1) The document discusses the isolation, extraction and identification of various phytoconstituents like terpenoids, glycosides, alkaloids and resins. 2) Extraction methods like solvent extraction, fractional crystallization and chromatography are described for menthol, citral, artemisin, glycyrrhetinic acid, rutin, atropine, quinine, reserpine, caffeine and podophyllotoxin. 3) Identification tests include chemical tests and thin layer chromatography. Rf values are provided for comparison with reference standards.

1. Isolation, Identification and Analysis
of Phytoconstituents
DR. SIDDHI UPADHYAY
H.O.D. & ASSOCIATE PROFESSOR
DEPT. OF PHARMACOGNOSY AND PHYTOCHEMISTRY
SIGMA INSTITUTE OF PHARMACY

2. CONTENT
a) Terpenoids:
1. Menthol
2. Citral
3. Artemisin
b) Glycosides:
1. Glycyrhetinic acid
2. Rutin
c) Alkaloids:
1.Atropine
2.Quinine
3.Reserpine
4.Caffeine
d) Resins:
1. Podophyllotoxin
2. Curcumin

3. 🠶Biological source –
🠶 Menthol is a monoterpene alcohol obtained from different variety
of mint oils or peppermint oils
🠶 Biological source – It consists of the fresh flowering tops of Mentha
piperita, Mentha officinalis
🠶Family- Labiatate
🠶 Granular substance or crystalline with peppermint taste and
odour, freely soluble in alcohol, chloroform, ether, petroleum
ether
MENTHOL

4. EXTRACTION AND ISOLATION
🠶 Take the accurately weighed quantity of coarse powder of
Mentha piperita parts just before flowering .
🠶Extract the peppermint oil by water distillation method.
🠶Separate the oil and allow cooling. Crystals of (-) menthol will
separate out.
🠶Collect the crystals by centrifugation.
🠶 Re- Crystallize menthol from acetone or any other low
boiling point solvent

5. Identification and Analysis
T.L.C Method
Sample preparation – Dissolved 1mg of menthol in 1ml of
methanol
Stationary phase - Silica gel –G
Standard sample - Menthol
Detecting agent – 1% vanillin – sulphuric acid reagent and heat
the plate 110 0C for 10 minutes
Mobile phase – Pure Chloroform
Rf Value – 0.48-0.62

6. 🠶Biological sources –
🠶 Citral is a monoterpene aldehyde found in variety of sources
like lemon grass, lemon and orange peels etc
🠶 Cymbopogon fleuosus ( Lemon grass) , Graminae . 75-85%
of citral present in the drug
🠶Citral obtained from a natural source is a mixture of two
geometric isomers geranial and neral
CITRAL

7. 🠶Properties
🠶Geranial and Neral both are light oily liquids with lemon odour
🠶 Citral is practically insoluble in water but miscible with
alcohol, ether, benzyl benzoate etc

8. EXTRACTION AND ISOLATION
🠶The fresh plant material is hydro- distilled to obtain lemon
grass oil.
🠶Purification by Fractional crystallization
🠶 To the total oil, first Sodium sulphite is added, the citral get
converted into its sulphite salt
🠶The salt crystallizes out of the solution
🠶The crystals are filtered and washed with ether or chloroform
🠶 The product is then subjected to sodium carbonate treatment
to recover citral

9. Identification and Analysis
T.L.C Method
Sample preparation – Dissolved 1mg of Citral in 1ml of methanol
Stationary phase - Silica gel –G Standard
sample - Citral
Detecting agent – 2,4,dinitrophenyl hydrazine reagent to produce
Yellow to orange Color spots
Mobile phase – Pure Chloroform RF Value –
0.51

10. 🠶Synonym – Santonica
🠶 Biological source – Artemisin is a sesquiterpenoid
lactone , obtained from the unexpanded flower- heads of
Artemisia annua
🠶Family - Compositae
🠶Medicinal use – Antimalarial drug
🠶White crystalline powder, soluble in organic solvents
ARTEMISIN

11. EXTRACTION AND ISOLATION
🠶 The leaves are air dried, coarsely powdered and extracted with
petroleum ether (40-60).
🠶 The extract is concentrated ,dried and re-dissolved in
chloroform. Add acetonitrile to precipitate sugars and
waxes.
🠶 Filter and collect the filtrate. Evaporate to dryness to yield
residue

12. 🠶 The chromatographic fractionation of the concentrate on silica
gel by eluting with Chloroform- ethyl acetate yields the fraction of
artemisin
🠶 The fractions containing artemesin could be crystallised from
cyclohexane or 50% Ethanol

13. Identification and Analysis
T.L.C Method
Sample preparation – Dissolved 1mg of Artemisin in 1ml of
Chloroform
Stationary phase - Silica gel –G
Standard sample - Artemisin
Detecting agent – p- dimethylaminobenzaldehyde and heat at
80C to produce color
Mobile phase – Petroleum ether - Ethyl acetate (1:2) RF
Value – Compare with standard Artemisin

14. 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.
GLYCYRHETINIC ACID

15. 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

16. • 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

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

18. 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 1100 C
Mobile phase – Toluene–Ethyl acetate-Glacial acetic acid (12.5:7.5:0.5)
Reference drug - Glycyrrhetinic acid RF
Value – Purplish – 0.41

19. • There are around 200 types of Quercetin,
Flavanoid glycosides, among this the rutin is the
one of most important type.
• It is chemically Quercetin-3- rutinoside . On
hydrolysis , it yields the aglycone quercetin and
the sugars glucose and rhamnose.
• It is used as a Vitamin P OR Capillary fragility
factor
RUTIN

20. • SOURCES OF RUTIN
• 1. Fagophyrum esculentum- ( Polygonaceae )
• 2. Rhubarb – ( Rheum emodi- ( Polygonaceae )
• 3. Tobacco – (Nicotiana tobaccum –( Solanaceae )
• 4. Ruta – (Ruta graveolens – ( Rutaceae )
• 5. Tea – Thea sinensis – ( Theaceae )
• 6. Eucalyptus macroryncha ( Myrataceae )

21. ISOLATION
• Source -
• Eucalyptus macroryncha ( Myrataceae )
• Boil the powder drug with boiling water.Filter while hot and
collect the filtrate . Cool for the precipitation of the rutin.
• Recrystallize it from boiling water , dry the product.
• Greenish yellow crystalline powder

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

23. 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

24. 🠶Atropine is a tropane alkaloid obtained from Atropa
belladonna ,Datura stramonium and Hyoscyamus niger
🠶Family – Solanaceae
🠶Used as Antispasmodic, Mydriatic etc
ATROPINE

25. EXTRACTION AND ISOLATION
🠶Take weighed quantity of coarse powder and moisten with
sodium carbonate solution.
🠶Extract the blended mixture in petroleum ether. Filter the
petroleum ether extract
🠶Extractthe filtrate with aqueous acetic acid ( alkaloids
extracted in aqueous layer)
🠶Extract the aqueous fraction with solvent ether and
separate both fraction. Discard solvent ether fraction

27. Identification and Analysis
🠶Chemical test –
🠶Vitalin –morin test -
🠶 Take small quantity of the solid atropine and add 2 drops of
Con.nitric acid in an evaporating dish and evaporated to
dryness on water bath. Then dissolve the residue in 1ml of
acetone. Add few drops of freshly prepared alcoholic potassium
hydroxide solution.
🠶Violet colouration takes place due to tropane nucleus

28. T.L.C Method
Sample preparation – Dissolved 1mg of Atropine in 1ml of Chloroform
Stationary phase - Silica gel –G Standard
sample - Atropine
Detecting agent – Drangendroffs reagent to produce yellow
orange Color spots
Mobile phase – Toluene - Ethyl acetate – Diethyl amine
(70:20:10)
RF Value – Compare with standard Atropine (0.70)

29. Synonym – Quinine
It is a quinoline alkaloid of cinchona bark. The other
important alkaloids of this drug are quinidine,
cinchonine, cinchonidine, cinchonamine etc.,
Biological sources : It consists of dried inner bark of
C.Calisaya, C.succirubra, C.officinalis, C.ledgeriana
and hybrids of this. Family – Rubiaceae
QUININE

30. 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.

31. ISOLATION
1. The dry powder bark material is first well mixed with about
30% of its weight of calcium hydroxide or calcium oxide and
sufficient quantity of sodium hydroxide solution to make a
paste. It is allowed to stand for few hours.
2. The mass is then transferred to a Soxhlet apparatus and
extraction is carried out with benzene.

32. 3. Subsequently the benzene extract is shaken with successive
portions of 5% sulphuric acid.
4. The aqueous acid extract is adjusted the pH 6.5 with dilute
sodium hydroxide, cool. Crystals of neutral quinine sulphate are
formed.
5. These crystals are freed from cinchonine and cinchonidine by
repeated recrystallization from hot water.

33. 6. Colouring matter is removed by activated charcoal.
7.Quinine sulphate crystals are dissolved in dilute sulphuric
acid and made alkaline with ammonia. Initially amorphous
quinine is formed , which becomes crystalline.
8.Finally washed to remove sodium and ammonium salts and
dried to 45- 55 o C.

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

35. 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

36. 🠶Biological source – Reserpine is an indole alkaloid
obtained from the roots of Rauwolfia serpentina
🠶Family – Apocyanaceae
🠶It is a white or pale buff to slightly yellow, odourless,
crystalline powder
🠶It is soluble in alcohol, acetone and chloroform.
🠶Reserpine is an antihypertensive and antipsychotic
agent
RESERPINE

37. EXTRACTION AND ISOLATION
🠶 Rauwolfia root powder is exhaustively extracted with 90%
alcohol by percolation
🠶 The alcoholic extract is concentrated and dried under reduced
pressure below 60c to yield Rauwolfia dry extract.
🠶 Rauwolfia dry extract is extracted with Ether-chloroform-
90%alcohol (20:8:2.5)
🠶 Collect the extract and add little dilute ammonia with
intermittent shaking. Add water and allow the drug to settle after
vigorous shaking.

39. Identification and Analysis
T.L.C Method
Sample preparation – Dissolved 1mg of Reserpine in 1ml of
methanol or chloroform
Standard sample - Reserpine Stationary
phase - Silica gel – G
Mobile phase – Chloroform: acetone :diethyl amine
(50:40:10)
Detecting agent – Dragendroffs reagent RF
Value – 0.72

40. 🠶Caffeine is a purine alkaloid obtained from T
ea leaves,
Coffee seeds, cocoa, and other species
🠶 Biological source -It consists of dried leaves of plant known
as Thea sinensis
🠶Family – Theaceae
🠶It is chemically 1,3,7, trimethyl xanthine. It is isolated from
tea and coffee seeds during decaffeination process.
🠶 Tea leaves contains 1-4% of caffeine and coffee contains 1-
2% of caffeine
🠶It is white powder or white ,glistering needles, odour less,
bitter in taste, Soluble in hot water.
🠶 Caffeine is a CNS stimulant and Diuretic
CAFFEINE

41. EXTRACTION AND ISOLATION
🠶The powder tea leaves is extracted with boiling water and the
aqueous extract is filtered while hot.
🠶The warm extract is treated with lead acetate to
precipitate tannins and filtered.
🠶The filtrate is treated with excess of dilute sulphuric acid to
precipitate lead in the form of lead sulphate.

42. 🠶Filter and collect the filtrate
🠶The filtrate is boiled with Activated charcoal to remove
colouring matter, if any and filtered to remove charcoal
🠶The filtered decolourized solution is extracted with
chloroform successively .
🠶Combined the chloroform extracts evaporate on water bath to
yield caffeine (white powder)
🠶It is recrystallized with alcohol

43. Identification and Analysis
🠶Chemical test –
🠶 Murexide test – To the caffeine add hydrochloric acid and
potassium chlorate, heated to dryness. A purple colour is
obtained by exposing the residue to vapours of dilute ammonia.
🠶Thin layer chromatography (TLC)

44. T.L.C Method
Sample preparation – Dissolved 1mg of caffeine in 1ml of
methanol or chloroform
Stationary phase - Silica gel –G Standard
sample - Caffeine
Mobile phase – Ethyl acetate: methanol : acetic acid
(80:10:10)
Detecting agent – Expose to vapors of iodine RF Value –
0.41

45. 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)

46. EXTRACTION
• Take the weighed quantity of rhizomes or roots of Podophyllum
emodi with methanol. Filter and evaporate to semisolid mass.
• Dissolve semisolid mass into acidic water. Precipitate is formed
which should be allowed for at least for 2hrs

47. • Filter and wash filtrate with cold water. Collect the residue, wash
with acidified water and dry to obtain dark brown amorphous
powder.
• Extract the residue with hot alcohol. Filter and evaporate to
dryness.
• Re- crystallise the residue in benzene to yield podophyllotoxin

48. IDENTIFICATION AND ANALYSIS
• 1. Chemical test –
Treat podophyllotoxin with 50% Sulphuric acid it will show violet –
blue colour.
• 2.Thin layer chromatography ( TLC )

49. 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.

50. 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 1100 C
RF Value – Yellow spot – 0.65

51. 🠶 Curcumin or Curcuminoids are the diaryl hepnoid
compounds obtained from the dried rhizomes of
Turmeric, Curcuma longa, Family – Zingiberaceae
🠶 Curcumin is the major colouring principle. It is a
mixture of curcumin, monodesmethoxycurcumin and
bisdesmethoxycurcumin
CURCUMIN

52. 🠶It as an orange yellow, crystalline powder
🠶Insoluble in water and ether, but soluble in alcohol
🠶 It is used as wound healing, ant-inflammatory, anti arthritic and
antimicrobial activities
🠶Used against peptic ulcer

54. Identification and Analysis
T.L.C Method
Sample preparation – Dissolved 1mg of Curcumin in 1ml of methanol
Stationary phase - Silica gel –G Standard
sample - Curcumin
Detecting agent – Observed under U.V light at 366nm Mobile phase –
Chloroform - Ethanol - Glacial acetic acid
(94:5:1)
RF Value – Curcumin – 0.79

55. THANK YOU !!!
siupa.pharma@gmail.com