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STANDARDIZATION OF HERBAL
DRUGS
Under the guidance of:
Dr.M.S.Doddamani
HOD & Professor
PG Dept of RS & BK
T.G.A.M.C, Ballari.
By
Dr.Lavanya.S.A
2nd Year P.G Scholar
Dept of RS & BK
T.G.A.M.C, Ballari.
INTRODUCTION
In the last few decades there has been an exponential growth in the
field of herbal medicine. It is getting popularized in developing as well as
in developed countries owing to its natural origin and lesser side effect.
In olden times, Vaidyas used to treat patients on individual
basis, and prepare drug according to the requirement of the patient. But
the scenario has changed now; medicines are being manufactured on the
large scale in Pharmaceutical units, where manufacturers come across
many problems such as availability of good quality raw material,
authentication of raw material, availability of standards, proper
standardization methodology of single drugs and formulation & quality
control parameters.
HERBS:
Crude plant material such as leaves, flowers, fruit,
seed, stems, wood, bark, roots, rhizomes or other plant
parts, which may be entire, fragmented or powdered.
HERBAL DRUGS:
Finished labeled products that contain active ingredients
such as aerial or underground parts of plant or other
plant material or combinations thereof, whether in the
crude state or as plant preparations
Standardization of herbal drug refers to
“confirmation of its identity and determination of its
quality, purity and detection of nature of adulterant
by various parameters like morphological,
microscopical, physical, chemical and biological
observations.”
Standardization is used to describe all measures,
which are taken during the manufacturing process and
quality control leading to a reproducible quality.
It also encompasses the entire field of study from
birth of a plant to its clinical application.
Standardization is the first step for the
establishment of a consistent biological activity,
chemical profile, or simply a quality assurance program
for production and manufacturing.
TOTAL 10000 SPECIES
325
425
550
1000
3500
8000
325 – Pesticides
425 – Gums, Resins & Dyes
550 – Fibre
1000 – Others
3500 – Edible
8000 – Medicinal Plants
NEED FOR STANDARDIZATION
In recent years there is a spurt in the interest regarding survival of
Ayurvedic forms of medication. In the global perspective, there is a shift
towards the use of medicine of herbal origin, as the dangers and the
shortcoming of modern medicine have started getting more apparent, majority
of Ayurvedic formulation are prepared from herbs.
It is the cardinal responsibility of the regulatory authorities to ensure
that the consumers get the medication, which guarantee, Purity, safety, potency
and efficacy. This duty is discharged by the regulatory authorities by rigidity
following various standards of quality prescribed for raw materials and finished
products in pharmacopoeias controlling manufacturing formulate through the
use of formularies and manufacturing operation through statutory imposed
“Good manufacturing practices”.
PROCEDURES FOR STANDARDIZATION
OF HERBAL DRUGS
In order to assure a consistent and acceptable quality herbal product,
care should be taken right from the identification and authentication of
herbal raw materials to the verification process of final products.
The following parameters are recommended:
• Authentication.
• Physical parameters.
• Quantitative and qualitative analysis.
• Microbiological Contamination.
• Pesticide residue.
• Heavy Metal analysis.
The first stage is identification of the plant species or
botanical verification by the currently accepted Latin binomial
name and synonyms. The steps involved in authentication are
taxonomic, macroscopic & microscopic studies. Records should
be maintained for stage of collection, parts of the plant
collected, regional status, botanical identity such as
phytomorphology, microscopial, histological analysis,
taxonomical identity etc…
Physical tests include organoleptic evaluation (
sensory characters such as taste, appearance, odour,
feel of the drug etc…), viscosity, moisture content, pH,
disintegration time, friability, hardness, flow ability,
sedimentation, extract values and ash value
Chromatographic and sophisticated modern techniques
of standardization such as spectroscopic evaluation,
spectrophotometry, TLC, HPTLC, HPLC, NMR, near infrared
spectroscopy provide quantitative and semi-quantitative
information about the main active constituents or marker
compounds present in the crude drug or herbal products.
Markers play an important role in fingerprinting of herbs.
Quality of drug can also be assessed by chromatographic
fingerprint.
It can be measured according to parameters or
methods described in the Ayurvedic Pharmacopoeia, as
well as in the Indian pharmacopoeia. Microbiological
analysis includes analysis of limits of E.Coli and molds,
total viable aerobic count, total enteriobacteria and
their count, aflatoxin analysis
Standard limits of pesticides have been set by
WHO and FAO ( Food and Agricultural Organization).
Some common pesticides that cause harm to human
beings are DDT, BHC, Toxaphene, aldrin etc should be
analysed.
Toxic metals such as Cu, Zn, Mn, Fe and
particularly Cd, As, Pb and Hg should be analysed.
In the analysis of metals their speciation is to be
taken into consideration.
The subject of herbal drug standardization is massively
wide and deep. The guidelines set by WHO can be summarized as
follows:
• Reference to the identity of the Drug.
• Reference to the Physicochemical Character of the Drug.
• Reference to the Pharmacological parameters.
• Toxicity Details.
• Microbiological Parameters.
• Radioactive Contamination.
Reference to the identity of the drug:
Botanical Evaluation: Botanical identity like phytomorphology,
microscopical and histological analysis ( characteristic of cell walls,
cell contents, starch grains, calcium oxalate crystals, trichomes, fibers,
vessels etc..), sensory characters, foreign organic matter,
histochemical evaluation, quantitative measurements etc…
Reference to the pharmocological properties:
Biological activity profiles, bitterness value, hemolytic index,
astringency, swelling factor, foaming index etc..
Reference to the physicochemical character of the drug:
Physical and chemical identity, chromatographic fingerprints,
ash values, extractive values, moisture content, volatile oil and
alkoloidal assays, quantitative estimation protocols etc…
Physical parameters include colour, odour, appearance, clarity,
viscosity, moisture content, pH, disintegration time, friability,
hardness, flow ability, flocculation, sedimentation, settling rate and
ash values.
Chemical parameters include limit tests, chemical tests,
chemical assays. Chromatographic analysis of herbals can be done
using TLC, HPLC, HPTLC, GC, UV, GC-MS, fluorimetry.
Toxicity Details:
Pesticide residue, heavy metals, microbial contamination like total viable
count, pathogens like E.Coli, salmonella, P.aeroginosa, S.aurea, Enterobacteria.
Microbiological parameters:
It include total viable count, total mold count, total entero-bacterial count.
Limiters can be utilized as a quantitative or semi-quantitative tool to ascertain
and control the amount of impurities like the reagents used during abstraction
of various herbs, impurities coming directly from the manufacturing vessels and
from the solvents.
First step Search for official &
vernacular names of the
drug.
Formulary
Pharmacopoeia
Monographs
Second step List the possible
adulterants and
substitutes.
Pharmacopoeia
Nighantu’s etc…
Other books on
controversial drugs like
book by Balwant singh etc.
Third step Taxonomically identify
crude drugs for reference.
Herbarium
Drug Museum
Collection
Fourth step Pharmacognostical
identification of official
adulterant /substitute
Botanical
Physicochemical
Chemical Identification
IDENTIFICATION OF CRUDE DRUGS
• Systematic study of crude drugs.
• Microscopic Identification.
• Determination of stomatal index.
• Determination of vein-islet number.
SYSTEMATIC STUDY OF CRUDE DRUGS
Study of the drugs of plant which are used in their natural form is
called crude drug. The study of these crude drugs made with a view to
recognise them is called Pharmacognosy.
Pharmakon = Drug ; Gignosco = To acquire knowledge of, meaning the
knowledge of drug.
In pharmacognosy a complete and systematic study of a drug is
done, which comprises of:
• Origin, common names, scientific nomenclature and family.
• Geographical source and history.
• Cultivation, collection, preservation and storage.
• Macroscopical, microscopical and sensory characters.
• Chemical composition.
• Identity, purity, strength and assay.
• Substitute and Adulterants.
Macroscopical Characters:
The morphological details of the respective part are given by observing it
with naked eyes or with the aid of magnifying lens. In this description general
conditions of the drug, size, shape, outer surface, inner surface etc.. Are
referred to. This study can only be done if plant parts are available in entire
condition.
Sensory or Organoleptic characters:
These describe colour, odour, taste, consistency etc…
Chemical constituents:
It can be identified by chemical and micro-chemical tests.
Eg : Strychnine present in Nux-vomica gives purplish red colour with
ammonium vanadate and Conc. Sulphuric acid.
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 reagents.
Appropriate processing for separation and isolation of botanical
debris from a formulation without loss of debris, by hand picking, shifting,
washing, sedimentation etc…, are the priliminary steps.
This is followed by clearing the debris in chemical reagents, reacting
with suitable regents and stains and finally mounting a little part on a
slide in a medium of suitable refractive index that helps to show the unit
structures.
MICROSCOPIC IDENTIFICATION
Methods of examining crude vegetable
drugs
Entire
material &
powder
Leaves and
flowers
Fruits and
seeds
Bark
Roots and
rhizomes
In the mature leaf, four significantly different types of stoma are
distinguished by their form and the arrangement of the surrounding
cells, especially the subsidiary cells.
1. Anomocytic or ranunculaceous type:
The stoma is surrounded by a varying number of cells,
generally not different from those of the epidermis.
2. Anisocytic or cruciferous type:
The stoma is usually surrounded by three or four
subsidiary cells, one of which is markedly smaller than the others.
TYPES OF STOMATA
3. Diacytic or Caryophyllaceous type:
The stoma is accompanied by two subsidiary cells, the
common wall of which is at right angles to the stoma.
4. Paracytic or rubiaceous type:
The stoma has two subsidiary cells, of which the long
axes are parallel to the axis of stoma.
The stomatal index is the percentage of the number
of stomata formed by the total number of epidermal cells,
including the stomata, each stoma being counted as one cell.
Stomatal index = S x 100
E + S
Where S = No of stomata in a given area of leaf.
E = No of epidermal cells in the same area of leaf.
Determination of Palisade Ratio:
Palisade ratio is the average number of palisade cells
under one epidermal cell.
E.g.- for the above picture, Palisade ratio = 18/4
= 4.5
Determination of Vein- Islet Number:
The mesophyll of a leaf is divided into small portions of
photosynthetic tissue by anastomosis of the veins and veinlets ; such
small portions or areas are termed as Vein-Islets.
The number of vein-islets per square millimetre is termed
the Vein-Islet number.
This value has been shown to be constant for any given
species and, for full grown leaves, to be unaffected by the age of the
plant or the size of the leaves.
QUANTITATIVE STUDY OF CRUDE DRUG:
• Sampling of drug
• Determination of foreign matter
• Determination of Total Ash
• Determination of Acid Insoluble Ash
• Determination of Water Soluble Ash
• Determination of Moisture content
• Determination of Volatile Oil in Drugs
• Determination of Alcohol soluble extractive
• Determination of Water soluble Extractive
• Determination of ether soluble Extractive
• Starch Estimation
• Sugar Estimation
• Determination of Foaming Index
• Protein Estimation
• Fatty oil estimation
• Thin layer Chromatography
• Limit Tests
• Microbial Limit Tests
• Pesticide Residue
• Test for Aflatoxins
Sampling of drug:
Original sample:
When the total weight of the drug to be
sampled is less than 100kg, at least 250g are
withdrawn to constitute an original sample.
Test Sample:
Withdraw as much as may be necessary of
the original sample by quartering, taking care to
see that the portion is representative of the gross
sample.
In the case of unground or unpowdered
drugs, grind the sample so that it will pass
through a no. 22 sieve.
Foreign Matter and its Determination:
Medicinal plant materials should be entirely free from visible
signs of contamination by insects and other animal contamination etc..
No abnormal odour, discoloration, slime or signs of deterioration
should be detected.
Sample size: Roots, rhizomes and bark – 500g
Leaves, flowers, seeds and fruit – 250g
The foreign matter should be detected by inspection with the
unaided eye or by the use of a lens. Separate and weigh it and
calculate the percentage present.
Determination of Total 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 (e.g. sand and soil) adhering to the
plant surface.
Determination of Acid Insoluble Ash:
Acid – Insoluble ash is the residue obtained after
boiling the total ash with dilute HCl, and igniting the
remaining insoluble matter.
This measures the amount of silica present,
especially as sand and siliceous earth.
Determination of Water soluble ash:
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 Moisture content:
An excess of water in medicinal plant materials will
encourage microbial growth, the presence of fungi or
insects, and deterioration following hydrolysis. Limits for
water content should therefore be set for every given plant
material. This is especially important for materials that
absorb moisture easily or deteriorate quickly in the presence
of water.
Determination of volatile oil in drugs:
volatile oils are characterized by their odour, oil-like
appearance and ability to volatilize at room temperature.
Chemically, they are usually composed of mixtures of for
example, monoterpenes, sesquiterpenes and their oxygenated
derivatives.
Aromatic compounds predominate in certain volatile
oils, because they are considered to be the essence of the plant
material, and are often biologically active, they are also known as
essential oils.
The determination of volatile oil in a drug is
made by distilling the drug with a mixture of water and
glycerin, collecting the distillate in a graduated tube in
which the aqueous portion of the distillate is
automatically separated and returned to the distilling
flasks, and measuring the volume of the oil. The
content of the volatile oil is expressed as a percentage
v/w.
Water & Alcohol soluble extractive:
It plays an important role in evaluation of crude drugs. Less
extractive value indicates addition of exhausted material,
adulteration or incorrect process during drying, or storage or
formulating.
Ether soluble extractive:
Ether soluble extractive signifies the presence of fats, lipids, and
some steroids in the drug. Less extractive value indicates addition
of exhausted material, adulteration or incorrect process during
drying, or storage or formulating.
STARCH AND SUGAR ESTIMATION:
Starch analysis or starch grain analysis is a technique that is useful
to determine plant taxa.
Plant starch grain analysis is a diagnostic feature of multiple
applications according to the peculiarities and to the origin of the plant
material.The size, shape and structure of grains from plant species, varies
little, which can lead to identification.
DETERMINATION OF FOAMING INDEX:
Many Medicinal plant materials contain saponins that can cause a
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.
Protein assay is the biochemical assay for determining the total level of protein in
a solution. The total protein concentration is exhibited by a colour change of the
sample solution in proportion to the protein concentration.
Homogenise 100mg plant material with 3ml of 10% trichloroacetic acid.
Centrifuge the homogenate at 10,000 rpm, discard the supernatant. Treat the pellets
obtained after centrifugation with 3ml 1N NaOH , heat on water bath for 7 minutes
and cool. Centrifuge the solution again for 5 to 10 minutes at 5,000 rpm. To 0.5 ml of
supernatant does obtained after centrifugation, add 5ml reagent containing 100 pads
of 2% solution of sodium carbonate and one part of 2% solution of sodium potassium
tartrate. Allow it to stand for 10 – 15 minutes. Then add 5ml Folin and Ciocalteu’s
phenol reagent and allow to stand for half an hour for development of colour and then
finally measure the absorbance at 700 nm.
Fatty oil Estimation:
To estimate fatty oils , extract accurately weighed air dried powdered
plant material with petroleum ether in soxhlet apparatus.
Dry the extract over anhydrous sodium sulphate and remove the solvent
under vacuum at 400 C.
Weigh the residue and calculate the percentage with reference to the
plant material used.
Thin layer Chromatography:
TLC is particularly valuable for the qualitative determination os small
amounts of impurities. It is a technique in which a solute undergoes
distribution between 2 phases, a stationary phase and a mobile phase.
Limit test is defined as quantitative or semi-quantitative test
designed to identify and control small quantities of impurity
which is likely to be present in the substance.
Limit test is generally carried out to determine the inorganic
impurities present in the compound
Permissible limits of heavy metals:
SL No Heavy Metal Contents Permissible Limits
1 Lead 10ppm
2 Arsenic 3ppm
3 Cadmium 0.3ppm
4 Mercury 1ppm
The microbial limit tests are designed to perform the qualitative
and quantitative estimation of specific viable micro-organisms
present in the samples.
Microbial contamination limits:
SL NO Parameters Permissible Limits
1 Staphylococcus aureus Absent
2 Salmonella Absent
3 Pseudomonas
aeruginosa
Absent
4 Escherichia coli Absent
5 Total Microbial plate
count
105/ g
6 Total yeast and mould 103/ g
A pesticide is a substance or a mixture of substances used
for killing pests: organisms dangerous to plants or animals.
Pesticide residue refers to the pesticides that may remain
on or in the plant material after they are applied to the plant.
Many of these chemical residues, especially derivatives of
chlorinated pesticides, exhibit bio-accumulation which could build
up to harmful levels in the body as well as in the environment.
Maximum limit of pesticide residues for medicinal plant
materials:
Substance Limit (mg/kg)
Alachlor 0.02
Aldrin and Dieldrin (sum of) 0.05
Azinphos-methy1 1.0
Bromopropylate 3.0
Chlordane (sum of cis-, trans
– and Oxythlordane)
0.05
Chlorfenvinphos 0.5
Chlorpyrifos 0.2
Deltamethrin 0.5
Etc…
Aflatoxin:
These are a group of toxins consisting primarily of aflatoxins
B1, B2, G1, G2 and M1.
Aflatoxins are produced by the common fungi Aspergillus
flavus and the closely related species A.parasiticus.
Permissible Limit of Aflatoxins:
B1 & G1 – 0.5ppm
B2 & G2 – 0.1 ppm
• Powder Fineness.
• Specific gravity.
• Determination of pH values.
• Determination of Viscosity.
• Determination of Saponification Value.
• Determination of Iodine value
• Determination of Peroxide value.
• Rancidity tests.
Powder fineness
The degree of coarseness of a powder is expressed by reference to the nominal
mesh aperture size of the sieves for measuring the size of the powders.
Coarse powder – All the particles will pass through a No.2000 sieve, and not more
than 40% through a No. 355 sieve.
Moderately coarse – All the particles will pass through a No.710 sieve, and not more
than 40% pass through a No. 250 sieve.
Moderately Fine – All the particles will pass through a No.355 sieve, and not more
than 40% through a No. 180 sieve.
Fine – All the particles will pass through a No.180 sieve.
Very Fine – All the particles will pass through a No.125 sieve.
Specific Gravity :
The specific gravity of a liquid is the weight of a given volume of the
liquid at 25 0 compared with the weight of an equal volume of water at
the same temperature, all weighing's being taken in air.
pH Value:
pH value fundamentally represents the value of hydrogen ion activity
in solutions.
It represents the acidity or alkalinity of an aqueous solution. In the
pharmacopoeia, standards and limits on pH have been provided for these
pharmacopoeial substances in which pH as a measure of the hydrogen
activity is important from the stand point of stability or physiological
suitability.
Viscosity:
Viscosity is the quantity that describes a fluid’s resistance to
flow. Fluids resist the relative motion of immersed objects through
them as well as to the motion of layers with differing velocities
within them.
Saponification Value:
Saponification value is the number of mg of potassium
hydroxide required to neutralize the fatty acids, resulting from the
complete hydrolysis of 1g of the oil or fat. It is a measure of the
average molecular weight of all the fatty acids present.
Iodine value:
Iodine value of a substance is the weight of iodine absorbed by 100
part by weight of the substance. Used to determine the amount of
unsaturation in fatty acids.
Peroxide Value :
Detection of peroxide gives the initial evidence of rancidity in
unsaturated fats and oils.
Peroxide value is the number of milli-equivalents of active oxygen that
expresses the amount of peroxide contained in 1000g of the substance. The
amount of iodine produced is directly proportional to the iodine value.
Rancidity Test:
Rancidity is the process which causes a substance to become
rancid, that is, having a rank, unpleasant smell or taste. Specifically, it
is the hydrolysis and/or auto-oxidation of fats into short chain
aldehydes and ketones which are objectionable in taste and odour.
The test depends upon the formation of a red colour when
oxidised fat is treated with conc. Hydrochloric acid and a solution
of phloroglucinol in ether. The compound in rancid fats responsible
for the colour reaction is epihydrin aldehyde. All oxidized fats
respond the rancidity test and the intensity of the colour produced
is roughly proportional to the degree of oxidative rancidity.
• Estimation of Total Phenolics.
• Estimation of Total Tannins ( Tannins are the organic substance
present in some galls, barks and other plant tissues, consisting
of derivatives of gallic acid).
• Determination of Aluminium, Borax, Calcium, Copper, Iron,
Magnesium, Mercury, Silica, Sodium chloride.
Plant materials are used throughout the developed and developing
world as home remedies, in over-the-counter drug products, and as raw
material for the pharmaceutical industry, and they represent a substantial
proportion of the global drug market. Therefore, it is essential to establish a
standard guidelines for assessing their quality.
In order to establish a standard guidelines, it is recommended that
various government agencies should follow a more universal approach to
herbal quality and also developing monographs using the various quality
parameters outlined above. This will strengthen the regulatory process and
minimize quality breach.
The assurance of the safety and efficacy of a herbal drug requires
monitoring of the quality of the product from collection through processing to
the finished packaged product.
Certain herbs have become popular over the years, but the
general public, medical practitioners and the media still have a poor
understanding of safe and effective use of herbal medicine. Evidence is
emerging on the dangers of indiscriminate use of some of these herbs.
As in most situations, the truth lies hidden under the media hype,
poorly understood science, an exaggerated claim. The need for
standardization of herbals is now very essential given the global acceptance of
herbal products as remedies for various diseases and ailments.
The subject of herbal drug standardization is massively wide and deep.
There is so much to know and so much seemingly contradictory theories on
the subject of herbal medicines and its relationship with human physiology
and mental function.
For the purpose of research work on standardization of herbal
formulations, a profound knowledge of the important herbs found in India
and widely used in Ayurvedic formulation is of utmost importance.
Even when the chemical composition of a plant extract is known, the
pharmacologically active moiety may not be. Environment, climate, and
growth conditions influence composition, as does the specific part of the
plant and its maturity.
Dr.Lavanya.S.A -  standardization of herbal drugs

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Dr.Lavanya.S.A - standardization of herbal drugs

  • 1. STANDARDIZATION OF HERBAL DRUGS Under the guidance of: Dr.M.S.Doddamani HOD & Professor PG Dept of RS & BK T.G.A.M.C, Ballari. By Dr.Lavanya.S.A 2nd Year P.G Scholar Dept of RS & BK T.G.A.M.C, Ballari.
  • 2. INTRODUCTION In the last few decades there has been an exponential growth in the field of herbal medicine. It is getting popularized in developing as well as in developed countries owing to its natural origin and lesser side effect. In olden times, Vaidyas used to treat patients on individual basis, and prepare drug according to the requirement of the patient. But the scenario has changed now; medicines are being manufactured on the large scale in Pharmaceutical units, where manufacturers come across many problems such as availability of good quality raw material, authentication of raw material, availability of standards, proper standardization methodology of single drugs and formulation & quality control parameters.
  • 3. HERBS: Crude plant material such as leaves, flowers, fruit, seed, stems, wood, bark, roots, rhizomes or other plant parts, which may be entire, fragmented or powdered. HERBAL DRUGS: Finished labeled products that contain active ingredients such as aerial or underground parts of plant or other plant material or combinations thereof, whether in the crude state or as plant preparations
  • 4. Standardization of herbal drug refers to “confirmation of its identity and determination of its quality, purity and detection of nature of adulterant by various parameters like morphological, microscopical, physical, chemical and biological observations.”
  • 5. Standardization is used to describe all measures, which are taken during the manufacturing process and quality control leading to a reproducible quality. It also encompasses the entire field of study from birth of a plant to its clinical application. Standardization is the first step for the establishment of a consistent biological activity, chemical profile, or simply a quality assurance program for production and manufacturing.
  • 6. TOTAL 10000 SPECIES 325 425 550 1000 3500 8000 325 – Pesticides 425 – Gums, Resins & Dyes 550 – Fibre 1000 – Others 3500 – Edible 8000 – Medicinal Plants
  • 7. NEED FOR STANDARDIZATION In recent years there is a spurt in the interest regarding survival of Ayurvedic forms of medication. In the global perspective, there is a shift towards the use of medicine of herbal origin, as the dangers and the shortcoming of modern medicine have started getting more apparent, majority of Ayurvedic formulation are prepared from herbs. It is the cardinal responsibility of the regulatory authorities to ensure that the consumers get the medication, which guarantee, Purity, safety, potency and efficacy. This duty is discharged by the regulatory authorities by rigidity following various standards of quality prescribed for raw materials and finished products in pharmacopoeias controlling manufacturing formulate through the use of formularies and manufacturing operation through statutory imposed “Good manufacturing practices”.
  • 8. PROCEDURES FOR STANDARDIZATION OF HERBAL DRUGS In order to assure a consistent and acceptable quality herbal product, care should be taken right from the identification and authentication of herbal raw materials to the verification process of final products. The following parameters are recommended: • Authentication. • Physical parameters. • Quantitative and qualitative analysis. • Microbiological Contamination. • Pesticide residue. • Heavy Metal analysis.
  • 9. The first stage is identification of the plant species or botanical verification by the currently accepted Latin binomial name and synonyms. The steps involved in authentication are taxonomic, macroscopic & microscopic studies. Records should be maintained for stage of collection, parts of the plant collected, regional status, botanical identity such as phytomorphology, microscopial, histological analysis, taxonomical identity etc…
  • 10. Physical tests include organoleptic evaluation ( sensory characters such as taste, appearance, odour, feel of the drug etc…), viscosity, moisture content, pH, disintegration time, friability, hardness, flow ability, sedimentation, extract values and ash value
  • 11. Chromatographic and sophisticated modern techniques of standardization such as spectroscopic evaluation, spectrophotometry, TLC, HPTLC, HPLC, NMR, near infrared spectroscopy provide quantitative and semi-quantitative information about the main active constituents or marker compounds present in the crude drug or herbal products. Markers play an important role in fingerprinting of herbs. Quality of drug can also be assessed by chromatographic fingerprint.
  • 12. It can be measured according to parameters or methods described in the Ayurvedic Pharmacopoeia, as well as in the Indian pharmacopoeia. Microbiological analysis includes analysis of limits of E.Coli and molds, total viable aerobic count, total enteriobacteria and their count, aflatoxin analysis
  • 13. Standard limits of pesticides have been set by WHO and FAO ( Food and Agricultural Organization). Some common pesticides that cause harm to human beings are DDT, BHC, Toxaphene, aldrin etc should be analysed.
  • 14. Toxic metals such as Cu, Zn, Mn, Fe and particularly Cd, As, Pb and Hg should be analysed. In the analysis of metals their speciation is to be taken into consideration.
  • 15. The subject of herbal drug standardization is massively wide and deep. The guidelines set by WHO can be summarized as follows: • Reference to the identity of the Drug. • Reference to the Physicochemical Character of the Drug. • Reference to the Pharmacological parameters. • Toxicity Details. • Microbiological Parameters. • Radioactive Contamination.
  • 16. Reference to the identity of the drug: Botanical Evaluation: Botanical identity like phytomorphology, microscopical and histological analysis ( characteristic of cell walls, cell contents, starch grains, calcium oxalate crystals, trichomes, fibers, vessels etc..), sensory characters, foreign organic matter, histochemical evaluation, quantitative measurements etc… Reference to the pharmocological properties: Biological activity profiles, bitterness value, hemolytic index, astringency, swelling factor, foaming index etc..
  • 17. Reference to the physicochemical character of the drug: Physical and chemical identity, chromatographic fingerprints, ash values, extractive values, moisture content, volatile oil and alkoloidal assays, quantitative estimation protocols etc… Physical parameters include colour, odour, appearance, clarity, viscosity, moisture content, pH, disintegration time, friability, hardness, flow ability, flocculation, sedimentation, settling rate and ash values. Chemical parameters include limit tests, chemical tests, chemical assays. Chromatographic analysis of herbals can be done using TLC, HPLC, HPTLC, GC, UV, GC-MS, fluorimetry.
  • 18. Toxicity Details: Pesticide residue, heavy metals, microbial contamination like total viable count, pathogens like E.Coli, salmonella, P.aeroginosa, S.aurea, Enterobacteria. Microbiological parameters: It include total viable count, total mold count, total entero-bacterial count. Limiters can be utilized as a quantitative or semi-quantitative tool to ascertain and control the amount of impurities like the reagents used during abstraction of various herbs, impurities coming directly from the manufacturing vessels and from the solvents.
  • 19. First step Search for official & vernacular names of the drug. Formulary Pharmacopoeia Monographs Second step List the possible adulterants and substitutes. Pharmacopoeia Nighantu’s etc… Other books on controversial drugs like book by Balwant singh etc. Third step Taxonomically identify crude drugs for reference. Herbarium Drug Museum Collection Fourth step Pharmacognostical identification of official adulterant /substitute Botanical Physicochemical Chemical Identification
  • 20. IDENTIFICATION OF CRUDE DRUGS • Systematic study of crude drugs. • Microscopic Identification. • Determination of stomatal index. • Determination of vein-islet number.
  • 21. SYSTEMATIC STUDY OF CRUDE DRUGS Study of the drugs of plant which are used in their natural form is called crude drug. The study of these crude drugs made with a view to recognise them is called Pharmacognosy. Pharmakon = Drug ; Gignosco = To acquire knowledge of, meaning the knowledge of drug. In pharmacognosy a complete and systematic study of a drug is done, which comprises of:
  • 22. • Origin, common names, scientific nomenclature and family. • Geographical source and history. • Cultivation, collection, preservation and storage. • Macroscopical, microscopical and sensory characters. • Chemical composition. • Identity, purity, strength and assay. • Substitute and Adulterants.
  • 23. Macroscopical Characters: The morphological details of the respective part are given by observing it with naked eyes or with the aid of magnifying lens. In this description general conditions of the drug, size, shape, outer surface, inner surface etc.. Are referred to. This study can only be done if plant parts are available in entire condition. Sensory or Organoleptic characters: These describe colour, odour, taste, consistency etc… Chemical constituents: It can be identified by chemical and micro-chemical tests. Eg : Strychnine present in Nux-vomica gives purplish red colour with ammonium vanadate and Conc. Sulphuric acid.
  • 24. 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 reagents. Appropriate processing for separation and isolation of botanical debris from a formulation without loss of debris, by hand picking, shifting, washing, sedimentation etc…, are the priliminary steps. This is followed by clearing the debris in chemical reagents, reacting with suitable regents and stains and finally mounting a little part on a slide in a medium of suitable refractive index that helps to show the unit structures. MICROSCOPIC IDENTIFICATION
  • 25. Methods of examining crude vegetable drugs Entire material & powder Leaves and flowers Fruits and seeds Bark Roots and rhizomes
  • 26. In the mature leaf, four significantly different types of stoma are distinguished by their form and the arrangement of the surrounding cells, especially the subsidiary cells. 1. Anomocytic or ranunculaceous type: The stoma is surrounded by a varying number of cells, generally not different from those of the epidermis. 2. Anisocytic or cruciferous type: The stoma is usually surrounded by three or four subsidiary cells, one of which is markedly smaller than the others. TYPES OF STOMATA
  • 27. 3. Diacytic or Caryophyllaceous type: The stoma is accompanied by two subsidiary cells, the common wall of which is at right angles to the stoma. 4. Paracytic or rubiaceous type: The stoma has two subsidiary cells, of which the long axes are parallel to the axis of stoma.
  • 28. The stomatal index is the percentage of the number of stomata formed by the total number of epidermal cells, including the stomata, each stoma being counted as one cell. Stomatal index = S x 100 E + S Where S = No of stomata in a given area of leaf. E = No of epidermal cells in the same area of leaf.
  • 29. Determination of Palisade Ratio: Palisade ratio is the average number of palisade cells under one epidermal cell. E.g.- for the above picture, Palisade ratio = 18/4 = 4.5
  • 30. Determination of Vein- Islet Number: The mesophyll of a leaf is divided into small portions of photosynthetic tissue by anastomosis of the veins and veinlets ; such small portions or areas are termed as Vein-Islets. The number of vein-islets per square millimetre is termed the Vein-Islet number. This value has been shown to be constant for any given species and, for full grown leaves, to be unaffected by the age of the plant or the size of the leaves.
  • 31. QUANTITATIVE STUDY OF CRUDE DRUG: • Sampling of drug • Determination of foreign matter • Determination of Total Ash • Determination of Acid Insoluble Ash • Determination of Water Soluble Ash • Determination of Moisture content • Determination of Volatile Oil in Drugs • Determination of Alcohol soluble extractive • Determination of Water soluble Extractive
  • 32. • Determination of ether soluble Extractive • Starch Estimation • Sugar Estimation • Determination of Foaming Index • Protein Estimation • Fatty oil estimation • Thin layer Chromatography • Limit Tests • Microbial Limit Tests • Pesticide Residue • Test for Aflatoxins
  • 33. Sampling of drug: Original sample: When the total weight of the drug to be sampled is less than 100kg, at least 250g are withdrawn to constitute an original sample. Test Sample: Withdraw as much as may be necessary of the original sample by quartering, taking care to see that the portion is representative of the gross sample. In the case of unground or unpowdered drugs, grind the sample so that it will pass through a no. 22 sieve.
  • 34. Foreign Matter and its Determination: Medicinal plant materials should be entirely free from visible signs of contamination by insects and other animal contamination etc.. No abnormal odour, discoloration, slime or signs of deterioration should be detected. Sample size: Roots, rhizomes and bark – 500g Leaves, flowers, seeds and fruit – 250g The foreign matter should be detected by inspection with the unaided eye or by the use of a lens. Separate and weigh it and calculate the percentage present.
  • 35. Determination of Total 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 (e.g. sand and soil) adhering to the plant surface.
  • 36. Determination of Acid Insoluble Ash: Acid – Insoluble ash is the residue obtained after boiling the total ash with dilute HCl, and igniting the remaining insoluble matter. This measures the amount of silica present, especially as sand and siliceous earth. Determination of Water soluble ash: Water soluble ash is the difference in weight between the total ash and the residue after treatment of the total ash with water.
  • 37. Determination of Moisture content: An excess of water in medicinal plant materials will encourage microbial growth, the presence of fungi or insects, and deterioration following hydrolysis. Limits for water content should therefore be set for every given plant material. This is especially important for materials that absorb moisture easily or deteriorate quickly in the presence of water.
  • 38. Determination of volatile oil in drugs: volatile oils are characterized by their odour, oil-like appearance and ability to volatilize at room temperature. Chemically, they are usually composed of mixtures of for example, monoterpenes, sesquiterpenes and their oxygenated derivatives. Aromatic compounds predominate in certain volatile oils, because they are considered to be the essence of the plant material, and are often biologically active, they are also known as essential oils.
  • 39. The determination of volatile oil in a drug is made by distilling the drug with a mixture of water and glycerin, collecting the distillate in a graduated tube in which the aqueous portion of the distillate is automatically separated and returned to the distilling flasks, and measuring the volume of the oil. The content of the volatile oil is expressed as a percentage v/w.
  • 40. Water & Alcohol soluble extractive: It plays an important role in evaluation of crude drugs. Less extractive value indicates addition of exhausted material, adulteration or incorrect process during drying, or storage or formulating. Ether soluble extractive: Ether soluble extractive signifies the presence of fats, lipids, and some steroids in the drug. Less extractive value indicates addition of exhausted material, adulteration or incorrect process during drying, or storage or formulating.
  • 41. STARCH AND SUGAR ESTIMATION: Starch analysis or starch grain analysis is a technique that is useful to determine plant taxa. Plant starch grain analysis is a diagnostic feature of multiple applications according to the peculiarities and to the origin of the plant material.The size, shape and structure of grains from plant species, varies little, which can lead to identification. DETERMINATION OF FOAMING INDEX: Many Medicinal plant materials contain saponins that can cause a 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.
  • 42. Protein assay is the biochemical assay for determining the total level of protein in a solution. The total protein concentration is exhibited by a colour change of the sample solution in proportion to the protein concentration. Homogenise 100mg plant material with 3ml of 10% trichloroacetic acid. Centrifuge the homogenate at 10,000 rpm, discard the supernatant. Treat the pellets obtained after centrifugation with 3ml 1N NaOH , heat on water bath for 7 minutes and cool. Centrifuge the solution again for 5 to 10 minutes at 5,000 rpm. To 0.5 ml of supernatant does obtained after centrifugation, add 5ml reagent containing 100 pads of 2% solution of sodium carbonate and one part of 2% solution of sodium potassium tartrate. Allow it to stand for 10 – 15 minutes. Then add 5ml Folin and Ciocalteu’s phenol reagent and allow to stand for half an hour for development of colour and then finally measure the absorbance at 700 nm.
  • 43. Fatty oil Estimation: To estimate fatty oils , extract accurately weighed air dried powdered plant material with petroleum ether in soxhlet apparatus. Dry the extract over anhydrous sodium sulphate and remove the solvent under vacuum at 400 C. Weigh the residue and calculate the percentage with reference to the plant material used. Thin layer Chromatography: TLC is particularly valuable for the qualitative determination os small amounts of impurities. It is a technique in which a solute undergoes distribution between 2 phases, a stationary phase and a mobile phase.
  • 44. Limit test is defined as quantitative or semi-quantitative test designed to identify and control small quantities of impurity which is likely to be present in the substance. Limit test is generally carried out to determine the inorganic impurities present in the compound Permissible limits of heavy metals: SL No Heavy Metal Contents Permissible Limits 1 Lead 10ppm 2 Arsenic 3ppm 3 Cadmium 0.3ppm 4 Mercury 1ppm
  • 45. The microbial limit tests are designed to perform the qualitative and quantitative estimation of specific viable micro-organisms present in the samples. Microbial contamination limits: SL NO Parameters Permissible Limits 1 Staphylococcus aureus Absent 2 Salmonella Absent 3 Pseudomonas aeruginosa Absent 4 Escherichia coli Absent 5 Total Microbial plate count 105/ g 6 Total yeast and mould 103/ g
  • 46. A pesticide is a substance or a mixture of substances used for killing pests: organisms dangerous to plants or animals. Pesticide residue refers to the pesticides that may remain on or in the plant material after they are applied to the plant. Many of these chemical residues, especially derivatives of chlorinated pesticides, exhibit bio-accumulation which could build up to harmful levels in the body as well as in the environment.
  • 47. Maximum limit of pesticide residues for medicinal plant materials: Substance Limit (mg/kg) Alachlor 0.02 Aldrin and Dieldrin (sum of) 0.05 Azinphos-methy1 1.0 Bromopropylate 3.0 Chlordane (sum of cis-, trans – and Oxythlordane) 0.05 Chlorfenvinphos 0.5 Chlorpyrifos 0.2 Deltamethrin 0.5 Etc…
  • 48. Aflatoxin: These are a group of toxins consisting primarily of aflatoxins B1, B2, G1, G2 and M1. Aflatoxins are produced by the common fungi Aspergillus flavus and the closely related species A.parasiticus. Permissible Limit of Aflatoxins: B1 & G1 – 0.5ppm B2 & G2 – 0.1 ppm
  • 49. • Powder Fineness. • Specific gravity. • Determination of pH values. • Determination of Viscosity. • Determination of Saponification Value. • Determination of Iodine value • Determination of Peroxide value. • Rancidity tests.
  • 50. Powder fineness The degree of coarseness of a powder is expressed by reference to the nominal mesh aperture size of the sieves for measuring the size of the powders. Coarse powder – All the particles will pass through a No.2000 sieve, and not more than 40% through a No. 355 sieve. Moderately coarse – All the particles will pass through a No.710 sieve, and not more than 40% pass through a No. 250 sieve. Moderately Fine – All the particles will pass through a No.355 sieve, and not more than 40% through a No. 180 sieve. Fine – All the particles will pass through a No.180 sieve. Very Fine – All the particles will pass through a No.125 sieve.
  • 51. Specific Gravity : The specific gravity of a liquid is the weight of a given volume of the liquid at 25 0 compared with the weight of an equal volume of water at the same temperature, all weighing's being taken in air. pH Value: pH value fundamentally represents the value of hydrogen ion activity in solutions. It represents the acidity or alkalinity of an aqueous solution. In the pharmacopoeia, standards and limits on pH have been provided for these pharmacopoeial substances in which pH as a measure of the hydrogen activity is important from the stand point of stability or physiological suitability.
  • 52. Viscosity: Viscosity is the quantity that describes a fluid’s resistance to flow. Fluids resist the relative motion of immersed objects through them as well as to the motion of layers with differing velocities within them. Saponification Value: Saponification value is the number of mg of potassium hydroxide required to neutralize the fatty acids, resulting from the complete hydrolysis of 1g of the oil or fat. It is a measure of the average molecular weight of all the fatty acids present.
  • 53. Iodine value: Iodine value of a substance is the weight of iodine absorbed by 100 part by weight of the substance. Used to determine the amount of unsaturation in fatty acids. Peroxide Value : Detection of peroxide gives the initial evidence of rancidity in unsaturated fats and oils. Peroxide value is the number of milli-equivalents of active oxygen that expresses the amount of peroxide contained in 1000g of the substance. The amount of iodine produced is directly proportional to the iodine value.
  • 54. Rancidity Test: Rancidity is the process which causes a substance to become rancid, that is, having a rank, unpleasant smell or taste. Specifically, it is the hydrolysis and/or auto-oxidation of fats into short chain aldehydes and ketones which are objectionable in taste and odour. The test depends upon the formation of a red colour when oxidised fat is treated with conc. Hydrochloric acid and a solution of phloroglucinol in ether. The compound in rancid fats responsible for the colour reaction is epihydrin aldehyde. All oxidized fats respond the rancidity test and the intensity of the colour produced is roughly proportional to the degree of oxidative rancidity.
  • 55. • Estimation of Total Phenolics. • Estimation of Total Tannins ( Tannins are the organic substance present in some galls, barks and other plant tissues, consisting of derivatives of gallic acid). • Determination of Aluminium, Borax, Calcium, Copper, Iron, Magnesium, Mercury, Silica, Sodium chloride.
  • 56. Plant materials are used throughout the developed and developing world as home remedies, in over-the-counter drug products, and as raw material for the pharmaceutical industry, and they represent a substantial proportion of the global drug market. Therefore, it is essential to establish a standard guidelines for assessing their quality. In order to establish a standard guidelines, it is recommended that various government agencies should follow a more universal approach to herbal quality and also developing monographs using the various quality parameters outlined above. This will strengthen the regulatory process and minimize quality breach.
  • 57. The assurance of the safety and efficacy of a herbal drug requires monitoring of the quality of the product from collection through processing to the finished packaged product. Certain herbs have become popular over the years, but the general public, medical practitioners and the media still have a poor understanding of safe and effective use of herbal medicine. Evidence is emerging on the dangers of indiscriminate use of some of these herbs. As in most situations, the truth lies hidden under the media hype, poorly understood science, an exaggerated claim. The need for standardization of herbals is now very essential given the global acceptance of herbal products as remedies for various diseases and ailments.
  • 58. The subject of herbal drug standardization is massively wide and deep. There is so much to know and so much seemingly contradictory theories on the subject of herbal medicines and its relationship with human physiology and mental function. For the purpose of research work on standardization of herbal formulations, a profound knowledge of the important herbs found in India and widely used in Ayurvedic formulation is of utmost importance. Even when the chemical composition of a plant extract is known, the pharmacologically active moiety may not be. Environment, climate, and growth conditions influence composition, as does the specific part of the plant and its maturity.