Phytochemical screening

It contains extraction, isolation methods and identifications of phyto-constituents.

1. Faculty of Pharmacy,
Integral University Lucknow
Phyto-chemical Screening of plants
Dr. Muhammad Arif
Associate Professor (Jr.)

2. INTRODUCTION:
•Plant is the biosynthetic laboratory
•Naturalbioactivecompoundsfoundindifferentpartsofplant
(fruit,flower,stem,leaf,root)andprovidedefinite
physiologicalactiononthehumanbody.
•Phyto-constituentshavegreatinterestinpharmaceutical
companiesfortheproductionofthenewdrugsforcuringof
variousdiseases.AlotofPhyto-constituentshavebeen
identifiedwiththeirpotentialtherapeuticactionsandutilized
bydifferentpharmaceuticalcompaniesforproductionof
variousformulations.

3. Types of Phyto-chemicals
Primary constituents (Primary metabolites)
Aprimarymetaboliteisakindofmetabolitethatisdirectly
involvedinnormalgrowth,development,andreproduction.It
usuallyperformsaphysiologicalfunctionintheorganism.A
primarymetaboliteistypicallypresentinmanyorganismsor
cells.
Examples:
Carbohydrates: Glucose, Fructose, Rhamnose, Sucrose
Chlorophylls : Carotenoids
Proteins: Amino acids
Lipids: Fatty acids and glycerol

4. Secondary metabolites
•Doesnotdirectlyinvolvedinthenormalgrowth,
development,orreproductionoftheorganism.
•Theyshowedspecificpharmacologicalactionin
animalbodyinminuteconcentrations.
Examples: Steroids, Triterpenoids, Alkaloids, Phenolic
compounds, Flavonoids, Saponins, Tannins,
Anthroquinones, Glycosides etc.

5. Phyto-chemical screening methods
Qualitative Quantitative
• Steroids,
• Reducing sugars,
• Triterpenoids,
• Alkaloids,
• Phenolic compounds,
• Flavonoids,
• Saponins,
• Tannins,
• Anthroquinones,
• Amino acids.
•Determination of total
alkaloids,
• Total flavonoids,
• Total phenolics,
• Total saponins,
• Total tannins,
• Total glycosides.

6. Qualitative analysis methods

7. Detection of carbohydrates
Molisch’s Test: Dissolve 2g extract in 5
ml distilled water & filter it. Filtrate was
treated with 2 drops of alcoholic α-
naphthol solution in a test tube, shake and
add conc. sulphuric acid from the side of
the test tube. Development of a violet ring
at the junction of two liquid confirmed the
presence of carbohydrates and glycosides.
Detection of reducing sugars
Benedict’s test:Filtrate was treated with
Benedict’s reagent & boil in a water bath
for 5 minutes. Formation of an orange red
precipitate indicated the presence of
reducing sugars.

8. Detection of reducing sugars
Fehling’s Test:Filtrate was acidified with
dil. Hydrochloric acid and neutralized
with alkali & heated with Fehling’s A & B
solutions. Formation of red precipitate
indicated the presence of reducing sugars.
Detection of alkaloids
General test:
The individual extract is dissolved in dilute
hydrochloric acid and filter. The filtrate was further
tested with following reagents.
Dragendroff’s Test: Filtrate was treated with
potassium bismuth iodide solution. Orange red
precipitate will form.

9. Detection of alkaloids
Hager’s Test:Filtrate was treated
with saturated aqueous solution of
picric acid. Yellow coloured
precipitate will form.
Mayer’s Test:Filtrate was treated
with potassium mercuric iodide
solution . Whitish yellow or cream
coloured precipitate will form.
Wagner’s Test:Filtrate was treated
with potassium iodide + iodine
solution . Brown red coloured
precipitate will form.

10. Specific test:
ThalleioquinTest:AcidifythechloroformextractwithHCL
andaddfewdropsofbromine-waterfollowedby0.5mlof
strongammoniasolution,adistinctandcharacteristic
emeraldgreencolourisproduced.Thecolouredproductis
termedasthalleioquin.

11. Specific test:

12. Specific test:

13. Detection of Saponins
Froth Test:Extract was diluted with distilled water to 20 ml &
shaken in a graduated test tube for 15 minutes. Formation of 1 cm
layer of foam indicated the presence of saponins.
They areamphipathicglycosides.

14. Detection of Steroids and Tri-terpenoids
Salkowski’sTest:Smallquantityofextractdissolvedin5mlof
Chloroform.Onaddingafewdropsofconc.Sulphuricacidred
brownatlowerlayerwillformindicatedthepresenceofsteroidsand
formationofyellowcoloureindicatestritepenoids.
LibermannBurchard’stest:Thechloroformextractedsolutionwas
treatedwithfewdropsofaceticanhydride.Boil&cool.Addconc.
sulphuricacid.Formationofabluishgreencolourconfirmedthe
presenceofsteroidsanddeepredcoloureindicatedtriterpenoids.

15. Detection of cardiac glycosides

16. Detection of cardiac glycosides

18. Detection of Tannins and phenolic compounds
Ferric Chloride Test: Treat the extract with 3-4 drops of ferric
chloride solution. Bluish black colour will form.
Lead Acetate Test:Treat the extract with 3ml of 10% lead acetate
solution. A bulky white precipitate will form.

19. Detection of flavonoids:
AlkalineReagentTest:Treattheextractwithfew
dropsofConc.NaOHsolution.Formationofintense
yellowcolour,whichbecomescolourlessonfurther
additionofdiluteacidduetothepresenceofflavonoids.
Lead acetate Test: Treat the extract with few drops of
lead acetate solution. Formation of yellow precipitate
indicated the presence of flavonoids.
Ferric chloride Test:Add a few drops of ferric chloride
solution to the extract solution. Development of intense
green colour indicates the presence of flavonoids.

20. Pathway from Plant to Pure Bioactive Constituents

21. Solvents used for active component extraction
WaterEthanol/Methanol Chloroform n-Hexane
•Anthocyanins
•Starches
•Tannins
•Saponins
•Terpenoids
•Polypeptides
•Lectins
•Tannins
•Polyphenols
•Flavonols
•Terpenoids
•Sterols
•Alkaloids
•Anthocyanins
•Saponins
•Anthraquinones
•Caroteins
•Terpenoids
•Anthraquinones
•Steroides
•Caroteins
•Terpenoids
•Steroides
•Caroteins
•Oils/Fats
Water > Ethanol/Methanol > n-Butanol> Chloroform > n-Hexane

22. Isolation of Pure Bioactive Constituents from Plants

23. 19
th
to 30
th
December, 05 FDP HaridwarFDP at Varanasi
THANKS