VALIDATION OF HERBAL THERAPIES:

The use of herbal medicine is the oldest form of healthcare. About 80% of the
world’s population has faith in traditional medicine, particularly herbal drugs
for their primary healthcare. India has a rich tradition of herbal medicine as
evident from Ayurveda. As growing public interest in use of herbal medicines,
it is necessary to development of modern and objective standards for evaluating
quality of herbal medicines. So that it is a need for validation in manufacturing
of herbal drugs for control the quality of herbal drugs.

The validation of herbal products is a major public health concern. In this

regard, there is no control by the government agencies, despite the existence of
certain guidelines in some individual countries and those outlined by the WHO.
If the herbal products are marketed as therapeutic agents, and irrespective of
whether the products really have any positive effects to cure and reduce the
severity of the disease, it is necessary to ensure scientific validation and
periodic monitoring of the quality and efficacy. This concept of validation is
getting well applied to manufacturing of synthetic drugs from long time back.
But this concept is not that much deeply or methodically studied and applied for
the manufacturing of herbal drugs. All international regulations like USFDA,
MCC, MHRA, TGA etc. shows the applicability of validation to pharmaceutical
manufacturing but no one regulation except WHO applies the validation
concept to manufacturing of herbal drugs9. WHO also emphasize on very little
part of validation. Therefore introduction of scientific validation would control
the production of impure and adulterated herbal product. Chemical profiling of
traditional herbal preparations is essential in order to assess the quality of drugs.
It deals with bioactive compound quantification, spurious drug determination,
comparative fingerprint analysis, standardization of herbs, stability of
formulations and quality consistency. Various chemical markers have been
described for the validation of TM including therapeutic components, bioactive
components, synergistic components, characteristic components, main
component, correlative components, toxic components and Chemical
fingerprints can be used to authenticate plant material, identification and
quantification of active compounds to relate the chemical composition to
biological activity for product standardization and validation. Quality of TMPs
can be defined as the status of a drug that is determined by identity, purity,
content, physical, and biological properties. QC is very importance for efficacy
and safety of herbal products.

PHARMACODYNAMIC AND PHARMACOKINETIC

INTERACTIONS:

Drugs may interact with other drugs or any diet or dietary supplement taken at
the same time. Interactions may be pharmacodynamic in which interaction is
close to the target organ and involves direct antagonism or addition of
pharmacological properties. Alternatively interaction may be pharmacokinetic
in which one drug, or dietary supplement, alters the absorption, distribution,
metabolism or excretion of another drug. The induction or inhibition of drug
metabolizing enzymes is a particularly important cause of clinically significant
interactions. However, although some interactions may be life threatening,
many are only theoretical or clinically trivial. Nevertheless it is always wise to
check interactions in an appropriate reference work.

PHARMACODYNAMIC INTERACTIONS: Pharmacodynamic

interactions can occur when a herbal product produces additive,
synergistic, or antagonist activity in relation to the conventional drug with
no change in the plasma concentration of either herbal product or drug.
Pharmacodynamic interactions are related to the pharmacologic activity
of interacting agents and can affect organ systems, receptor sites, or
enzymes. A pharmacodynamic interaction may occur when herbals that
 possess antiplatelet activity are administered with anticoagulant drugs,
thus increasing the risk of bleeding. The anticoagulant action of warfarin
is enhanced by ginkgo (Ginkgo biloba) and possibly by many other herbs,
such as Danshen (Salvia miltorrhiza), Dong quai (Angelica sinensis),
Galic (Allium sativum) and Panax Ginseng. Regarding effects of angelica
root with warfarin, angelica root affects not the pharmacokinetics but the
pharmacodynamics in rabbits. Other examples of animal studies with
barbiturates, pentobarbital and hexobarbital, and other non-barbiturate
CNSdepressants like urethane and glutethimide suggest not simply
additive but synergistic effects with kavalactone and G. biloba may act as
an antagonist on gamma-aminobutyric acid (GABA) activity at
benzodiazepine binding sites.
Pharmacodynamics looks at the effects of an agent at active sites in the
body.

 PHARMACOKINETIC INTERACTIONS:
Pharmacokinetic herb-drug interactions are due to altered absorption,
metabolism, distribution and excretion of drugs. The primary mechanism
of absorption is passive diffusion of nonionized drug molecules via the
lipophilic gastrointestinal (GI) mucosa. Therefore, drugs that change pH,
gastric emptying time, or GI motility will interact with the absorption of
other agents. The most popular stimulant laxative herbs of the anthranoid-
containing senna (Cassia senna and C. angustifolia), cascara sagrada
(Rhamnus purshiana), aloe vera (Aloe barbadensis), frangula (Rhamnus
frangula), yellow dock (Rumexcrispus), and Chinese rhubarb (Rheum
officinale) will accelerate intestinal transit, and thus may interfere with
the absorption of almost any intestinally absorbed drugs . It is well-
known that metal ions and tannic acid present in herbal medicine form
insoluble chelates or complexes with some western drugs such as
antibiotics, isoniazid and levodopa, resulting in reduction of drug
absorption. It is also reported that alkaloids or other flavonoids form
precipitates with some western drugs containing aluminium, bismuth,
calcium, ferrous and magnesium ions. More recently, induction and
inhibition of intestinal P-glycoprotein have been described in significant
drug interactions. The P-glycoprotein drug transporter is a glycoprotein
encoded by the MDR1 gene and functions as a transmembrane efflux
transporter that pumps drugs out of cells. P-glycoproteins are found in
many tissues and especially in organs responsible for drug absorption or
elimination, such as the intestine, liver, and kidneys. P-glycoprotein is
vulnerable to inhibition, activation, or induction by herbs and herbal
constituents. Curcumin, ginsenosides, piperine, some catechins from
green tea, and silymarin from milk thistle were found to be inhibitors of P
glycoprotein . Drug metabolism is divided into 2 categories of phase I
and phase II transformation reactions. Phase I reactions include oxidation,
hydrolysis, and reduction, resulting in a compound that is generally less
toxic and more hydrophilic, allowing for easy excretion. Phase II
reactions primarily result intermination of biologic activity of the drug.
Phase II transformation reactions include glucuronidation, sulfation,
acetylation, and methylation. The CYP450 is the most important phase I
drug-metabolizing enzyme system and it is a family of monooxygenase
enzymes that are mainly found in intestinal and liver cells and catalyzes
several Phase I metabolic processes including oxidation, hydroxylation,
Sand O-demethylation, and oxidative deamination. Many herbs and
natural compounds isolated from herbs (e.g., flavonoids, coumarins,
furanocoumarins, anthraquinones, caffeine and terpenes) have been
identified as substrates, inhibitors or inducers of various CYP enzymes .
An investigation of pharmacokinetic interaction between the active
compound and other drug or substance is of particular importance for
synthetic drugs with narrow therapeutic window. If it comes to herbal
medicines, we have to take into account that those products have a
relatively wide therapeutic window probably because the human CYO
system was originally conditioned to work with compounds of natural
origin. The study of herbal pharmacokinetic is a unique field, which is
extraordinarily complex for the following reasons:
 The chemical complexity of plant medicines and thud potential
interactions between constituents.
 The different bioavailability of different compounds
 The active components are not known, so the components in the
plant which should be studied cannot be identified.
 Herbal medicines are not designed for predictable pharmacokinetic
characteristics and in particular, natural compounds are often
metabolised in the digestive tract that is, they act like pro- drugs.
 Often large polar molecules are involved, which might be expected
to have poor and unpredictable bioavailability.
For evaluating the herbal pharmacokinetics the following
parameters need to be considered:
 Information to further assess the traditional and anecdotal
uses of medicinal plant and better information on which to
base rational dosages.
 A better appreciation of the safety and toxicity of plant and
anticipation of potential herbal-drug interactions.
 Supporting evidence for the synergistic nature of herbal
medicines.
 The fat solubility of the molecule- the more fat-soluble, the
better the bioavailablilty.
 Specific factors related to crossing the gut wall. eg- active
transport.
  The presence or absence of food may also influence the
absorption and bioavailability of plant constituents.

HERBAL DRUG STANDARDISATION:

 WHO GUIDELINES:

Standardization of herbal formulations is essential in order to assess of quality

drugs, based on the concentration of their active principle, physical, chemical,
physcio-chemical standardization and in vitro, in-vivo parameters[1]. Natural
products have been our single most successful source of medicines. Each plant
is like factory capable of synthesizing unlimited no of highly complex and
unusual chemical substance whose structure covered otherwise escape the
imagination forever. It is necessary to maintain reproducible efficacy and safety
of phyto pharmaceutical therefore if phytopharmaceutical have to regard as
rational drug should be standardized and pharmaceutical quality must be
approved. World Health Organization (WHO) has individual herbal drugs as
whole, labelled medicinal products that have robust ingredients, aerial or secret
parts of the whole plant or other plant material or mixture of them. World
Health Organization (WHO) has a set of specific Guidelines for the evaluation
of the safety, efficacy and Quality of herbal drugs or herbal medicines. WHO
find out that 80% of the world people currently use herbal medicine or drugs for
the most important health ares. Herbs are usually measured as safe toxicity, side
effects of allopathic drugs, has led to more increased in number of herbal drugs
manufacturers. For the past few years, herbal drugs have been mostly used by
the people with no prescription, Leaves, stem, bark, flower, seeds, roots and
extract of all these have been used in herbal drugs over the thousands of their
use.
  Parameters for standardization and Quality Control of
herbal drugs Morphological or Organoleptic evaluation :
It includes the evaluation of herbal drugs by size, shape color, odor,
taste and particular characteristics like touch, texture etc. This is a
technique of qualitative evaluation related to the study of
morphological and sensory report of whole drugs.eg. Fractured
surfaces in cascara, cinchona, and quillia bark and quassia wood are
essential characteristics. Umbelliferous fruits have aromatic odour and
liquorice have sweet taste are the example of this type of evaluation.
Shape of drug may be conical (aconite), subcylindrical
(podophyllum), cylindrical (sarsapilla), fusiform (jalap).Size
represents thickness, length, breadth and diameter. Color represents
external color which various from white to brownish black are
essential diagnostic features. Taste which is a specific type of
sensation feel by epithelial layer of tongue. taste may be sweetish
(saccharic),sour (acidic),salt like (saline),and bitter or tasteless. It is
regularly used for qualitative analysis of organized crude drugs in total
and powder form with the help of microscope. The inner
pseudoparenchyma cells are round or oval shape. They

Contain protein and fixed oil. Crude drugs are microscopically identified by
taking thin TS (Transverse section), LS (Longitudinal Section) in a bark, wood
and leaf. The various parameters included in microscopy are given bellow. I.
Stomata II. Trichomes III. Leaf Content IV. Quantitative Microscopy.

 Chemical Evaluation: The most of drug contain definite chemical

constituents to which their pharmacological and Biological activity
depended. Qualitative chemical test used to identify drug quality and
purity. The identification, isolation and purification of active chemical
 constituents is depends chemical methods of evaluation. Preliminary
phytochemical investigation is also a part of chemical evaluation.

Srno Name of constituents Identification test

1 volatile oil 1. ester value
2. acetyl value
2 Balsams 1. acid value
2. saponification
value 3.ester value
3 Resins 1. Sulphated ash
2. Acid value
4 Gums 1. Methoxy
determination
2. Volatile acidity

Determination of Foreign Matter: Herbal drugs should be prepared from

the confirmed part of the plant. They should be totally free from insects or
moulds, including visible and excreta contaminant such as stones, sand, harmful
and poisonous foreign matter and chemical residues. Animal objects such as
insects and invisible microbial contaminants, which produces toxins, as well as
the potential contaminants of herbal medicines

% foreign organic matter = n×w×94100×100/s×m×p

Where; n = No. of chart particles in 25 field. S = No. of spores in the same area
of 25 fields. W = Weight in mg of lycopodium taken. M= weight in mg of the
sample P= number of characteristics particles per mg of the pure foreign matter.
94,000= number of spores per mg of lycopodium.

Determination of Total Ash Value The residue after incineration is

the total ash content of the crude drug, which simply represents inorganic
salts,
 naturally found in drug or adhering to it or deliberately added to it, in the form
of adulteration. Two types of total Ash value:

1. Water soluble Ash value 2. Acid insoluble Ash value.

Sl no drugs Total ash(%w/w) Acid insoluble
ash(%w/w)
1 Agar - 1.00
2 Bael 3.50 -
3 Cannabis 15.00 5.00
4 ginger 6 1.57
Determination of Extractive Values: The extracts obtained by
exhausting crude drugs are indicative of approximate measure of their chemical
constituents. The various solvent are used for determination of extractives.
These are classified as fallows.

1. Water Soluble extractives. 2. Alcohol Soluble extractives. 3. Ether

Soluble extractives.
Sl no drugs water Alcohol Ether
soluble soluble soluble
extractives extractives extractives
1 ALOE NLT 25 NLT 10 -
2 GINGER NLT 10 NLT 4.50 -
3 CAPSICUM - - NLT 12
4 NUTMEG - - NLT 25

Determination Of heavy Metals In general, quantitative and limit tests

accurately determine the concentration of heavy metals in the form of impurities and
contaminants. The heavy metals like Arsenic, mercury, lead, thalium, cadmium
have been shown to be contaminants of few herbal ingredients.
A simple determination of heavy metals can be found ia many pharmacopeia and it
is based on color reaction with special reagents such as diethyldithio carbonate or
thioacetamide and amount is determined by comparison with a standards. The
methods commonly used for analysis are inductive coupled plasma (ICP), Neutron
activation analysis(NAA), Atomic Absorption Spectrophotometry(AAS).
Determination of specific optical rotation [19] Specific rotation
determination formula -D25= 100 × φlc Where: φ = corrected observed rotation in
drug at-25° D = d line of sodium light l = length of the Polari meter tube in done. c
= concentration of substance in per cent w/v.

Radioactive contamination The microbial growth in herbal drugs is usually

avoided by irradiation. Dangerous contamination 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 wide spread contamination
by radio nuclides resulting from major nuclear accidents. Examples of such
radionuclides include long lived and short lived fission products, actinides and
activation products. Therefore, at current no limits are proposed for radioactive
contamination.

Pesticides Residue Pesticides residue are any particular substance in food,

agriculture commodities or animal feed resulting from the use of a pesticides.
Herbal drugs are prone to contain pesticide residue, which gather from agricultural
practices, such as Spraying, behavior of soil during cultivation and addition of
fumigants during storage. The Pesticides contain chlorine in the molecules, which
can be determined by analysis of chlorine; insecticides containing phosphate can be
detected by measuring total organic phosphorus. The various methods are used to
measure pesticides by GC, MS, OR GCMS. Some simple methods are also
published by the WHO and European pharmacopeia has in general limits for
pesticides residue in medicine.
Physical evaluation: Physical constants are sometimes taken into consideration
to evaluate certain drugs. These include moisture content, specific gravity, optical
rotation, refractive, melting point, viscosity and solubility in different solvents. All
these physical properties are useful in identification and detecting of constituents
present in plants.

Biological evaluation: Some drugs have specific biological and pharmacological

activity which is utilized for their evaluation. Actually this activity is due to specific
type of constituents present in the plant extract. For evaluation the experiments were
carried out on both intact and isolation organs of living animals. With the help of
bioassays, strength of drug in its preparation can be evaluated.

Chromatography techniques: TLC (Thin layer chromatography):

TLC was the most common, versatile methods of choice for herbal analysis before
instrumental chromatography methods like gas chromatography and HPLC were
established. Even now a day’s TLC is still frequently used for the analysis of herbal
medicines since various pharmacopeias such as Indian herbal pharmacopeia,
Ayurvedic pharmacopeias, American herbal pharmacopeias, and Chinese drugs
monographs. Rather TLC is used as an easier method of initial screening with a
semi-qualitative evaluation together with other chromatography techniques as there
is relative less change in the simple TLC separation of herbal medicines the with
instrumental chromatography. For example the four sample of cordyceps sinensis
from that joint product of china and Japan cooperation has more valuable medicinal
effect compared to other as they contain the most effective component
“cordycepine” more over with the help of imagine analysis and digitized technique
developed in computer science, evaluation of similarities between different samples
is also possible.:
Electrophoretic method: Capillary electrophoresis was introduced in early
1980s as a powerful analytical and separation technique and has been developed
almost explosively. It allows an efficient way to document the purity/complexity of
a sample and can handle virtually every kind of charged sample components ranging
from simple inorganic ions to DNA. Thus, there was an obvious increase of
electrophoretic methods, especially capillary electrophoresis, used in the analysis of
herbal medicines. In general, CE is a versatile and powerful separation tool with
high separation efficiency and selectivity when analyzing mixtures of low-
molecular- mass components. However, the fast development in capillary
electrophoresis causes improvement of resolution and throughout rather than
reproducibility and absolute precision.

GAS CHROMATOGRAPHY:
It is well-known that many pharmacologically active components in herbal
medicines are volatile chemical compounds. Thus, the analysis of volatile
compounds by gas chromatography is very important in the analysis of herbal
medicines. The GC analysis of the volatile oils has a number of advantages.
Firstly, the GC of the volatile oil gives a reasonable “finger print” which can be
used to identify the plant. The composition and relative concentration of the
organic compounds in the volatile oil are the characteristic of the particular
plant and the presence of impurities in the volatile oil can be readily detected.
Secondly, the extraction of the volatile oil is relatively straight forward and can
be standardized and the components can be readily identified using the GC-MS
analysis.
HPLC:
HPLC is a popular method for the analysis of herbal medicines. Because it is easy to
learn and use and is not limited by the volatile or stability of the sample compound.
In general, HPLC can be used to analyse almost all the compounds in the herbal
medicines. Reversed- phase (RP) columns may be most popular columns used in the
analytical separation of herbal medicines. the increasing usage of LC- MS and
HPLC-DAD in the analysis of herbal medicines is quite obvious. Several good
reviews have been published for the analysis of the bioactive chemical compounds
in plants and medicines, in which the technique used most in HPLC, especially the
hyphenated HPLC technique. Moreover, combined HPLC-DAD-MS technique take
advantage of chromatography as a separation method and both DAD and MS as an
identification method. DAD and MS can provide on- line UV and MS information
for each individual peak in a chromatography. With the help of this hyphenation, in
most cases, one could identify the chromatography peaks directly online by
compression with literature data. Recently, the hyphenation between HPLC and
NMR also available, which might become a vital and an attractive analytical tool for
the analysis of herbal medicines.
Chromatographic fingerprinting: Chromatographic fingerprinting is the most
powerful approach for the quality ontrol of herbal medicines. Chromatographic
fingerprint of Herbal Medicine is a chromatographic pattern produced from extract
of some common chemical components which may be pharmacologically active or
have some chemical characteristics. This chromatographic profile should be
featured by the fundamental attributions of - integrity and - fuzziness or - sameness
and - differences so as to chemically represent the herbal medicines investigated.
This suggest that chromatographic fingerprint can successfully demonstrate both
sameness and differences between various samples and the authentication and
identification of herbal medicines can be accurately conducted even if the number
and/or concentration of chemically characteristic constituents are not very similar in
different samples of herbal medicine. Thus chromatographic fingerprint should be
considered to evaluate quality of herbal drugs.
DNA fingerprinting: DNA analysis has been proved as an important tool in
herbal drug standardization which is useful for the identification of
phytochemically indistinguishable genuine drug from substituted or adulterated
drug. DNA fingerprint genome remains the same irrespective of the plant part
used while the phytochemical constituents will vary with the part of plant used,
physiology and environment. The other useful application of DNA
fingerprinting is the availability of intact genomic DNA specificity in
commercial herbal drugs which helps in distinguishing adulterants even in
processed samples.

AYUSH GUIDELINES: India is a mother hub for development of

Ayurveda, Unani, Siddha; Homoeopathy and other natural herbs based health
science (Ayush). Ayush Pharmaceutical industry is having great potential and
opportunities for development in future. Mainly in following herbal medicinal
plants and their value added products well accepted in domestic and
international market e.g. Ayurvedic medicines, Unani medicines, Siddha
medicines, Homoeopathic medicines, herbal nutraceuticals, herbal
cosmoceutical, herbal health drinks, dietary health supplements, medicinal
plants / crude drugs, herbal extracts / concentrates
Morphological or Organoleptic Evaluation: It includes the evaluation of
herbal drugs by size, shape colour, odour, taste and particular characteristics like
touch, texture etc. This is a technique of qualitative evaluation related to the study of
morphological and sensory report of whole drugs. eg. Fractured surfaces in cascara,
cinchona, and quillia bark and quassia wood are essential characteristics.
Umbelliferous fruits have aromatic odour and liquorice have sweet taste are the
example of this type of evaluation. Shape of drug may be conical (aconite),
subcylindrical (podophyllum), cylindrical (sarsapilla), fusiform (jalap). Size
represents thickness, length, breadth and diameter. Color represents external color
which various from white to brownish black are essential diagnostic features. Taste
which is a specific type of sensation feel by epithelial layer of tongue. Taste may be
sweetish (saccharic), sour (acidic), salt like (saline) and bitter or tasteless .

Macroscopic and Microscopic Examination: Medicinal plant materials are

categorized according to sensory, macroscopic and microscopic characteristics. An
examination to determine these characteristics is the first step towards establishing
the identity and the degree of purity of such materials, and should be carried out
before any further tests are undertaken.
Macroscopic identity of medicinal plant materials is based on shape, size, colour,
surface characteristics, texture, fracture characteristics and appearance of the cut
surface. Microscopic inspection of medicinal plant materials is indispensable for
the identification of broken or powdered materials; the specimen may have to be
treated with chemical regents. An examination by microscopy alone cannot always
provide complete identification, though, when used in association with other
analytical methods, it can frequently supply invaluable supporting evidence.

Physical Evaluation: Each monograph contains detailed botanical,

macroscopic and microscopic with detailed illustrations and photographic images
which provide visual descriptions documentation of accurately identified material.
A microscopic analysis assures the identity of the material and as an initial
screening test for impurities.
Determination of ash: The ash remaining following ignition of medicinal plant
materials is determined by three different methods which measure total ash, acid-
insoluble ash and water-soluble ash. The total ash method is designed to measure
the total amount of material remaining after ignition. This includes both
“physiological ash”, which is derived from the plant tissue itself, and “non-
physiological” ash, which is the residue of the extraneous matter adhering to the
plant surface. Acid-insoluble ash is the residue obtained after boiling the total ash
with dilute hydrochloric acid, and igniting the remaining insoluble matter.
This measures the amount of silica present, especially as sand and siliceous earth.
Water soluble ash is the difference in weight between the total ash and the residue
after treatment of the total ash with water.

Determination of extractable matter: This method determines

amount of active constituents extracted with solvents from a given amount
of medicinal plant material .
1. Water Soluble extractives
2. Alcohol Soluble extractives
3. Ether Soluble extractives

Determination of Foreign Matter: Herbal drugs should be prepared from the

confirmed part of the plant. They should be totally free from insects or moulds,
including visible and excreta contaminant such as stones, sand, harmful and
poisonous foreign matter and chemical residues. Animal objects such as insects and
invisible microbial contaminants, which produces toxins, as well as the potential
contaminants of herbal medicines. Macroscopic evaluation can easily used to
determine the presence of foreign matter, although microscopy is essential in
certain special cases for example starch intentionally added to “dilute” the plant
material

Chemical Evaluation: The most of drug contain definite chemical

constituents to which their pharmacological and Biological activity depended.
 Qualitative chemical test used to identify drug quality and purity. The
identification, isolation and purification of active chemical constituents is
depends chemical methods of evaluation. Preliminary phytochemical
investigation is also a part of chemical evaluation. Some Qualitative chemical
tests for chemical evaluation crude drug are Saponification value and acid
value etc.

Chromatographic Fingerprinting and Marker Compound

Analysis: A chromatographic fingerprint of an Herbal Medicine (HM) is a
chromatographic pattern of the extract of some common chemical components
of pharmacologically active and or chemical characteristics. This
chromatographic profile should be featured by the fundamental attributions of
“integrity” and “fuzziness” or “sameness” and “differences” so as to chemically
represent the HM investigated. It is suggested that with the help of
chromatographic fingerprints obtained, the authentication and identification of
herbal medicines can be accurately conducted (integrity) even if the amount
and/or concentration of the chemically characteristic constituents are not exactly
the same for different samples of this HM (hence, “fuzziness”) or, the
chromatographic fingerprints could demonstrate both the “sameness” and
“differences” between various samples successfully.

TLC: 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, TLC is being
employed extensively for the following reasons:
1. It enables rapid analysis of herbal extracts with minimum sample clean-up
requirement,
2. It provides qualitative and semi quantitative information of the resolved
compounds.
3. It enables the quantification of chemical constituents.
Fingerprinting using HPLC and GLC is also carried out in specific cases In
TLC fingerprinting, the data that can be recorded using ma high-performance
TLC (HPTLC) scanner includes the chromatogram, retardation factor (R f)
values, the color of the separated bands, their absorption spectra, λ max and
shoulder inflection/s of all the resolved bands.
HPTLC: HPTLC technique is widely employed in pharmaceutical
industry in process development, identification and detection of
adulterants in herbal product and helps in identification of pesticide
content, mycotoxins and in quality control of herbs and health foods . It
has been well reported that several samples can be run simultaneously by
use of a smaller quantity of mobile phase than in HPLC . It has also been
reported that mobile phases of pH 8 and above can be used for HPTLC.
They have developed HPTLC method for phytoconstituents in crude
drugs or herbal formulations such as bergenin, catechine and gallic acid
in Bergenia cilliata and Bergenia lingulata .

Liquid Chromatography - Mass Spectroscopy: (LC-MS) LC-MS has become

method of choice in many stages of drug development. Recent advances
includes electrospray, thermo spray, and ionspray ionization techniques which
offer unique advantages of high detection sensitivity and specificity, liquid
secondary ion mass spectroscopy, later laser mass spectroscopy with 600 MHz
offers accurate determination of molecular weight proteins, peptides. Isotopes
pattern can be detected by this technique.

Gas Chromatography (GC-MS): GC equipment can be directly interfaced

with rapid scan mass spectrometer of various types. GC and GC-MS are
unanimously accepted methods for the analysis of volatile constituents of herbal
medicines, due to their sensitivity, stability and high efficiency. Especially, the
hyphenation with MS provides reliable information for the qualitative analysis
of the complex constituent. The flow rate from capillary column is generally
 low enough that the column output can be fed directly into ionization chamber
of MS. The simplest mass detector in GC is the Ion Trap Detector (ITD).

DNA Fingerprinting: DNA analysis has been proved as an important tool

in herbal drug standardization. This technique is useful for the identification of
phytochemically indistinguishable genuine drug from substituted or adulterated
drug. It has been reported that DNA fingerprint genome remain the same
irrespective of the plant part used while the phytochemical content will vary
with the plant part used, physiology and environment.

Genetic Marker: A genetic marker is a gene or DNA sequence with a known

location on a chromosome and associated with a particular gene or trait. It can
be described as a variation, which may arise due to mutation or alteration in the
genomic loci that can be observed. A genetic marker may be a short DNA
sequence, such as a sequence surrounding a single base-pair change (single
nucleotide polymorphism SNP), or a long one, like mini satellites.
Some commonly used types of genetic markers are
 RFLP (or Restriction fragment length polymorphism)
 AFLP (or Amplified fragment length polymorphism)
 RAPD (or Random amplification of polymorphic DNA)
 VNTR (or Variable number tandem repeat)
 Micro satellite polymorphism
 SNP (or Single nucleotide polymorphism)
 STR (or Short tandem repeat)
 SFP (or Single feature polymorphis
Biological Evaluation:
Determination of Bitterness Value: Medicinal plant materials that
have a strong bitter taste are employed therapeutically, mostly as
appetizing agents. Their bitterness stimulates secretions in the
gastrointestinal tract, especially of gastric juice. Bitter substances can be
determined by taste. However, since they are mostly composed of two or
more constituents with various degrees of bitterness, it is first necessary
to measure total bitterness by taste. The bitter properties of plant material
are determined by comparing the threshold bitter concentration of an
extract of the materials with that of a dilute solution of quinine
hydrochloride. The bitterness value is expressed in units equivalent to the
bitterness of a solution containing 1 gm of quinine hydrochloride in
2000ml. Safe drinking water should be used as a vehicle for the
extraction of plant materials and for the mouth wash after each tasting.
Taste buds dull quickly if distilled water is used. The hardness of water
rarely has any significant influence on bitterness.
Determination of Haemolytic Activity: Many medicinal plant
materials, especially those derived from the families Caryophyllaceae,
Araliaceae, Sapinaceae, Primulaceae, and Dioscoreaceae contain
saponins. The haemolytic activity of plant materials, or a preparation
containing saponins, is determined by comparison with that of a reference
material, saponin, which has a haemolytic activity of 1000 units per gm.
A suspension of erythrocytes is mixed with equal volumes of a serial
haemolysis and is determined after allowing the mixtures to stand for a
given period of time. A similar test is carried out simultaneously with
saponin.
Determination of Swelling Index: The swelling index is the
volume in ml taken up by the swelling of 1 gm of plant material under
specified conditions. Its determination is based on the addition of water or
a swelling agent as specified in the test procedure for each individual
plant material. Using a glass stoppered measuring cylinder, the material is
shaken repeatedly for 1 hour and then allowed to stand for a required
period of time. The volume of the mixture is then read. The mixing of
whole plant material with the swelling agent is easy to achieve, but cut or
pulverized material requires vigorous shaking at specified intervals to
ensure even distribution of the material in the swelling agent.
Determination of Foaming Index: Many medicinal plant materials
contain saponins that can cause persistent foam when an aqueous
decoction is shaken. The foaming ability of an aqueous decoction of plant
materials and their extracts is measured in terms of a foaming index.
Determination of Pesticide Residues: Limits for pesticide
residues should be established following the recommendations of the
Food and Agriculture Organization of the United Nations (FAO) and the
World Health Organization (WHO) which have already been established
for food and animal feed. These recommendations include the analytical
methodology for the assessment of specific pesticide residues. Pesticides
residue are any particular substance in food, agriculture commodities or
animal feed resulting from the use of a pesticides. Herbal drugs are prone
to contain pesticide residue, which gather from agricultural practices,
such as Spraying, behaviour of soil during cultivation and addition of
fumigants during storage. The Pesticides contain chlorine in the
molecules, which can be determined by analysis of chlorine , insecticides
containing phosphate can be detected by measuring total organic
phosphorus. The various methods are used to measure pesticides by GC,
MS, OR GCMS. Some simple methods are also published by the WHO
and European pharmacopeia has in general limits for pesticides residue in
medicine.
REFERNCES:
1. Pulok K Mukherjee*, Ranjit K Harwansh, Shiv Bahadur, Subhadip
Banerjee and Amit Kar., EVIDENCE BASED VALIDATION ON
TRADITIONAL MEDICINES
2. Desai S R*, Disouza J I, Shirwadkar B B., Process Validation: An
Approach for Herbal Tablet Standardization
3. Shulammithi R1, Sharanya M2, Tejaswini R3, Kiranmai M.,
Standardization and quality evaluation of herbal drugs
4. Kunle, Oluyemisi Folashade1*, Egharevba, Henry Omoregie1 and
Ahmadu, Peter Ochogu., Standardization of herbal medicines - A review
5. Mr. Shailesh. L Patwekar, Suryawanshi Arvind B, Gaikwad Manoj S,
Pedewad Snehal R, Potulwar Ashwini P., Standardization of herbal drugs:
An overview
6. Rohit Kumar Bijauliya, Shashi Alok, Dilip Kumar Chanchal and
Mayank Kumar., A COMPREHENSIVE REVIEW ON
STANDARDIZATION OF HERBAL DRUGS