Transcript - ISpatula

9. Determination of Bitterness value
 Bitters are the medicinal plant materials
that have a strong bitter taste and
employed therapeutically, mostly as
appetizing agent.
 Their bitterness stimulate secretion in the
GIT especially of gastric juice.
 The total bitterness need to be measured
by taste, which is main physiological
parameters.
• As prescribed by WHO 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 HCl.
10. Determination of Hemolytic activity
 Saponin have characteristic of frothing
property and have ability to cause haemolysis
when added to a suspension of blood.
 Many medicinal plant material especially
derived from family: Caryophyllacea, Aralacae,
Sapindacea, Primulaceae & Dioscoreacea
contain saponin.
 Method of determination: by comparing with
reference material Saponin which have
hemolytic activity 1000 unit per g.
11. Determination of Swelling index
• Swelling factor: measuring the volume of
mucilage produced in 24 hours for 1 g of
seed.
• Many medicinal plant materials are of
specific therapeutic utility because of their
swelling properties.
• The swelling index is the volume in ml taken
up by the swelling of 1 g of plant material
under specified condition.
12. Determination of foaming index
• The saponin are high Mwt containing
phytoconstituents having detergent activity.
• Many medicinal plant materials contain saponin
that can cause a persistent foam when an
aqueous decoction is shaken.
• The foaming ability of an aqueous decoction of
plant material is measured in term of foaming
index.
13. Determination of Tannins
• Tannins are widely distributed in plants and
occur in solution in cell sap, often in the
distinct vacuoles.
• Tanins are substance capable of turning
animal hides into leather by binding
proteins to form water insoluble substance
that are resistant to proteolytic enzyme.
14. Determination of Arsenic and Heavy metals
 The medicinal plant material can be
contaminated with arsenic and heavy metals
which can be attributed to many causes
including environmental pollution and traces of
pesticides.
 As these components even in trace amounts
are dangerous, they have to be removed from
the herbal drugs.
 Limit tests for these material have been
prescribed in almost all the pharmacopoeia.
15. Determination of pesticides
 Pesticides: simple substances or mixtures used to eliminate
undesirable vegetable and animal life in agricultural and upon
ecosystem.
 Chemical pesticide mainly organochlorine, organophosphorous,
carbamate & triazine have pronounced physiological activity and
cause unpleasant S/E, such as disturbance of biological
equilibrium.
 e.g. Organochlorine: over stimulation of CNS
Organophosphorous: potent cholinesterase inhibitors
(CNS symptoms such as restlessness, depression of
respiratory and cardivascular system.
 Standardized phytopharmaceuticals must have a known content
of active characteristics substance, exogenous substances must
be below specified limits recommended.
Methods for determination of pesticide residues
 Chromatography (GC/HPLC) is the principle method
for determination of pesticide residues.
 Other method also available:
Measuring total organic phosphorous and total
arsenic or total lead.
 As prescribed by WHO if the pesticide to which the
plant are exposed are known or cab be identified by
suitable method, a well established method for
determination of this pesticide residue should be
employed.
 When pesticide are unknown , it may be necessary
to measure total organic chlorine, phosphorous,
arsenic and lead.
Removal of pesticide from phytopharmaceuitcals
• Many alternative methodology have to be
developed for the removal of pesticide from
crude drug.
• DDT in Cassia angustifolia leaves and
podscan be removed by supercritical fluid
extraction with CO2 at 100 bar.
• Preserve herbal drug using CO2 pressurized
up to 40 bar at R.T for 1.2 hour, this is
harmless and effective.
Analysis of crude drug extracts and isolates
 Quantitative chemical analysis by determine the
purity of the isolated substance or the concentration
of a single substances or a group of substances in a
mixture by finger printing and different other
techniques.
 The nature of the substance (chemical group,
specific identity, polarity,..etc) in the mixture can be
determined by a number of means including
chromatographic and wet chemical test.
 For isolated compound whose structure is less
certain , additional methods such as MS, UV, NMR
can be applied to elucidate unambiguously the
molecular structure of compound.
Analytical Methods
 The quantitative determination of constituents has been made
easy by recent developments in analytical instrumentation.
Recent advances in the isolation, purification, and structure
elucidation of naturally occurring metabolites have made it
possible to establish appropriate strategies for the
determination and analysis of quality and the process of
standardization of herbal preparations. Classification of plants
and organisms by their chemical constituents is referred to as
chemotaxonomy. TLC, HPLC, GC, quantitative TLC (QTLC), and
high-performance TLC (HPTLC) can determine the homogeneity
of a plant extract. Over-pressured layer chromatography
(OPLC), infrared and UV-VIS spectrometry, MS, GC, liquid
chromatography (LC) used alone, or in combinations such as
GC/MS, LC/MS, and MS/MS, and nuclear magnetic resonance
(NMR), are powerful tools, often used for standardization and
to control the quality of both the raw material and the finished
product. The results from these sophisticated techniques
provide a chemical fingerprint as to the nature of chemicals or
impurities present in the plant or extract.
Parameters required for quality evaluation of
herbal drugs
Standardization & Quality Evaluation of Herbal drugs
QUALITY EVALUATION OF
HERBAL DRUGS
Example on standardization
 Paronychia argentea
 Paronychia argentea has many folk names: Rijl El Hmameh in
Jordan,Mountain Knot Grass in England, Bessat Al- Ard in North Africa, and
other common names as Silver nailroot, and Silvery whitlow Wort.
 Edible Uses: Tea. An infusion of the flowers is used as a tea.
 Folkloric uses:
Paronychia argentea has many uses in folk medicine, infusion of entire plant,
except the roots, is used to expel renal stones, it has also been used as a
diuretic, febrifuge, appetizer, or as mild aphrodisiac. It is also used in the
treatment of TB.
 Medicinal uses:
Many studies showed that Paronychia argentea has hypoglycemic activity, and
it has been proved to be useful as gastric analgesic, bladder, prostate,
abdominal ailments treatment, and stomach ulcers treatment, it also showed
significant alpha amylase and acetyl cholinesterase (AChE) enzyme inhibitory
activity, the plant extract of Paronychia argentea showed antioxidant activity.
Other in vivo and invitro studies on different extracts from Paronychia
argentea revealed the immunomodulating activity of the plant.
• Chemical Constituents:
• Many chemical compounds have been
identified and isolated from Paronychia
argentea as: jaceosidin, tricin, nepetin,
octadecanoic acid, 1-docosanol, glycerol
octadecanoate monoester, β- sitosteryl
glucoside, β- sitosterol, luteolin-3- methyl
ether, ethyl-α- D- galacto- pyranoside,
Dpinitol and vanillic acid, luteolin
OH
O
OH
OH
HO
O
OMe
OH
Vanillic acid
OH
O
Luteolin
Standardized using vanillic acid:
Mobile phase: Methanol : Water 1%Acetic acid (30:70) ,
flow rate =1.0 ml/min, at 260 nm
Example 2: Artemisia dracunulus
Example 3: Ginkgo biloba dosage
forms
• High-performance liquid chromatographic
(HPLC) - photodiode array (PDA) detection
method to quantitate five flavonol components
as markers; rutin, quercitrin, quercetin,
kaempferol and isorhamnetin for use in the
quality control of Ginkgo biloba dosage forms.
•
Figure 1: HPLC chromatogram of reference standards, rutin, quercitrin, quercetin, kaempferol
and isorhamnetin with corresponding retention times at l = 350nm. Separation of the
flavonols was achieved at 45°C on a minibore Phenomenex Luna 5mm C18 (2) column with
dimensions 250 x 2.00mm using a one step linear gradient and flow rate of 0.4 ml/min. Mobile
phase A (acetonitrile) and B (0.3% formic acid) ratios where changed after 15 minutes from
15:85 to 25:75 and total run time was 33 minutes.
Figure 2: Overlaid HPLC chromatograms of the reference standards including
the profiles of the analysed Ginkgo biloba dosage forms (λ = 350nm).
Adulteration
 Adulteration: Substitution of the original crude drugs
partially or fully with other substances which is either
free from or inferior in therapeutic and chemical
properties, may be deliberate or accidental
 Admixture: addition of one article to another through
accident, ignorance or carelessness, e.g. soil in
underground organ, co-collection of two similar
species.
 Sophistication: deliberate addition of superior or
inferior material with intent to defraud, e.g. powder
ginger may be diluted with starch with addition of little
coloring material to give yellow color.
 Substitution: addition of an entirely different article in
place of that which required, e.g. cotton seed oil in
place of olive oil.
Types of adulteration or substitution of herbal drug
1. Substitution with inferior commercial verities due to
morphological resemblance to authentic drugs, e.g.
capsicum annum fruits and Japanese chilies for fruit of
capsicum minimum.
2. Adulteration by artificially manufactured substitutes, e.g.
artificial invert sugar for honey.
3. Substitution by Exhausted drugs, e.g. volatile oil contain
clove, fennel etc.
4. Substitution by superficially similar but cheaper natural
substance, e.g. leaves of species-Ailanthus are
substituted for belladonna, senna.
5. Adulteration by addition of worthless heavy materials,
e.g. pieces of limestone are found in asafetida.
6. Addition of synthetic principles, e.g. add citral to oil of
lemon.
7. Use of vegetative matter from the same plant.
Causes and measures of adulteration
 Adulteration have caused a major problem in the
research on commercial natural products. Adulteration
is implicitly by manufacturers who purchase only low
priced ingredient by accepting certificate of purity as
sole proof of quality and by common practice of
employing “Label claims” in cosmetic industry.
 Detection of adulteration using many different means of
detection which will establish (1) identity of adulterant,
(2) determine quality of drug.
 Guarantee purity & quality of natural product:
Appropriate level of testing must be carefully assessed
before using raw material based on the monographs
available in different official books as will as regulatory
guidelines.
Deterioration
 Deterioration: impairment of the quality on value of an
article due to destruction or abstraction of valuable
constituents by bad treatment or aging or to the
deliberate extraction of the constituents and the sale of
the residue as the original drug.
 Crude drug are prone to deterioration on storage.
 The shelf life of crude drug influenced by many factors,
include not only the quality of storage condition but
also the stability of the secondary metabolite.
 Primary factors:
1. Light 2. Moisture/Humidity 3. Temperature
4. Airic oxidation, e.g. volatile oil with terpenoid resinified
& fixed oil rancidification
 Secondary factors
Living organism usually develop in stored drugs where
the condition are satisfactory for them.
1. Bacteria & Moulds:
dried herbs are liable to contamination by spore of
bacteria and moulds present in air.
2. Mites & Nematode worms:
If found, mits are present in counless number up to 1.0
mm length, examined by microscope.
3. Insects & Moths:
The moths involved are unspectacular in appearance ,
22-30 mm with off-white wings, besides some insects ,
cockroaches, ants are found cause deterioration of
product.
4. Coleoptera or Beetles:
These are the insects that constitutes the largest order of
the animal kingdom, e.g. stegobion paniceum in
liquarice.
Control measure for Deterioration
 The container used for storage and its closure must not interact
physically or chemically with material within in any way would
alter its composition.
 A well closed container must protect the contents from
extraneous matter or from loss of material while handling and
tightly closed container must protect the material from
deliquescence or evaporation under normal condition.
 Storage area should be kept clean and spillages not allowed to
enter cracks or accessible crevices.
 Periodic spraying of the premises with insecticide prevent the
spread of infestation.
 Elimination effect of deterioration due to both the primary &
secondary factors are desirable.
 Cool & dry condition is most suitable condition of living
organism.
• Crude drugs purchased by the herbalist may
already have been sterilized by treatment of
bulk consignment with ethylene oxide or
methyl bromide under controlled
conditions, dry treatment like this should
comply with acceptable limit of toxic
residues,
e.g. senna pods 50ppm ethylene oxide