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
• Natural bioactive compounds found in different parts of plant
(fruit, flower, stem, leaf, root) and provide definite
physiological action on the human body.
• Phyto-constituents have great interest in pharmaceutical
companies for the production of the new drugs for curing of
various diseases. A lot of Phyto-constituents have been
identified with their potential therapeutic actions and utilized
by different pharmaceutical companies for production of
various formulations.

3. Types of Phyto-chemicals
Primary constituents (Primary metabolites)
A primary metabolite is a kind of metabolite that is directly
involved in normal growth, development, and reproduction. It
usually performs a physiological function in the organism. A
primary metabolite is typically present in many organisms or
cells.
Examples:
Carbohydrates: Glucose, Fructose, Rhamnose, Sucrose
Chlorophylls : Carotenoids
Proteins: Amino acids
Lipids: Fatty acids and glycerol

4. Secondary metabolites
• Does not directly involved in the normal growth,
development, or reproduction of the organism.
• They showed specific pharmacological action in
animal body in minute concentrations.
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:
Thalleioquin Test: Acidify the chloroform extract with HCL
and add few drops of bromine-water followed by 0.5 ml of
strong ammonia solution, a distinct and characteristic
emerald green colour is produced. The coloured product is
termed as thalleioquin.

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 are amphipathic glycosides.

14. Detection of Steroids and Tri-terpenoids
Salkowski’s Test: Small quantity of extract dissolved in 5 ml of
Chloroform. On adding a few drops of conc. Sulphuric acid red
brown at lower layer will form indicated the presence of steroids and
formation of yellow coloure indicates tritepenoids.
Libermann Burchard’s test: The chloroform extracted solution was
treated with few drops of acetic anhydride. Boil & cool. Add conc.
sulphuric acid. Formation of a bluish green colour confirmed the
presence of steroids and deep red coloure indicated triterpenoids.

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:
Alkaline Reagent Test: Treat the extract with few
drops of Conc. NaOH solution. Formation of intense
yellow colour, which becomes colourless on further
addition of dilute acid due to the presence of flavonoids.
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
Water Ethanol/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. 19th to 30th December, 05 FDP HaridwarFDP at Varanasi
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