Unit-III Pharmacognosy and Phytochemistry II.pdf

Unit-III: Isolation, Identification and Analysis of
Phytoconstituents
a) Terpenoids: Menthol, Citral, Artemisin
b) Glycosides: Glycyrhetinic acid & Rutin
c) Alkaloids: Atropine, Quinine, Reserpine, Caffeine
d) Resins: Podophyllotoxin, Curcumin
O
O
OH
OH
OH
OH
HO
O
OH
OH
OH

Isolation, Identification and Analysis of Phytoconstituents
a) Terpenoids: Menthol, Citral, Artemisin
1. Menthol:
 Menthol is a monoterpene alcohol obtained from diverse type of mint oils or peppermint.
 The sources of mint oil include black peppermint (Mentha piperita; M. Vulgaris)
 White peppermint (Mentha piperita; Mentha officinalis, M. avensis, M. canadensis and piperascens etc. )
Isolation:
 Mentha oil is obtained from the hydrodistillation or steam distillation of fresh above-ground parts just before
flowering.
 The oil is subjected for cooling.
 The crystals of menthol crystallizes out from the oil which is separated by centrifugation.
Melting point: 41 to 44ºC
Menthol

Clavenger Apparatus
Fractional Distillation
Unit-III: Isolation, Identification and Analysis of Phytoconstituents

Identification and Analysis of Menthol:
Thin Layer Chromatography:
Preparation of sample: Dissolve about 1 gm of menthol in about 1 ml of methanol.
Stationary Phase: Silica Gel G
Mobile phase: Chloroform
Spraying /detection agent: 1% vanillin sulphuric acid reagent and heat the plate at 110 ºC for 10 min.
Rf value: 0.48 to 0.62

Pharmacognosy & Phytochemistry-II (BP504T) 5
FT-IR Spectra of Menthol
UV-Visible Spectra of Menthol

FT-IR Spectra of Menthol

2. Citral:
1. Take 100 ml of lemon juice and 15% sodium hydroxide with continuous stirring to make solution slightly
alkaline.
2. Filter the mixture and add 50 ml of 10 % calcium chloride solution. Boil for few minutes and filter off to
obtain precipitate of calcium citrate.
3. Wash the precipitate with small volumes of boiling water.
4. Dissolve the precipitate in sufficient quantity of cold water, boil and filter.
5. Allow the salt to air dry.
6. Calculate the yield on dry weight basis.
Citral

Identification and Analysis of Citral:
Chemical test: To a neutral solution of substance being examined a white ppt is formed
when HCL is added which is soluble in acetic acid.
Stationary Phase: Siliga Gel G
Mobile Phase: 1) 70:30 (v:v) Acetonitrile : methanol,
2) 5 mM Phosphate monobasic in water adjusted to pH 2.3 with
phosphoric acid; alternate A: 0.02% formic acid in water
Std. Citral
Sample Citral

3. Artemisin:
Artemisinin is sesquiterpennoid lactone present in flower heads of Artemisia annuna, of family Asteraceae. It is an
antimalerial drug found to be effective against chloroquine resistance strains P. falciparum.
Isolation:
1. Take accurately weighed quantity of powder of Artemisia leaves.
2. Extract the powder with petroleum ether.
3. Filter and evaporate filtrate to dryness. Dissolve the residue in chloroform.
4. Add acetonitrile to precipitate the impurities.
5. Separate the precipitate and discard. Concentrate the filtrate to dryness.
6. Load this residue on a column chromatography by using chloroform: Ethyl acetate mixture.
7. Monitor the elution by TLC.
8. Combine the fraction containing artemisinin.
9. Purify the crude artemisinin by using aqueous alcohol.

Artemisinin
TLC profile of Artemisin
TLC profile of Artemisin

UV Spectra of Artemisin FT-IR Spectra of Artemisin

b) Glycosides: Glycyrrhetinic acid & Rutin
1. Glycyrrhetinic acid : It is a pentacyclic triterpenoid acid obtained from the roots and stolones of Glycyrrhiza glabra;
family Leguminosae commonly known as Liquorice.
a) The crude drug is extracted with chloroform. The chloroform extract is discarded.
b) The marc is extracted with 0.5 M sulphuric acid.
c) The acid extract is cooled and shaken with chloroform.
d) The combined chloroform extract is concentrated and dried to yield glycyrrhetinic acid. Glycyrrhizin is hydrolyzed to
glycyrrhetinic acid during extraction with sulphuric acid.
e) Melting point: 300ºC

TLC profile of Glycyrrhetinic acid :
 Preparation of sample: Dissolve 1 mg of glycyrrhetinic acid in about 1 ml of methanol: Chloroform (1:1) mixture.
 Stationary Phase: Silica Gel-G
 Mobile Phase: Toluene: Ethyl acetate: Glacial acetic acid
 (12.5:7.5:0.5)
 Spraying reagent: 1% vanillin-sulphuric acid or anisaldehyde-sulphuric acid and heat for 10 min at 110ºC.
 Glycyrrhetinic acid gives purplish spot corresponding to Rf value: 0.41

2. Rutin:
Rutin is a flavonoid obtained from various citrus fruits. It can be extracted from the leaves of Fagopyrum esculentum also known as
buckwheat belonging to family Polygonaceae. It is used as capillary fragility factor.
Fagopyrum esculentum
Rutin

Extraction of Rutin:
1. Take the accurately weighed quantity of coarse powder of drug. Defat the powder with n-hexane. Extract
the marc with 78% alcohol for 1 hour.
2. Filter and evaporate the solvent to obtain dry residue. Dissolve this residue in sufficient quantity of 30 %
acetone
3. Filter and evaporate filtrate to 1/4th of its original volume
4. Add sufficient quantity of 5 % aqueous solution of borax (pH-7.5) Add sufficient quantity of solid Nacl
with stirring
5. Filter and acidify the clear filtrate with phosphoric acid to bring pH to 5.5. Stir for 15 min. Filter and wash
the residue with 20 % Nacl solution.
6. Again filter and evaporate the filtrate to 500ºC to 1/4th of its original volume.
7. Add HCL in hot condition to bring pH to 1.5. Cool and keep in refrigerator overnight. Crystals of rutin
will separate out. Collect and dry to calculate percentage yield.

TLC profile of Rutin:
 Preparation of sample: Dissolve 1 mg of Rutin in about 1 ml of methanol
 Stationary Phase: Merck TLC plates precoated with silica gel 60F254, 10×10
 Mobile Phase: Ethyl acetate: Glacial acetic acid: Formic acid: Water (100:11:11:25)
 Spraying reagent: 1% vanillin-sulphuric acid or anisaldehyde-sulphuric acid and heat for 10 min at 110ºC.
 Rf value: 0.54

Alkaloids: Atropine, Quinine, Reserpine, Caffeine
Atropine

1. Atropine: Atropine is a tropane alkaloid from the members of the Solanaceae family. It is present in Atropa belladona,
Datura stramonium and Hyoscyamus niger. Atropine is an optically inactive laevorotatory isomer of hyoscyamine.
Isolation:
Moist the powder drug with an aqueous solution of sodium carbonate
Extract the above mixture with ether or benzene.
The alkaloidal free bases are acidified with acetic acid
The acid solution is then shaken with solvent ether to remove colouring matter
The alkaloids are ppt. with sodium carbonate, filtered off, washed and dried. The dried mass is dissolved in
solvent ether or acetone dehydrated with sodium sulphate before filtration
The filtrate after concentration and cooling yields crude crystals of hyoscyamine and atropine
from the solution

Melting point: 115-116ºC
Identification:
Vitali Morin reaction: Dilute solution of atropine, when treated with concentrated nitric acid and the mixture evaporated
to dryness on the steam bath, produces a pale yellow residue. The residue gives a violet colouration when drop of freshly
prepared solution of potassium hydroxide is added.
TLC Profile of Atropine and Atropine sulphate:
Preparation of sample: 1% of atropine dissolved in 2N acetic acid
Stationary Phase: Silica gel G
Mobile Phase: Strong amonia solution:methanol (1.5:100)/ Acetone: 0.5Msodium chloride
Spraying agent: Acidified iodoplantine solution/ Dragendorff’s reagent.
Atropine Rf value: 0.18
Atropine sulphate: 0.7

FT-IR Profile of Atropine Sulphate

2. Quinine: Cinchona is the dried bark of stem or of the root of Cinchona calisaya, Cinchona ledgeriana, Cinchona
officinalis and Cinchona succirubra; family Rubiaceae
Quinine is laevorotatory.
Isolation:
1. The powder cinchona bark is mixed with about 30 % of its calcium hydroxide or calcium oxide and sufficient
quantity of 5% sodium hydroxide solution.
2. The moistened mass is then transferred into soxhlet and extracted with benzene.
3. To the benzene extract, add 5% sulphuric acid and mix well.
4. The benzene layer is separated from that of the aqueous layer.
5. To the aqueous layer sodium hydroxide is added to adjust the pH to 6.5
6. On cooling, ppt of quinine sulphate is formed.
7. Melting point: Quinine 177ºC

TLC profile of Quinine:
 Preparation of sample: Alkaloids are dissolved in methanol
Stationary Phase: Silica gel-G plate
Mobile phase: Chloroform: diethyl amine (9:1) for Quinine
Chloroform:acetone:diethyl amine (5:4:1) Quinidine
•Spraying agent: Dragendorff’s reagent.
•Rf value: 0.17 for quinine and 0.28 for Quninidine
Identification Test:
Thalleioquin test: To the sample add one drop of dilute sulphuric acid and 1 ml of water. Add bromine
water drop-wise till the solution acquires permanent yellow colour and add 1 ml of dilute ammonia solution,
emerald green colour is produced.

3. RESERPINE: Reserpine is an indole alkaloid obtained from the roots of Rauwolfia serpentina family
Apocynaceae.
Extraction/Isolation:
1. Powder drug of rauwolfia is exhaustively extracted with 90% alcohol by suitable method of extraction
such as percolation.
2. The alcoholic extract is concentrated and dried under reduced pressure below 60ºC to yield rauwolfia dry
extract containing about 4 % of total alkaloid.
3. Rauwolfia dry extract is extracted further with proportions of ether: Chloroform: 90% alcohol (20:8:2.5).
4. To the extract obtained, add little dilute ammonia with intermittent shaking.

5. Alkaloid is converted to water-insoluble base. Add water and allow the drug to settle after few vigorous shakings. Filter
the solution and extract the residue with 4 volumes of 0.5 N sulphuric acid in separating funnel. Combine the total
acid extract which contains the alkaloidal salt.
6. The extract is filtered, made alkaline with dilute ammonia to liberate alkaloid.
7. Finally, it is extracted with chloroform. The total chloroform extract is filtered; chloroform is removed by distillation
and the total alkaloidal extract is dried under vaccum to yield total rauwolfia alkaloids.
8. Total rauwolfia alkaloid consists of the mixture of over 30 different components.
9. It is subjected to column chromatographic fractionation for the separation of reserpine.

Reserpine: Identification by TLC
Sample preparation: Dissolve about 1 mg of the sample in methanol.
Stationary Phase: Immerse a 20X20 sheet of Whatman N0. 1 filter paper in the immobile solvent
formamide : acetone (3:10). Blot the paper between filter paper toweling and allow the acetone to dry.
Apply the spots of the sample solution and standard on the filter paper and dry the spots.
Immerse the spotted papers in the chromatographic chamber containing the mobile solvent isooctane:
carbontetrachloride : piperidine: ter-butyl alcohol (90:60:4:2) and elute the paper by ascending
chromatography. Remove the chromatogram when the mobile solvent has raised approximately 7/8th of the
height of the paper.
Dry the chromatogram at 90ºC for 10 min. Samples yield spots corresponding in position and colour to those
of the standard solution.

Reserpine: Identification by TLC
Dissolve 1 mg of rauwolfia alkaloidal extract or pure reserpine in methanol.
Apply the spots over the TLC plate.
In case of silica gel G-plates elute the plate in solvent system Chloroform : acetone :
diethylamine (50:40:30).
In case of alumina-G plate, elute in the solvent system cyclohexane : chloroform
(30:70).
Dry the eluted plates and spray with Dragendorff’s reagent.
Rf: 0.72
While in case of alumina G plate, it gives Rf value 0.35

Alkaloids: Isolation and Identification of Caffeine: Caffeine or a purine base present along with other
related bases like theophylline and theobromine in coffee, tea, cocoa, kola etc. Caffeine is isolated from tea
leaves or recovered from coffee seeds during decaffeination process.
Isolation:
The coarse powder of 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 excess of lead acetate present in the solution is precipitated as lead sulphur with dilute sulphuric acid.
The filtered solution is boiled with charcoal to remove colouring matter, if any, and filtered to remove
charcoal.
The filtered decolourized solution is extracted with chloroform.
Evaporation of chloroform affords caffeine as a white material. It is recrystallied with alcohol.

Isolation and Identification of Caffeine:
TLC profile of caffeine
Sample preparation: Dissolve 1 mg of caffeine in 1 ml of chloroform or methanol.
TLC Plate: Silica Gel G
Mobile phase: Ethyl acetate : methanol : acetic acid (80:10:10)
Visualize: Iodine vapour.
Caffeine develops a spot at Rf value 0.41

D) Resins: Podophyllotoxin, Curcumin
(i) Podophyllotoxin: Indian podophyllum is the root and rhizome of Podophyllum hexandrum Royle
(Berberidaceae)
Isolation:
Extract the powdered rhizome/roots of P.emodi with methanol.
Then it is reduced under vacuum. Semisolid mass is put into acidulated water (10 ml HCL in 100 ml water).
The precipitates are allowed to settle. Filtrate is decanted and then washed with cold water.
Resin obtained is dried and upon drying , it gives dark brown amorphous powder called podophyllin.

Isolation of Podophyllotoxin: Resin obtained is dried and upon drying , it gives dark brown amorphous powder called
podophyllin.
The obtained powder is extracted with chloroform and further purification is done by repeated recryatallisation from
benzene alone or alcohol benzene mixture followed by washing with petroleum ether yield podophyllotoxin.
Meting point: 114 to 118ºC
TLC profile:
Preparation of sample: 1 mg dissolved in methanol
Stationary phase: Silica gel Gf
Mobile phase: Toluene:ethyl acetate (5:7)
Detecting agent: Sulphuric acid
Rf: 0.39

(ii) Isolation and Identification of Resin [Curcumin]:
Curcumin or curcuminoids are the diarrylheptanpoid compounds obtained from the dried rhizome of turmeric
Curcuma longa family Zinziberaceae.
Isolation:
•Turmeric powder is extracted with alcohol in soxhlet apparatus/extractor. The alcoholic
extract is concentrated under reduced pressure and dried.
•In another procedure, turmeric powder is first extracted with hexane followed by acetone.
The acetone extract is concentrated and dried to yield curcumin.
•M.P: 183ºC= Curcumin

Isolation and Identification of Resin [Curcumin]:
TLC profile:
Sample preparation: Dissolve 1 mg of curcumin in 1 ml methanol.
Apply the spots on silica gel G plate
Mobile phase: Chloroform : ethanol : galcial acetic acid (94:5:1)
Dry the eluted plate and visualize under 366 nm light.
Curcumin exhibits a bright yellow flourescent spot at Rf value 0.79
The other spots at 0.6 and 0.43 correspond to desmethoxycurcumin and
bisdemethoxycurcumin.

Unit-III Pharmacognosy and Phytochemistry II.pdf

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