Screening Phytochemistry

of Extract
(Penapisan Fitokimia
Ekstrak)
Antonius Padua Ratu, M.Farm., Apt.
STTIF Bogor

SCREENING PHYTOCHEMISTRY 1
 Tests for Alkaloids
1. Dragendroff’s test:
Methods : 1 ml of extract, add 1 ml of Dragendroff’s
reagent (potassium bismuth iodide solution).
Result : An orange-red precipitate indicates the
presence of alkaloids.
2. Mayer’s test:
Methods : 1 ml of extract, add 1 ml of Mayer’s reagent
(potassium mercuric iodide solution).
Result : Whitish or cream colored precipitate
indicates the presence of alkaloids.
SCREENING PHYTOCHEMISTRY 2
3. Hager’s test:
Methods : 1 ml of extract, add 3 ml of Hager’s
reagent (saturated aqueous solution of
picric acid).
Result : Yellow colored precipitate indicates
the presence of alkaloids.
4. Wagner’s test:
Methods : 1 ml of extract, add 2 ml of Wagner’s
reagent (iodine in potassium iodide).
Result : Reddish brown colored precipitate
indicates the presence of alkaloids.
SCREENING PHYTOCHEMISTRY 3
 Tests for Glycosides
1. Tests for free sugars:
The extract is hydrolyzed with mineral acid and then tested
for the glycone and aglycone moieties.
2. Raymond’s test:
Test solution when treated with dinitrobenzene in hot
methanolic alkali, gives violet color.
3. Legal’s test:
Treat the extract with pyridine and add alkaline sodium
nitroprusside solution, blood red color appears.
4. Bromine water test
Test solution when treated with bromine water gives yellow
precipitate.
SCREENING PHYTOCHEMISTRY 4
 Test for Saponin Glycosides
❑ Froth Test:
Place 1ml solution of drug in water in a semi-micro
tube and shaken well and noted for a stable froth.

❑ Hemolysis test:
Add 0.2 ml solution of saponnin (prepared in 1%
normal saline) to 0.2ml of v/v blood in normal saline
and mix well, centrifuge and note the red supernatant
compare with control tube containing 0.2 ml of 10%
blood in normal saline diluted with 0.2 ml of normal
saline.
SCREENING PHYTOCHEMISTRY 5
 Test for Anthraquinone Glycosides
Borntrager's test:
Methods : Boil the test material with 1ml of dilute sulphuric acid in
a test tube for 5 min (anthracene glycosides are hydrolyzed to
aglycone and sugars by boiling with acids) centrifuge or filter while
hot, filtrate, cool and shake with an equal volume of
dichloromethane (the aglycones will dissolve preferably in
dichloromethane) separate the lower dichloromethane layer and
shake with half its volume with dilute ammonia.
Result : A rose pink to red color is produced in the ammonical
layer (aglycones based on anthroquinones give red color in
the presence of alkali).
SCREENING PHYTOCHEMISTRY 6
 Test for Cardiac Glycosides
Kedde’s test:
Extract the drug with chloroform, evaporate to dryness,
add one drop of 90% alcohol and 2 drops of 2% 3,5-
dinitro benzoic acid(3,5-dinitro benzene carboxylic acid -
Kedde's reagent) in 90%alcohol.
Make alkaline with 20% sodium hydroxide solution. A
purple color is produced.
The color reaction with 3, 5-diinitrobenzoic acids depends
upon the presence of an β- unsaturated-O-lactones in the
aglycone.
SCREENING PHYTOCHEMISTRY 7
Keller killiani test [test for Deoxy sugars]:
Methods : Extract the drug with chloroform
and evaporate it to dryness. Add 0.4 ml of
glacial acetic acid containing a trace amount
of ferric chloride. Transfer to a small test tube;
add carefully 0.5 ml of concentrated sulphuric
acid by the side of the test tube.

Result : blue color appears in the

acetic acid layer.
SCREENING PHYTOCHEMISTRY 8
 Tests for Flavanoids
❑ Shinoda test:
Methods : Dry powder/extract + 5 ml 95%
ethanol + few drops conc. HCl + 0.5 g
magnesium
Result : turnings Pink colour.
❑ Lead acetate test:
Methods : Small quantity of extract + lead
acetate solution
Result : Yellow colour precipitated.
SCREENING PHYTOCHEMISTRY 9
❑Sodium hydroxide test:
Methods : Plant extract + NaOH
Result : Yellow colour which decolorize after
addition of glacial acetic acid.
❑Ferric chloride test:
Methods : 2-3 ml of alcoholic extract + 5% FeCl3 P
Result : deep blue – black colour
❑Gelatin test :
Methods : 2-3 ml of alcoholic extract + Gelatin
10% + NaOH (10%)
Result : white at lower level formed.
SCREENING PHYTOCHEMISTRY 10
 Test of Triterpenoids
Liebermann -Burchard’s test:
Methods : 2 mg of dry extract was dissolved in
acetic anhydride, heated to boiling, cooled and
then 1 ml of concentrated sulphuric acid was
added along the sides of the test tube.
Result : Formation of a pink colour indicates the
presence of triterpenoids.

SCREENING PHYTOCHEMISTRY 11
 Tests of Steroids
❑ Liebermann-Burchard’s test:
Methods : 2 mg of dry extract was dissolved in acetic
anhydride, heated to boiling, cooled and then 1 ml of
concentrated sulphuric acid was added along the
sides of the test tube.
Result : Formation of green colour indicates the
presence of steroids.
❑ Salkowski reaction:
Methods : 2 mg of dry extract was shaken with chloroform, to
the chloroform layer sulphuric acid was added
slowly by the sides of test tube.
Result : Formation of red colour indicated the presence
SCREENING PHYTOCHEMISTRY 12
of steroids.
 Test of Tannins
Methods : To 1-2 ml of the ethanolic
extract, few drops of 5% w/v
FeCl3 solution was added.
Result :
• A green colour indicated the presence
of gallotannins,
• While brown colour indicates the
presence of pseudotannins.
SCREENING PHYTOCHEMISTRY 13
THANK YOU

SCREENING PHYTOCHEMISTRY 14