Isolation,identification and analysis of phytoconstituents. Dr.U.Srinivasa, Professor and Head, Srinivas college of Pharmacy, Mangalore-Karnataka

Useful for B.Pharm students

2. QUININE
Synonym–Quinine
Itisaquinolinealkaloidofcinchonabark.Theother
importantalkaloidsofthisdrugarequinidine,
cinchonine,cinchonidine,cinchonamineetc.,
Biologicalsources:Itconsistsofdriedinnerbarkof
C.Calisaya,C.succirubra,C.officinalis,C.ledgeriana
andhybridsofthis.Family–Rubiaceae

3. Quinine and quinidine are stereo-isomers .
Quinine is levorotatory and quinidine is dextrorotatory
Uses :
Quinine is antimalarial
Quinidine is a cardiac depressant therefore used in
cardiac arrhythmias.

4. ISOLATION
1.Thedrypowderbarkmaterialisfirstwellmixed
withabout30%ofitsweightofcalciumhydroxide
orcalciumoxideandsufficientquantityofsodium
hydroxidesolutiontomakeapaste.Itisallowedto
standforfewhours.
2.ThemassisthentransferredtoaSoxhlet
apparatusandextractioniscarriedoutwith
benzene.

5. 3.Subsequentlythebenzeneextractisshakenwith
successiveportionsof5%sulphuricacid.
4.TheaqueousacidextractisadjustedthepH6.5with
dilutesodiumhydroxide,cool.Crystalsofneutral
quininesulphateareformed.
5.Thesecrystalsarefreedfromcinchonineand
cinchonidinebyrepeatedrecrystallizationfromhot
water.

6. 6.Colouringmatterisremovedbyactivatedcharcoal.
7.Quininesulphatecrystalsaredissolvedindilute
sulphuricacidandmadealkalinewithammonia.
Initiallyamorphousquinineisformed,which
becomescrystalline.
8.Finallywashedtoremovesodiumandammonium
saltsanddriedto45-55
o
C.

7. IDENTIFICATION AND ANALYSIS
1.Chemical tests
2. Thin layer chromatography (TLC)

8. T.L.C Method
Sample preparation –Dissolved about 1mg of Quinine
or
Cinchona alkaloid in 1ml of methanol
Stationary phase -Silica gel –G
Detecting agent –Dragendroffs reagent
Mobile phase –Chloroform –Diethylamine (9:1)
RF Value –Quinine –0.17, Quinidine -0.26

10. GLYCYRRHETINIC ACID
Biological source -It is obtained from the roots and
subterranean stems of Glycyrrhiza glabra
Family –Leguminosae
Chemical Constituents –A major component is sweet
triterpenic saponin glycoside , glycyrrhizin
Glycyrrhizin –It is a potassium and calcium salt of
Glycyrrhizic acid
Glycyrrhetinic acid is a Pentacyclic triterpenoid
aglycone. It is used as an antiulcer.

11. ISOLATION
•The Liquorice / Glycyrrhiza coarse powder is
extracted with chloroform.
•Filter and discard the filtrate.
•Extract the marc with 0.5 M Sulphuric acid for a few
hours

12. •Filter and extract the filtrate with three portions of
chloroform
•Separate and combine the chloroform layers
•Distill off the chloroform extract to yield a dry
residue of glycyrrhetinic acid.
•White crystalline powder, insoluble in water,
soluble in chloroform, benzene, ether etc

13. IDENTIFICATION AND ANALYSIS
1.Chemical tests –Liebermann test and
Liebermann –Burchardtest
2. Thin layer chromatography (TLC)

14. T.L.C Method
Sample preparation –Dissolved about 1mg of
Glycyrrhetinic acid in 1ml of methanol-Chloroform (1:1)
Stationary phase -Silica gel –G
Detecting agent –1% vanillin-Sulphuric acid and heat for
10 minutes at 110
0
C
Mobile phase –Toluene–Ethyl acetate-Glacial acetic acid
(12.5:7.5:0.5)
Reference drug -Glycyrrhetinic acid
RF Value –Purplish –0.41

16. RUTIN
•Therearearound200typesofQuercetin,
Flavanoidglycosides,amongthistherutinisthe
oneofmostimportanttype.
•ItischemicallyQuercetin-3-rutinoside.On
hydrolysis,ityieldstheaglyconequercetinand
thesugarsglucoseandrhamnose.
•ItisusedasaVitaminPORCapillaryfragility
factor

17. SOURCES OF RUTIN
•1.Fagophyrumesculentum-(Polygonaceae)
•2.Rhubarb–(Rheumemodi-(Polygonaceae)
•3.Tobacco–(Nicotianatobaccum–(Solanaceae)
•4.Ruta–(Rutagraveolens–(Rutaceae)
•5.Tea–Theasinensis–(Theaceae)
•6.Eucalyptusmacroryncha(Myrataceae)

18. ISOLATION
•Source -
•Eucalyptus macroryncha ( Myrataceae )
•Boilthepowderdrugwithboilingwater.Filterwhile
hotandcollectthefiltrate.Coolfortheprecipitation
oftherutin.
•Recrystallizeitfromboilingwater,drytheproduct.
•Greenishyellowcrystallinepowder

19. IDENTIFICATION AND ANALYSIS
1.Chemical tests –Shinoda test
2.Thin layer chromatography (TLC)

20. T.L.C Method
Sample preparation –Dissolved about 1mg of
Rutin in 1ml of methanol
Stationary phase -Silica gel –G
Mobile phase –10 % aqueous sodium chloride
solution
Standard drug -Rutin
RF Value –Yellow spot –0.43

22. PODOPHYLLOTOXIN
•Podophyllotoxin is the Lactone resin present in the
root and rhizome of Podophyllum hexandrum
•Family –Berberidaceae
•It is used as anti-proliferative agent (Anti cancer
agent)

23. EXTRACTION
•Taketheweighedquantityofrhizomesorrootsof
Podophyllumemodiwithmethanol.Filterand
evaporatetosemisolidmass.
•Dissolvesemisolidmassintoacidicwater.Precipitate
isformedwhichshouldbeallowedforatleastfor2hrs

24. •Filterandwashfiltratewithcoldwater.Collectthe
residue,washwithacidifiedwateranddryto
obtaindarkbrownamorphouspowder.
•Extracttheresiduewithhotalcohol.Filterand
evaporatetodryness.
•Re-crystallisetheresidueinbenzenetoyield
podophyllotoxin

25. IDENTIFICATION AND ANALYSIS
•1.Chemicaltest–
Treatpodophyllotoxinwith50%Sulphuricaciditwill
showviolet–bluecolour.
•2.Thinlayerchromatography(TLC)

26. T.L.C Method
Sample preparation –Dissolved about 1mg of
podophyllotoxin in 1ml of methanol
Stationary phase -Silica gel –G
Mobile phase –
Chloroform : Methanol (90:10) for about 6 cm (Only
glycosides are separated but aglycone like
podophyllotoxin
remains in the region of the front.

27. The same plate is again eluted with more weakly polar
Solvent
Chloroform : Acetone (65:35) upto 12cm
Standard drug –Podophyllotoxin
Detection –Spray with methanol Sulphuric acid and
heat
10 minutes at 110
0
C
RF Value –Yellow spot –0.65