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

Evaluation of Natural Products


Evaluation of Natural Products

Morphological evaluation

Microscopical evaluation

Chemical evaluation

Physical evaluation

Spectroscopic methods

Biological methods


• 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


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






Special features like touch, texture, fracture

Examples: Shapes

Ovoid tears – Acacia

Ribbon shape – Tragacanth

Disc shape- Nuxvomica seed

Conical – Aconite

Quills – Barks

Wavy - Rauwolfia


Brown colour- Cinnamon


Aromatic odour – Umbelliferous fruits


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,


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


Cell wall

Cell contents - Starch grains, Crystals




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 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


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



Vein termination number

Cassia acutifolia                       

32.7 - 40.2

Cassia angustifolia                   

25.9 – 32.8


Palisade ratio

Atropa belladona                    


Cassia angustifolia

Upper: 5.5-10

Lower: 4-7.4



Vein islet number

D. purpurea                                   

2.5 - 3

D. lanata                                          

2- 8



- Stomatal pore

- Guard cells

- Subsidiary cells

Types of Stomata

- Moss

- Gymospermous

- Gramineous

- 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


• 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



Unicellular: Stalk absent – betel, vasaka


• 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                                                                    







• 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


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,


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






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


Barfoed test : Filtrate + Barfoed’s reagent, heat on boiling water bath

Red ppt

Presence of carbohydrates


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


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:





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


Biuret test: Test solution + few drops of 0.7% copper sulphate solution + Sodium hydroxide

Purplish violet colour

Presence of proteins


Ninhydrin test : Test solution + few drops of ninhydrin reagent, heat

Bluish colour

Presence of proteins

Test for Lipids

S. No:





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


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:





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:





Mayer’s test: Filtrate + potassium mercuric iodide (Mayer’s reagent)

Cream coloured precipitate


Presence of alkaloids


Dragendorff’s test: Filtrate + potassium bismuth iodide (Dragendorff’s reagent)

Reddish brown precipitate

Presence of alkaloids


Wagner’s test: Filtrates + solution of iodine in potassium iodide (Wagner’s reagent)

Reddish brown precipitate

Presence of alkaloids


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:





Shinoda test: Test solution + a few fragments of magnesium metal+con.Hcl, heat

Formation of Magenta colour


Presence of flavanoids


Alkaline reagent test: Test solution + few drops of sodium hydroxide solution

intense yellow colour, become less intense on addition of acid

Presence of flavanoids


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:





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



Gelatin test: Test solution + few drops of 1% gelatin solution in 10% sodium chloride

Formation of white precipitate

Presence of tannins


Lead acetate test: Test solution + few drops of 10% lead acetate solution

Formation of white precipitate

Presence of tannins


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:





Legal’s test: Hydrolysates + sodium nitroprusside in pyridine and methanolic alkali. 

Formation of blood red colour


Presence of cardiac glycosides


Baljet Test : Section of digitalis + sodium picrate solution

Yellow to orange colour

Presence of cardiac glycosides

Test for Anthraquinone Glycosides

S. No:





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


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:





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


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


• 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


Melting point


75- 85

okum butter


Cocoa butter


Bees wax


Wool fat



• 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

• 25OC –sodium lamp –source of light


Angles of optical rotation

Caraway oil

+ 75 to + 80

Castor oil

+3.5 to + 6

Clove oil

0 to – 1.5


+ 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



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


Total ash

Acid insoluble ash














• 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


Water soluble extractive value


NLT 10


NLT 15


NLT 20


NLT 25

Senna leaf

NLT 30

Alcohol soluble extractives – Tannins, resins

• 20 - 95% alcohol used

• Dilute alcohol may also be used


Alcohol soluble extractive value


NLT 50


NLT 4.5


NLT 70


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


% Moisture content


NMT 10






NMT 15


NMT 15

Loss on drying

• 1050C to a constant weight

• Volatile actives – toluene distillation method

Azeotropic distillation method

• Volatile oil content

• Hydro distillation method


Water soluble extractive value


NLT 2.5

Fresh lemon peel

NLT 2.5


NLT 2.5


NLT 15


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


• 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


• 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


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


• 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


• 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


• 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


- 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

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