Evaluation of Natural Products, Discuss the various parameters involved in Morphological, Microscopical, Chemical, Physical, Spectroscopic and Biological evaluation

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

Eye piece micrometer division	Stage micrometer division
3	4
11	15
14	19

• 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

Drug	Stomatal number		Stomatal index
Atropa belladona	Upper - 7-10                    Lower - 77-115	D. purpurea	Upper – 1.3 to 3.5 Lower – 17.9 – 19.5
Cassia angustifolia	Upper - 220-260              Lower - 240-265	D. lanata	Upper – 13.9 – 14.7 Lower – 14.9 – 17.6

                

Drug	Vein termination number
Cassia acutifolia	32.7 - 40.2
Cassia angustifolia	25.9 – 32.8

Drug	Palisade ratio
Atropa belladona	5-70
Cassia angustifolia	Upper: 5.5-10 Lower: 4-7.4

            

Drug	Vein islet number
D. purpurea	2.5 - 3
D. lanata	2- 8

                           

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

Solanaceous leaves	Type of crystals
Belladonna	Microspenoidal
Hyosyamus	Prism
Stramonium	Cluster

• 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 = (NxWx94,000x100)/(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

Sl.No	Test	Observation	Inference
1.	Molisch’s test: Filtrate + alcoholic solution of α-Naphthol + few drops of conc. sulphuric acid along the sides of the test tube	Formation of violet ring at the junction of the liquids	Presence of carbohydrates
2.	Barfoed test : Filtrate + Barfoed’s reagent, heat on boiling water bath	Red ppt	Presence of carbohydrates
3.	Fehling’s test: Filtrate + few ml of dilute HCl and heat on a water bath for 30 min+  sodium hydroxide solution, add equal quantities of  Fehling’s A & Fehling’s B solutions, heat on a water bath for a few min	Red-orange precipitate	Presence of carbohydrates
4.	Benedict’s test : Filtrate + Benedicts reagent, heat on boiling water bath for few min	Red-orange precipitate	Presence of carbohydrates

Test for Proteins

S. No:	Test	Observation	Inference
1.	Millon’s test: 2 ml of the test solution + 2 ml of Millon’s reagent, heat	Formation of white precipitate that gradually turns red	Presence of proteins
2.	Biuret test: Test solution + few drops of 0.7% copper sulphate solution + Sodium hydroxide	Purplish violet colour	Presence of proteins
3.	Ninhydrin test : Test solution + few drops of ninhydrin reagent, heat	Bluish colour	Presence of proteins

Test for Lipids

S. No:	Test	Observation	Inference
1.	Spot test : Press a small quantity of petroleum ether and benzene extracts separately between two filter papers	Formation of oil stains on the filter paper	Presence of fixed oils/fats
2.	Saponification test: A small quantity of petroleum ether or benzene extract + 0.5 N alcoholic KOH + drop of phenolphthalein. Mixture was heated on a water bath for 1 to 2 hrs	Formation of soap or partial neutralization of alkali	Presence of fixed oils/fats

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

S. No:	Test	Observation	Inference
1.	Liebermann-Burchard’s test Ethereal residues + few drops of acetic anhydride, boil and cool + Add 1 ml of sulphuric acid through the sides of the test tube	Formation of brown ring at the junction of two liquids and green colour in the upper layer	Presence of steroids and triterpenoids

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

S. No:	Test	Observation	Inference
1.	Mayer’s test: Filtrate + potassium mercuric iodide (Mayer’s reagent)	Cream coloured precipitate	Presence of alkaloids
2.	Dragendorff’s test: Filtrate + potassium bismuth iodide (Dragendorff’s reagent)	Reddish brown precipitate	Presence of alkaloids
3.	Wagner’s test: Filtrates + solution of iodine in potassium iodide (Wagner’s reagent)	Reddish brown precipitate	Presence of alkaloids
4.	Hager’s test : Filtrates + saturated solution of picric acid (Hager’s reagent)	Yellow precipitate	Presence of alkaloids

Test for Flavonoid

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

S. No:	Test	Observation	Inference
1.	Shinoda test: Test solution + a few fragments of magnesium metal+con.Hcl, heat	Formation of Magenta colour	Presence of flavanoids
2.	Alkaline reagent test: Test solution + few drops of sodium hydroxide solution	intense yellow colour, become less intense on addition of acid	Presence of flavanoids
3.	Lead acetate test: Test solution + few drops of 10% lead acetate Presence of solution	Formation of Yellow precipitate	Presence of flavanoids

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

S. No:	Test	Observation	Inference
1.	Ferric chloride test: Test solution + few drops of 5% ferric chloride solution	Formation of a bluish-black or greenish-black colour	Presence of phenolic compounds and tannins
2.	Gelatin test: Test solution + few drops of 1% gelatin solution in 10% sodium chloride	Formation of white precipitate	Presence of tannins
3.	Lead acetate test: Test solution + few drops of 10% lead acetate solution	Formation of white precipitate	Presence of tannins
4.	Aqueous bromine test: Test solution + few drops of aqueous bromine solution	Decolouration of bromine	Presence of tannins

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

S. No:	Test	Observation	Inference
1.	Legal’s test: Hydrolysates + sodium nitroprusside in pyridine and methanolic alkali.	Formation of blood red colour	Presence of cardiac glycosides
2.	Baljet Test : Section of digitalis + sodium picrate solution	Yellow to orange colour	Presence of cardiac glycosides

Test for Anthraquinone Glycosides

S. No:	Test	Observation	Inference
1.	Borntrager’s test: Filtrate +10ml of benzene, shake. Filtered and 5 ml of 10 % ammonia solution is added to the filtrate	Formation of pink colour in ammonical layer	Presence of anthraquinone glycosides
2.	Modified Borntrager’s test: Filtrate + FeCl3 +10 ml of benzene, shake. Filtered and 5 ml of 10 % ammonia solution is added to the filtrate	Formation of pink colour in ammonical layer	Presence of anthraquinone C-glycosides

Test for Saponin Glycosides

S. No:	Test	Observation	Inference
1.	Foam test: About 1 ml of alcohol and aqueous extracts were diluted separately with distilled water to 20ml and shaken in a graduated cylinder for 15 min	Formation of froth	Presence of Saponin glycosides
2.	Hemolytic test : Aqueous extract + Ox red blood cells	Lysis of blood cells	Presence of 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

Drug	Melting point
Colophony	75- 85
okum butter	39-42
Cocoa butter	30-33
Bees wax	62-65
Wool fat	34-44

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

Drug	Angles of optical rotation
Caraway oil	+ 75 to + 80
Castor oil	+3.5 to + 6
Clove oil	0 to – 1.5
Honey	+ 3 to -15
Eucalyptus oil	0 to + 10

Refractive index

• Purity, constant

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

• Varies – wavelength, temperature, pressure

Drug	RI
Arachis oil	1.4678 – 1.470
Caraway oil	1.4838 – 1.4858
Castor oil	1.4758 – 1.527
Clove oil	1.527 – 1.535

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

Drug	Total ash	Acid insoluble ash
Cannabis	15	5
Cardamom	6	3.5
Clove	7	0.75
Ginger	6	-

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

Drug	Water soluble extractive value
Ginger	NLT 10
Linseed	NLT 15
Liquorice	NLT 20
Aloe	NLT 25
Senna leaf	NLT 30

• Alcohol soluble extractives – Tannins, resins

• 20 - 95% alcohol used

• Dilute alcohol may also be used

Drug	Alcohol soluble extractive value
Asafoetida	NLT 50
Ginger	NLT 4.5
Myrrh	NLT 70
Aloe	NLT 10

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

Drug	% Moisture content
Aloes	NMT 10
Digitalis	NMT 5
Ergot	NMT 8
Acacia	NMT 15
Starch	NMT 15

Loss on drying

• 105⁰C to a constant weight

• Volatile actives – toluene distillation method

Azeotropic distillation method

• Volatile oil content

• Hydro distillation method

Drug	Water soluble extractive value
Caraway	NLT 2.5
Fresh lemon peel	NLT 2.5
Dill	NLT 2.5
Clove	NLT 15
Fennel	NLT 1.4

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