluation
These are important in both whole drug and powdered drug. It mainly includes
study of characteristics like parenchyma, trichomes, calcium oxalate crystals,
vascular bundle arrangements, stomata and fibres. Microscopic determination
such as vein islet number, stomatal index, stomatal number, vein termination
number.
 Physical evaluation
It includes the various physical parameters like moisture content, solubility,
viscosity, refractive index, melting point, optical rotation, ash values, extractives
and foreign organic matter.
 Chemical evaluation
Qualitative chemical evaluation
Qualitative tests comprise of various chemical tests to identify the nature of
compounds present in the crude drugs.
1. Test for Alkaloids: Mayer's test, Dragendroff's test, Hager's test, Wagner's Test
2. Glycosides and sugars: Bontrager's test, Molisch's test, killer killiani test,
legal test drugs
3. Test for Phytosterols: Liebermann's and Burchard tests.
4. Test for Tannins and Phenols: Ferric chloride test.
5. Test for protein and amino acid: Millen's test, Ninhydrin test, Biuret test
Etc.
6. Test for gums and mucilages: Swelling index

Quantitative chemical evaluation

It includes chemical assays and chromatographic methods which are used to
quantify the chemical compounds present in the crude drug.
Chromatographic Techniques The Chromatographic techniques are the new and
most common methods. Used to separate, identify and quantify the plant
constituents. It consists of various methods which are as follows:
1. TLC (Thin Layer Chromatography)
2. HPLC (High Performance liquid chromatography)
3. COLUMN CHROMATOGRAPHY
4. HPTLC (High Performance Thin Layer Chromatography)
5. GAS-LIQUID CHROMATOGRAPHY
 Biological parameters
It contsist of following evaluation methods
 Bitterness value
 Hemolytic activity
 Swelling index
 Foaming index
 Pesticides residue
 Heavy metals
 Microorganisms
 Aflatoxins
 Radioactive substances
  Toxicological studies
This helps to determine the pesticide residues, potentially toxic elements, safety
studies in animals like LD50 and Microbial assay to establish the absence or
presence of potentially harmful microorganisms. [37]

References
1.Worldth Organization (WHO). (2011). The world Traditional medicines situation.
In: Traditional Medicines: Global Situation, Issues and Challenges, 3, p. 1-14.
Geneva, Switzerland: World Health Organization.
2. Yudharajsmine Priyadarshini, R., Ashok Naik, E., Shankar, M., Sowjanya, R. and
Sireesha, B. (2016). Importance and uses of medicinal plant-an overview.
International Journal of Preclinical and Pharmaceutical Research, 7(2), 67-73.
3.Kalpana Joshi*, Preeti Chavan, Dnyaneshwar Warude and Bhushan
Patwardhan.,Molecular markers in herbal drug technology, CURRENT SCIENCE,
VOL. 57. NO. 2.25 JULY 2004. 4.Sumathi A*, Senthamarai. R ;A Review on Herbal
Medicines and Herbal Drug Technology Asian Journal of Pharmaceutical and
Health Sciences, 2015,5,1,1172-1178.Published: February 2015. 5.Saikat Sena Raja
Chakrabortya Biplab Deb., Challenges and opportunities in the advancement of
herbal medicine: India's position and role in a global context, Journal of Herbal
Medicine Volume 1, Issues 3-4, December 2011, Pages 67-75.
6.Sumathi A*, Senthamarai. R., A Review on Herbal Medicines and Herbal Drug
Technology,Asian Journal of Pharmaceutical and Health Sciences, 2015,5,1,1172-
1178.
7.Nadia Haider., IDENTIFICATION OF PLANT SPECIES USING TRADITIONAL AND
MOLECULAR-BASED METHODS., In: Wild Plants: Identification, Uses and
Conservation Editor: Ryan E. Davis, pp. ISBN 978-1- 61209-966-8 ©2011 Nova
Science Publishers, Inc.
8.Gavali pratiksha suresh, garute komal somnath, Advance herbal
technology,©2022 IJCRT | Volume 10, Issue 1 January 2022 | ISSN: 2320-2882
9.Jana Wäldchen, Patrick Mäder, Plant species identification using computer vision
techniques: A systematic literature review., Archives of Computational Methods in
Engineering 25 (2), 507-543, 2018
10.Showkat Hussain Ganie, Priti Upadhyay, Sandip Das, Maheshwer Pra. ad
Sharma, Authentication of medicinal plants by DNA markers, Plant Gene 4, 83-99,
2015
11.Mihael Cristin Ichim1*, Annette Häser2 and Peter Nick2., Microscopic
Authentication of Commercial Herbal Products in the Globalized Market: Potential
and Limitations, MINI REVIEW Article Front. Pharmacol., 09 June 2020 Sec.
Ethnopharmacology https://doi.org/10.3389/fphar.2020.00876.
12.JEGATHEESH U,BHAGHAWAN RK,A textbook of herbal drug technology, solution
pharmacy, Page.
No.72.
13.Sukhdev Swami Handa Suman Preet Singh Khanuja Gennaro Longo Dev Dutt
Rakesh. Extraction Technologies for Medicinal and Aromatic Plants,
INTERNATIONAL CENTRE FOR SCIENCE AND HIGH TECHNOLOGY Trieste, 2008
14..Stéphane, F. F. Y., Jules, B. K. J., Batina, G. E., Ali, I., & Bruno, L. N. (2021).
Extraction of Bioactive Compounds from Medicinal Plants and Herbs. In (Ed.),
Natural Medicinal Plants. Intec Open. https://doi.org/10.5772/intechopen.98602
15.Soxhlet, F. (1879). "Die gewichtsanalytische Bestimmung des Milchfettes".
Dingler's Polytechnisches Journal (in German). 232: 461-465.
16.Cecilia Sparr Eskilsson, Erland Björklund., Analytical-scale microwave-assisted
extraction., Journal of chromatography A 902 (1), 227-250, 2000.
17.Vivekananda Mandal, Yogesh Mohan, S. Hemalatha* .,Microwave Assisted
Extraction - An Innovative and Promising Extraction Tool for Medicinal Plant
Research., Pharmacognosy Reviews Vol 1, Issue 1, Jan-May, 2007.
18. Altemimi, A.W.; Watson, D.G.; Kinsel, M.; Lightfoot, D.A. Simultaneous
extraction, optimization, and analysis of flavonoids and polyphenols from peach
and pumpkin extracts using a tlc-densitometric method. Chem. Cent. J. 2015, 9, 1-
15. [CrossRef] [PubMed]
19.Altemimi, A.; Lightfoot, D.A.; Kinsel, M.; Watson, D.G. Employing response
surface methodology for the optimization of ultrasound assisted extraction of
lutein and B-carotene from spinach. Molecules 2015, 20, 6611-6625. [CrossRef]
[PubMed]
20.Mulinacci, N.; Prucher, D.; Peruzzi, M.; Romani, A.; Pinelli, P.; Giaccherini, C.;
Vincieri, F.F. Commercial and Laboratory extracts from artichoke leaves: Estimation
of caffeoyl esters and flavonoidic compounds content.J. Pharm. Biomed. Anal.
2004, 34, 349-357. [CrossRef]
21.Sarajlija, H.; Ckelj, N.; Novotni, D.; Mrši'c, G.; Curi'c, M.; Brncic, M.; Curic, D.
Preparation of flaxseed for Lignan determination by gas chromatography-mass
spectrometry method. Czech J. Food Sci. 2012, 30,
45-52.
22.Popova, I.E.; Hall, C.; Kubátová, A. Determination of lignans in flaxseed using
liquid chromatography with time-of-flight mass spectrometry. J. Chromatogr. A
2009, 1216, 217-229. [CrossRef] [PubMed]
23.Shahverdi, A.R., Abdolpour, F., Monsef-Esfahani, H.R. and Farsam, H.A. (2007).
TLC bioautographic assay For the detection of nitrofurantoin resistance reversal
compound. J. Chromatogr. B 850: 528-530.
24.Cannell, R.J.P. (1998). Natural Products Isolation. Human Press Inc. New Jersey,
pp. 165-208
25.Fan, X.H., Cheng, Y.Y., Ye, Z.L., Lin, R.C. and Qian, Z.Z. (2006). Multiple
chromatographic fingerprinting and its application to the quality control of herbal
medicines. Anal. Chim. Acta 555:
217-224
26.Li, H.B., Jiang, Y. and Chen, F. (2004). Separation methods used for Scutellaria
baicalensis active components. J. Chromatogr. B. 812: 277-290.
27.Ozlem Coskun .,Separation North Clin Istanbul 2016;3(2):156-60 Doi:
10.14744/nci.2016.327
28.Bandameedi Ramul, *, Kishore Babu Chittela2,High Performance Thin Layer
Chromatography and It's Role Pharmaceutical Industry: Review.,Open Science
Journal of Bioscience 5(3): and Bioengineering2018; 29-34
http://www.openscienceonline.com/journal/bio,ISSN: 2381-3806 (Print); ISSN:
2381-3814 (Online)
29. Ammar Altemimi, Naoufal Lakhssassi, [...], and David A. Lightfoot.,
30.Kunle, Oluyemisi Folashade1*, Egharevba, Henry Omoregiel And Ahmadu,
Peter Ochogu2Ochogu2.,Standardization of herbal medicines
A review, International Journal of
Biodiversity and Conservation Vol. 4(3), pp. 101-112, March 2012.
31.Waldesch FG, Konigswinter BS&Remagen HB, Herbal medicinal products-
Scientific And Regulatory Basis For Development Quality Assurance And Marketing
Authorization, Published by Medpharmstuttagart and CRC press, Washington DC,
(2003) 37-52.
32.Sharma AK, Gaurav SS, Balkrishna A. A rapid and simple scheme for the
standardization of polyherbal drugs. Int J Green Pharm 3, (2009), 134-140
33. Ahmad I, Aqil F&Owais M. Turning medicinl plants into drugs. Modern
Phytomed.384, (2006),
67-72.
34.] Bauer R. Quality criteria and standardization of phytopharmaceuticals: Can
acceptable drug standard can be achieved. J Drug Inform32 (1998), 101-110
35.Prashant Yadav*, Kanhiya Mahour and Ashok Kumar., Standardization and
Evaluation of Herbal Drug Formulations, Journal of Advanced Laboratory Research
in Biology, Volume 2, Issue 4, October 2011.
36.Kunle, Oluyemisi Folashade1*, Egharevba, Henry Omoregiel And Ahmadu,
Peter Ochogu2Ochogu2.,Standardinternational Journal of Biodiversity and
Conservation Vol. 4(3), pp. 101-112, March 2012 Available online at
http://www.academicjournals.org/JBC DOI: 10.5897/UBC11.163 ISSN 2141-243X
©2012 Academic Journals.
37.Krunali Thakkar 1*, Vijya Parmar1, Dhara Patel1, Dhananjay Meshram1.,
RECENT ADVANCES IN HERBAL DRUG STANDARDIZATION - A REVIEW, IJAPR
/August 2013/ Vol. 4 /Issue 08/2130 - 2138.