Evaluation of Natural Products

Content

Evaluation of Natural Products

• Morphological
evaluation

• Microscopical
evaluation

• Chemical
evaluation

• Physical
evaluation

• Spectroscopic
methods

• Biological
methods

Objective

• At the end of this
lecture, students will be able to

• Discuss the
importance of evaluation of crude drugs

• Discuss the
various parameters involved in Morphological, Microscopical, Chemical, Physical,
Spectroscopic and Biological evaluation

Evaluation of Natural Products

Evaluation

• Confirmation of
identity,

• Determination
of quality and purity

• Detection of
adulterants

Need of Evaluation

• Biochemical
variation in the drug

• Detoriation due
to treatment and storage

• Substitution
and adulteration – carelessness, ignorance

Type of Evaluation

• Morphological
evaluation

• Microscopical
evaluation

• Physical
evaluation

• Chemical
evaluation

• Spectroscopical
evaluation

• Biological
evaluation

Morphological or organoleptic evaluation

Qualitative
evaluation based on morphology and sensory profile of drugs

• Colour

• Odour

• Taste

• Size

• Shape

• Special
features like touch, texture, fracture

Examples: Shapes

• Ovoid tears –
Acacia

• Ribbon shape –
Tragacanth

• Disc shape-
Nuxvomica seed

• Conical –
Aconite

• Quills – Barks

• Wavy – Rauwolfia

Colour:

• Brown colour-
Cinnamon

Odour:

• Aromatic odour
– Umbelliferous fruits

Taste:

• Bitter Taste –
alkaloids containing drugs

• Sweet taste –
Liquorice

• Pungent taste-
Capsicum, Ginger

• Astringent
taste- asafoetida, black pepper, nutmeg, caraway, cumin

• Acrid taste –
Castor oil, Chaulmoogra oil

• Bland taste –
Arachis oil, sesame oil,

Fracture

• Short –
Cinchona

• Splintery –
Cinnamon bark

• Short and
granular – Cascara bark

• Laminated –
Quillaia bark

• Over drying-
brittle- morphological evaluation – difficult task

Microscopical Evaluation

• Evaluation of
crude drugs – microscopical / histological characters

• Qualitative
evaluation of organized drugs in whole and powder form

• Microscopical
evaluation can be performed by

– Sectioning of the drug – T.s

– Histochemical test

– Powder microscopy

– Quantitative microscopy – Leaf constants

                                              
–  Lycopodium spore method

Histological
studies- thin sections

Characteristics-

• Cell wall

• Cell contents –
Starch grains, Crystals

• Trichomes

• Fibres

• Vessels

• Lignified
trichome

• Warty trichome

• Wavy medullary
rays of cascara bark

• Glandular
trichome of mint

Histochemical
test

• Lignin –
Phloroglucinol and Conc. HCl

• Mucilage –
Ruthenium red

• Starch – N/50
iodine

• Phenolic
compounds – Ferric chloride

• Alkaloids –
Dragendroff’s reagent

• V. oil – Sudan
red III

Powder
microscopy

Helps in the identification of crude drugs and detecting
adulterants

1. Identification – senna – Paracytic stomata, unicelluar
covering trichomes

2. Detection of adulterants – Clove powder

• Powdered clove
stalk- sclereids, prism calcium oxalate cr

• Mother clove –
Powdered clove fruits – starch

• Powdered clove
flower bud:  No starch

                            
                    No
sclereids

                                                 Cluster crystals

Powder
microscopy – Digitalis

1. Comfrey leaves – Symphytum officinale – Hook at the top

2. Primrose leaves – Primula vulgaris – 6-12 celled

3. Mullein leaves – Verbascum thapsus – Cadelabra trichomes

Microscopical
linear measurements/quantitative microscopy

• Dimension of the cell (fiber, stone cell, trichome), cell
contents (starch grains), pollen grains of crude drug both in entire form or powder
form

1. Micrometry   Eye
piece micrometer

                                Stage
micrometer

2. Camera lucida              

• Calibrate eye piece micrometer – calibration factor

• 0 – 0 coincidence

• Next consecutive three coincidence – noted down

• Calibration factor

• 3 division of eye piece micrometer = 4 divisions of stage
micrometer

• 1 division stage = 0.01 mm = 10 µ

• 4 divisions = 40 µ

• 1 division of eye piece micrometer = 40/3 = 13.3

• Average calibration factor 
= 13

Camera
lucida

1. Swift Ives camera lucida

2. Abbe’s camera lucida with sliding mirror

Leaf
constants

• Stomatal number

• Stomatal index

• Palisade ratio

• Vein islet number

• Vein termination number

Stomata

– Stomatal pore

– Guard cells

– Subsidiary cells

Types of Stomata

– Moss

– Gymospermous

– Gramineous

– Dicot

 Dicot

–  Paracytic

–  Diacytic

–  Anamocytic

–  Anisocytic

• Paracytic / Parallel celled / Rubiaceous

• Diacytic / Cross celled (Diagnol) / Caryophyllaceous

• Anamocytic / Irregular celled (anamos=many) /
Ranunculaceous

• Anisocytic / Unequal celled / cruciferous

Microscopical
Evaluation – Trichomes

• Elongated tubular outgrowth of epidermal cell

• Plant hairs

• Any part – leaf, seed, fruit etc/ absent in roots

• Function- Protective, secertion of V.oil (Mentha spp),
absorption or secretion of water (Piper betel)

1.   Covering / Non
glandular / Clothing

A.  Unicellular                   

B. Multicellular

Un branched

• Uniseriate

• Biseriate

• Multiseriate

Branched

• Stellate

• Peltate

• Candelabra

• T- shaped

2. Glandular

3. Hydathodes / Special type

Unicellular
covering trichomes

• Lignified – Nuxvomica

• Short, sharp, curved – Cannabis Large, conical, shrunken –
Lobelia Short, conical, unicellular – Tea

• Strongly waved, thick walled – Yerba santa

Multicellular
covering trichomes

• Uniseriate

– Bi cellular, conical – Datura

– Three celled – Stramonium

– Three to four celled – Digitalis

– Four to five celled – Belladona

• Biseriate- Calendula officinalis

• Multiseriate – Male fern

Multicellular
Branched trichomes

• Stellate – Hammamelis

• Peltate – Humulus

• Candelabra – Verbascum thapsus

• T shaped – Pyrethrum

     

GLANDULAR
TRICHOMES

Unicellular:
Stalk absent – betel, vasaka

Multicellular:

• Unicellular head/unicellular stalk – Digitalis purpurea

• Unicellular head / uniseriate multicellular stalk –
Digitalis thapsi, Belladona

• Multicellular head / multicellular biseritae stalk –
Sunflower, compositae

• Unicellular stalk / biseriate head – Digitalis purpurea

• Short stalk / rosette head – Mentha

• Multicellular multiseriate cylindrical stalk / rosette
secretory head – Cannabis

• Multicellular uniseritae stalk / Multicellular
multiseriate head – Indian hemp,

Microscopical
Evaluation – Mineral Crystals

• Crystalline deposits

• Present in any part of the plant

• Insoluble in water

• Calcium oxalate, calcium carbonate and silica

• Occur in various forms like prisms, acicular, raphides,
clusters, rosettes, druses etc

• Have – diagnostic value

• Significance – identification and detection of adulterants

• Identification

• Prisms:
Quillaia, Senna, Liquorice, Wild Cherry

• Acicular/Raphides:
Cinnamon, Squill, Gentian, Andrographis

• Rosettes or
clusters: Cascara, Clove, Arjuna, Eucalytpus

• Microsphenoidal or
sandy: Cinchona, tobacco, henbane

Examples: Detection
of adulterants and substitutes

Clove Stalk – prism type, flower bud – no crystal

• Absent – digitalis, nutmeg, linseed, colchicum

• Powdered drugs

Lycopodium
spore method

• When chemical and other methods fail

• Inexpensive method- official

• Lycopodium spores-

– Characteristic in shape,

– Uniform in size 25 micron

– 94,000 spores per mg of powdered lycopodium

• Well defined particles that can be counted (starch, pollen
grains)

• Single layered cells/ tissues and the area can be traced

• Objects with uniform thickness- length of which can be
measured

[(NxWx94,000)/(SxMxP)]*100

N=No. f characteristic structures in 25 fields

W= Wt in mg of lycopodium taken

S = No. of lycopodium spores in the same 25 fields

M= wt of sample in mg

P = 2,86,000 in case of ginger

• 100 mg of ginger powder and 50 mg of lycopodium powder

• Suspending fluid – Glycerine, tragacanth gum and water
(2:1:2)

• Dilute till about 15-20 spores are observed in a single
field

• Add iodine, count the number of spores and starch grains
in 25 different fields

• Determine the percentage purity of ginger

• % purity = (NxWx94,000×100)/(SMP)

Chemical Evaluation

• Chemical tests and assays

• Chemical test – Preliminary phytochemical screening

Preliminary
Phytochemical screening – Successive Solvent Extraction

• Air dried plant material is made into coarse powder

• Powder is extracted in Soxhlet assembly successively with
solvents in order of increasing polarity,

i.e

Pet ether/n-hexane à
Toluene/Benzene à
Chloroform à
Acetone à
Ethyl acetate à
ethanol Water (maceration)

• Each time before extracting with next solvent, the powder
is dried below 50 ⁰C

• Each extract is concentrated by distilling of solvent

• Evaporated to dryness on water bath

• Extracts with different solvents can also be prepared by
maceration

Qualitative
Chemical Examination

Test for
Carbohydrates

Dissolve small quantity of alcohol and aqueous extracts
separately in distilled water and filter. Subject the filtrate to various tests

Test for
Proteins

Test for
Lipids

Test for
Phytosterols

Reflux petroleum ether, benzene and alcohol extracts
separately with alcoholic KOH till complete saponification takes place. Dilute
the saponified mixtures with distilled water and extract with solvent ether.

Evaporate ethereal extract to dryness and subject the residue
to Liebermann-Burchard’s test

Test for
Alkaloids

Stirr the small portions of solvent-free chloroform, alcohol
and aqueous extracts separately with a few drops of dilute hydrochloric acid
and filter. The filtrate was tested with various alkaloid reagents

Test for
Flavonoid

Dissolve small quantity of alcohol and aqueous extracts
separately in distilled water and filter. Subject the filtrate to various tests

Test for
Tannins and Phenolic Compounds

Dissolve small quantity of alcohol and aqueous extracts
separately in distilled water and filter. Subject the filtrate to various tests

Test for
Glycosides

Small quantity of alcohol and aqueous extracts were
dissolved separately in distilled water and filtered. It is then hydrolysed
with dilute hydrochloric acid for a few hrs (2 to 4 h) on a water bath and
subjected to various tests to detect the presence of different glycosides

• Cardiac glycosides

• Anthraquinone glycosides

• Saponin glycosides

Test for
Cardiac Glycosides

Test for
Anthraquinone Glycosides

Test for
Saponin Glycosides

 • Qualitative
chemical test- detection of adulteration

– Halphens test – Cotton seed oil

– Van urk test  –
Ergot

– Vitalis morin test – Tropane alkaloid

– Murexide  test –
Purine base

Quantitative
analysis/ Chemical assays

• Lipid analysis – Acid value, Sap value etc

• Resin – sulphated ash, acid value

• Balsams – acid, saponification, ester

• Volatile oil – acetyl, ester

• Gums – methoxy

• Cineole in eucalyptus oil

• Aldehyde in lemon oil

• Carvone in caraway, dill oil

• Balsamic acid, cinnamic acid in balsam of tolu, balsam of
peru, prepared storax

Titrimetric
estimations

Total alkaloid
content

• Opium (M)

• Belladona (A)

• Ipecacuanha (E)

• Nux vomica (S)

• Cinchona (Q)

• Rauwolfia (R)

Physical Evaluation

• Identify, determine purity and quality

Viscosity

• Used to determine the purity and quality

• Constant at given temp

• Index of its chemical composition

• Liquid drugs

• Eg. Liq. Paraffin – NLT 64 Centistokes

Melting
point

• Purity

• Sharp and constant – Pure chemicals/ phytochemical

Solubility

• Identify and Presence of adulterants- solubility studies

• Castor oil

• Colophony in light petroleum

• Alkaloidal bases

• Alkaloidal salts

• Glycosides

Optical
rotation

• Rotating plane polarized light in pure state or in
solution – optically active, Property- Optical rotation

• Right- dextro

• Left -Laevo

• 25^OC –sodium lamp –source of light

Refractive
index

• Purity, constant

• Ratio of velocity of light in vacuum to its velocity in
the substance

• Varies – wavelength, temperature, pressure

Ash Values

• Remain after incineration – inorganic salts – natural or
added

• Physiological ash – tissues itself

• Non physiological ash – extraneous materials- sand dirt
etc

– Total ash – Carbonates, oxides, phosphates, silicates,
silica

– Acid insoluble ash – HCl- adhering dirt and sand

– Water soluble ash

Extractives

• Extracting by exhausting crude drugs – indication of
approximate measures of the chemical constituents

• Various solvents used – nature of chemical compounds

• Water soluble
extractives – tannins, sugars, plant acids, mucilage, glycosides etc

• Alcohol soluble
extractives – Tannins, resins

• 20 – 95% alcohol used

• Dilute alcohol may also be used

Moisture
content

• Active chemical content expressed on air dried basis

• Decompose due to chemical or microbial contamination

– Loss on drying

– Azeotropic distillation method

– Karl Fischer method

Loss on drying

• 105⁰C to a constant weight

• Volatile actives – toluene distillation method

Azeotropic
distillation method

• Volatile oil content

• Hydro distillation method

Foreign
organic matter

• Parts of organ or organs other than those mentioned in the
definition and description of drug

• Maximum limit – monographs

Spectroscopic Methods

UV- visible Spectroscopic 

• Based on the light absorption by the substances

• 190-380 nm UV

• 380-900 nm Visible

• Electronic transition

• Single beam: Light through monochromator- sample –
detector

• Double beam: light through sample and through blank –
ratio

• Rapid scanning spectrophotometers – Multichannel detectors

• Differential spectroscopy – Presence of extraneous
materials

• Dual wavelength spectroscopy – two monochromatic beam of
different wavelengths

– Lobeline – 249 nm

– Reserpine 268 nm

– Morphine 286 nm

– Colchicine 360 nm

– Vannilin 301 nm

Visible
range

• Morphine 442 nm

• Anthraquinone 505 nm

• Ergot alkaloids – 550 nm

• Cardio active glycosides 590 nm by keller kiliani reaction

• Cyanogenetic glycosides 630 nm by pyridine pyrazolone
reaction

Sample

• 1 N NaOH

• 1 N HCl / Nitric acid with equal volume of water

IR
Spectroscopy

• Reflected, absorbed or transmitted radiant energy in the
electromagnetic spectrum ranging from 0.8 -500 cm

• Commonly used measurement is frequency –wave number

IR range

• Near IR -12500 -4000 cm^-1

• Mid IR – 4000 – 400 cm^-1

• Far IR 400 – 20 cm^-1

• Mid IR – commonly referred IR

• Single or double beam

• Fourier transform IR (FT-IR)- recent advancement in IR,
coupled with GC/LC

• Sensitive – drugs, polymorphic modifications, exipients,
raw materials

• Sample type- any; insoluble solids, polymers, solutions or
gases

• Identical spectra – two samples with same chemical
structure

• IR- detection of functional groups- structural elucidation

• IR – Quantitative analysis

Fluorescence
Analysis

• Absorbance and re emission – Luminescence

• Reemission during receiving only – Fluorescence

• Fluorescence in visible range

– Wild cherry bark

– Belladona leaf, root

– Gambier catechu

– Aloes

• Fluorescence in UV range

– Cinchona: luminous yellow, with light blue patches

– Calumba: Yellow, phloem and cambium – dark green
fluorescence

– Hydrastis: golden yellow

– Ergot: red

– Olive oil: deep golden yellow

• Quantitative analysis – flouorimeter/ spectrofluorimeter

Nuclear
Magnetic Resonance (NMR)

• Absorption of radio frequency radiation when the sample is
kept in magnetic field

• Absorption – interaction of radiation with magnetic moment
of nuclei in the sample and it occurs at different frequencies for nuclei with
chemically different environment within a molecule

• Important tool for elucidation, stereochemistry,
configuration

• Position of proton in a complex

• Reference standard is not required

Applications:
impurities, minor components in mixtures

• Ease, speed, specificity in analysis

NMR-MS

• Mass Spectrometry

• Electron ionization

• Subsequent fragmentation

• Determination of m/e and relative abundance of ions

Application: MW

• Alone or in combination with other techniques – effective
method of identification

• LC-MS / GC-MS

X-ray
Diffraction Method

• Capable of crystallizing in more than one crystal lattice

– Calcium oxalate, mica, sulphur etc

• At a specific temp and pressure: only one crystalline form
is thermodynamically stable

Chromatographic
Techniques

• Thin Layer Chromatography (TLC)

• High Performance Thin Layer Chromatography (HPTLC)

• High Performance liquid chromatography (HPLC)

• Gas chromatography / Gas liquid chromatography (GC / GLC)

• Column Chromatography

• Gel permeation, etc

Biological Evaluation

• Estimation of potency of drug/preparation on living
organism, fungi bacteria, tissue or entire animal – Bioassay

• Bioassay – measure of sample being tested capable of
producing same response as that of standard

• Activity represented as IU

– Digitalis: 1 IU is contained in 76 mg of Std prepn

– Vit A: 1 IU 0.344 micrograms of std preparn

– Vit D: 1 IU 0.025 micrograms of std preparn

– Heparin: 1 IU 7.7 micrograms of std preparn

Bioassays:

• Toxic – animals

• Symptomatic – animals

• Tissue – isolated organs or tissues

Biological
testing:

Hepatoprotective
activity

• CCl4, Paracetamol, Isoniazid,

• Liver enzymes

• Histopathology

Antidiabetic activity

• Alloxan

• Measurement of blood glucose- O toluidine method, glucose
strips

• Insulin levels – RIA or ELISA

Antiinflammatory
activity

• Carageenan induced paw edema – mucopolysaccharide –Irish
sea moss Chondrus cripus

• Paw volume is measured

Neuropharmacological
activity

• CNS acting drugs: Stimulants, depressants, hallucinogens,
antidepressants, tranquillisers

• Locomotor activity in mice – activity cage – Stimulant /
depressant

• Locomotor co-ordianation

• Pentylenetetrazole convulsions in mice

ANS:

• Guinea pig ileum: nonspecific antispasmodic activity

• Isolated rat jejunum: adrenergic activity

• Rat Phrenic nerve: Muscle relaxant activity

• Frog Rectus: activity on skeletal muscle

Microbiological
assays:

• Suppress or influence the growth of microorganisms

• Cylinder/cup plate method

• Turbidimetric method

Summary

• Evaluation is a process of confirmation of identity, determination
of quality and purity, and detection of nature of adulterants

• Evaluation is a process of confirmation of identity, determination of quality and purity, and detection of nature of adulterants

• Microscopical evaluation includes the application
histological studies, linear measurements etc

• Microscopical evaluation can be applied to crude drugs as
such, and also in powdered form

• Chemical method includes preliminary phytochemical
screening, chemical assays to determine the purity of the crude drugs and also
the nature of adulterants in crude drugs

– Physical evaluation includes

– Moisture content

– Viscosity

– Melting point

– Solubility

– Optical rotation

– Refractive index

– Ash values

– Extractive values

– Volatile oil content

– Foreign organic matter etc

• Spectroscopic
methods includes

– UV- Visible

– IR

– NMR

– Fluoresence analysis

– X ray diffraction studies etc

• Biological evaluation (Bioassay) is the estimation of
potency of drug/preparation on living organism, fungi bacteria, tissue or
entire animal

– Bioassays

– Toxic

– Symptomatic

– Tissue

– Toxic and symptomatic: animals