Qualitative & Quantitative phytochemical screening of herbs.

1. Phytochemical Screening of plants
Dr. SINDHU K.
M. V. Sc. SCHOLAR,
DEPT. OF VPT,
COVAS,
Pookode.

2. • Phytochemicals have two categories:
Primary & Secondary constituents.
• The phytochemical analysis  Commercially
value.
• Great interest in pharmaceutical companies for the
production of the new drugs for curing of various
diseases.

3. Qualitative Quantitative
• Steroids,
• Reducing sugars,
• Determination of total
alkaloids,
• Triterpenoids,
• Total flavonoids,
• Sugars,
• Total phenolics,
• Alkaloids,
• Total saponins,
• Phenolic compounds,
• Total tannins,
• Flavonoids,
• Total glycosides.
• Saponins,
• Tannins,
• Anthroquinones,
• Amino acids.

4. Standard procedures
• Sofowara (1993).
• Trease and Evans (1989).
• Harborne (1973).

5. Qualitative analysis methods

6. Detection of alkaloids
• The individual extract is dissolved in dilute hydrochloric acid
and filter.
• The filtrate was further tested with following reagents for the
presence of alkaloids.

7. Dragendroff’s Test:
• Filtrate was treated with potassium bismuth iodide solution (Dragendroff’s reagent).
• Formation of orange red precipitate indicated the presence of alkaloids.
Hager’s Test:
• Filtrate was treated with saturated aqueous solution of picric acid (Hager’s reagent).
• Presence of alkaloids were confirmed by the formation of yellow coloured
precipitate.
Mayer’s Test:
• Filtrate was treated with potassium mercuric iodide solution (Mayer’s reagent).
• Formation of a whitish yellow or cream coloured precipitate indicated the presence
of alkaloids.

8. Detection of carbohydrates
Dissolve 2g
extract in 5
ml distilled
water & filter
it.
The filtrates
were used to
test for the
presence of
carbohydrates.
Molisch’s Test:
• Filtrate was treated with
2 drops of alcoholic α-
naphthol solution in a
test tube, shaken
• Add conc. sulphuric acid
from the side of the test
tube.
• Development of a violet
ring @ the junction of
two liquid confirmed the
presence of
carbohydrates

9. Detection of reducing sugars
Benedict’s test:
• Filtrate was treated with Benedict’s reagent & boil in a thermostatic
• water bath for 5 minutes.
• Formation of an orange red precipitate indicated the presence of reducing
sugars.
Fehling’s Test
• Filtrate was acidified with dil. Hydrochloric acid, neutralized with
alkali & heated with Fehling’s A & B solutions.
• Formation of red precipitate indicated the presence of reducing
sugars.

10. 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.
Foam Test:
• Small quantity of the extract was shaken with 2 ml of water.
• Persistence of foam produced for ten minutes indicated the
presence of saponins.

11. Detection of phytosterols
Small quantity of
extract dissolved
in 5 ml of
chloroform
Salkowski’s Test:
On adding a few drops of
conc. Sulphuric acid.
Allow the solution to
stand
Formation of brown ring
indicated the presence of
phytosterols
LibermannBurchard’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 solution
confirmed the presence of
phytosterols.

12. Detection of phenolic compounds:
Ferric Chloride Test:
• Treat the extract with 3-4 drops of ferric chloride solution.
• Formation of bluish black colour indicated the presence of phenols.
Lead Acetate Test:
• Treat the extract with 3ml of 10% lead acetate solution.
• A bulky white precipitate indicated the presence of phenolic
compounds.

13. Detection of tannins:
Take 0.5 g of
the dried
powdered
plant
Boil 0.5g
sample in
20 ml of
water in a
test tube.
Filter the
above
mixture
Add few drops
of 0.1% ferric
chloride.
Development of a
brownish green or a
blue-black
colouration indicated
the presence of tannins

14. Detection of flavonoids:
• Treat the extract with few drops of sodium
hydroxide solution.
• Formation of intense yellow colour, which
becomes colourless on further addition of dilute
acid, indicated the presence of flavonoids.
Alkaline Reagent
Test
• Treat the extract with few drops of lead acetate
solution.
• Formation of yellow precipitate indicated the
presence of flavonoids.
Lead acetate Test:
• Add a few drops of ferric chloride solution to
the extract solution.
• Development of intense green colour indicates
the presence of flavonoids.
Ferric chloride
Test:

15. Detection of proteins and amino acids:
Millon’s Test:
• Treat the test solution with few drops of Millon’s reagents.
• when warmed , a white precipitate is formed which changes to a brick red
or disappears: indicates the presence of proteins & A.A.
Biuret Test:
• Treat the test solution with few drops of 2% of copper sulphate solution
• Add 1ml of ethanol followed by excess of potassium hydroxide pellets
• formation of pink colour in the extract layer indicates the presence of Pr.
Ninhydrin Test:
• Add Ninhydrin reagent to the test solution & boiled for few minutes.
• Formation of blue colour indicated the presence of amino acids.

16. Detection of terpenoids:
Salkowski test:
Mix 2 ml of chloroform
to extract solution
carefully added conc.
Sulphuric acid (3 ml) to
form a layer.
A reddish brown
colouration of the
interface indicated
the presence of
terpenoids.

17. Detection of cardiac glycosides
Keller-Killani test
Add 1ml of conc.
sulphuric acid,
Appearance of brown ring @
the interface indicate the
deoxysugar characteristic of
cardenolides
Appearance of a violet ring
below the brown ring & a
greenish ring in the acetic acid
layer confirmed the results.
Treat the extract with 2 ml of
glacial acetic acid containing
one drop of ferric chloride
solution.

18. Test for fixed oils and fats:
Spot Test:
• Place small quantity of the extract in between two filter papers.
• Oil stain produced with any extract showed the presence of fixed
oils and fats in the extracts.
Saponification test:
• Add few drops of 0.5N alcoholic potassium hydroxide extract
with few drops of phenolphthalein solution.
• Heat on a water bath for 1-2 hours.
• Formation of soap indicated the presence of fixed oils and fats in
the extracts.

19. Test for gums and mucilages
Dilute small
quantity of the
ethanolic extract
with water
Add ruthenium
red solution.
A pink colour
production
showed the
presence of gums
and mucilages.

20. Quantitative determination of phytochemicals
• Total phenols determination: Hagerman A., Muller I., Makkar
H. (2000).
• Total alkaloid determination: Harborne.J. (1973).
• Total flavonoids determination: Kumaran A, Karunakaran R.
(2006).
• Total tannins determination: Van-Burden T, Robinson W.
(1981).
• Total saponins determination: Obdoni B, Ochuko P. (2001).

21. Determination of total phenolic compounds(Hagerman A,
Muller I, Makkar H, 2000)
• Weigh accurately 100 mg of the extract of the sample &
dissolved in 100 ml of triple distilled water (TDW).
• Transfer 1 ml of this solution to a test tube & add 0.5 ml 2N of
the Folin-Ciocalteu reagent.
• Add 1.5 ml 20% of Na2CO3 solution & make volume up to 8 ml
with TDW followed by vigorous shaking.
• Finally allow to stand for 2 hours.
• Take the absorbance at 765 nm. (Spectroscopic determination).
• Data use: To estimate the total phenolic content using a standard
calibration curve obtained from various diluted concentrations of
gallic acid.

22. Determination of total alkaloids (Harborne J, 1973)
• Weigh 5 g of the sample & add 5g sample into a 250 ml beaker.
• Add 200 ml of 10% acetic acid in ethanol & cover the beaker with aluminum
foil
• Allow to stand for 4 hour.
• Filter the extract & concentrated on a water bath to one-quarter of the original
volume.
• Add concentrated ammonium hydroxide drop wise to the extract until the
precipitation was complete.
• The whole solution was allowed to settle
• Collect the precipitate & wash with dilute ammonium hydroxide and then filter.
• The residue is the alkaloid, which was dried and weighed

23. Determination of total flavonoids
(Kumaran A, Karunakaran R. 2006)
• The method is based on the formation of the flavonoids - aluminium
complex which has an absorptivity maximum at 415nm.
• Mix 100μl of the plant extracts in methanol (10 mg/ml) with 100 μl of
20 % aluminum trichloride in methanol
• Add a drop of acetic acid, and then diluted with methanol to 5ml.
• After 40 minutes read the absorption @ 415 nm.
• Blank samples were prepared from 100 ml of plant extracts and a
drop of acetic acid, and then diluted to 5ml with methanol.
• The absorption of standard rutin solution (0.5 mg/ml) in methanol was
measured under the same conditions.
• All determinations were carried out in triplicates.

24. Determination of total tannins
(Van-Burden T, Robinson W. 1981).
• Weigh 500 mg of the sample & transfer to a 50 ml plastic bottle.
• Add 50 ml of distilled water & shaken for 1 hour in a mechanical
shaker.
• Filter the above mixture into a 50 ml volumetric flask & make up to
the mark.
• Pipette out 5 ml of the filtrate into a test tube & mix with 2 ml of 0.1
M FeCl3 in 0.I N HCl & 0.008 M potassium ferrocyanide.
• Measure the absorbance @ 120 nm within 10 min.

25. Determination of total saponins
(Obdoni B, Ochuko P. 2001)
• Ground the samples & 20 g of each were put into a conical flask
• Add 100 cm3 of 20% aqueous ethanol.
• Heat the samples over a hot water bath for 4 hour with continuous
stirring @ about 55°C.
• Filter the mixture.
• Re-extract the residue with another 200 ml 20% ethanol.
• The combined extracts were reduced to 40 ml over water bath @
about 90°C.
• The concentrate was transferred into a 250 ml separatory funnel &
add 20 ml of diethyl ether; shake vigorously.

26. • The aqueous layer was recovered while the ether layer was discarded.
• Repeat the purification process.
• Add 60 ml of n-butanol & wash twice with 10 ml of 5% aqueous sodium
chloride.
• Heat the remaining solution in a water bath.
• After evaporation the samples were dried in the oven to a constant weight.
• The saponins content was calculated using standard formulae.

27. Qualitative Quantitative
• steroids,
• reducing sugars,
• Determination of total
alkaloids,
• triterpenoids,
• Total flavonoids,
• sugars,
• Total phenolics,
• alkaloids,
• Total saponins,
• phenolic compounds,
• Total tannins,
• flavonoids,
• Total glycosides.
• saponins,
• tannins,
• Anthroquinones,
• amino acids.

28. Steps Involved in the Extraction of Medicinal Plants
1. Size reduction
2. Extraction
3. Filtration
4. Concentration
5. Drying
6. Packing
7. Storage

29. Size reduction

30. Different extraction methods
maceration,
infusion,
percolation,
digestion,
decoction,
hot continuous extraction (Soxhlet),
aqueous-alcoholic extraction by fermentation,
counter-current extraction,
microwave-assisted extraction,
ultrasound extraction (sonication),
supercritical fluid extraction,
phytonic extraction (with hydrofluorocarbon solvents).

31. Different methods of drying
I. Natural drying  Sun drying
 Shade drying
II. Artificial drying  Tray drying
 Vacuum dryers
 Spray dryers

32. GARBLING / DRESSING
• Process of removal of sand & foreign organic part
from same plant.
Eg: removal of excessive stem.
removal of excessive stalk in case of cloves.
careful removal of rhizomes from roots & rootlets.
removal of iron piece using magnet in caster beans.
bark removal from gum acacia.

33. Packing
• Pressed & balded  leaf drugs like senna, vinca.
• Amber color bottle  cod liver oil
(protect from direct sunlight)
• Closed container  asfoetida (prevent loss of volatile oil)
• Kerosine tins  colophony, balsam of tolu.
• Goat skin  aloe preparations.
• Packing in big masses  colophony (control auto oxidation).
• Packing one side the other  cinnamon bark /quill (prevent
volatilization of oils).
• Simple packing  crude drugs from root & seeds.

34. Storage & preservation of crude drugs
Sterile, Closed containers, Moisture free, air tight.
• Physical & chemical damages
• Insect & mould attacks

35. References
I. HERIN SHEEBA D. GRACELIN A. JOHN DE BRITTO & P. BENJAMIN JEYA RATHNA KUMAR.2013. QUALITATIVE AND
QUANTITATIVE ANALYSIS OF PHYTOCHEMICALS IN FIVE PTERIS SPECIES. Int J Pharm Pharm Sci, Vol 5(1): 105-107 .
II. Adarsh Krishna T.P., Ajeesh Krishna T.P., Sanyo Raj V.N., Juliet S., Nair S.N., Ravindran R. and Sujith S. 2013.Evaluation of
phytochemical constituents and proximate contents of the ethanolic leaf extract of Tetrastigma leucostaphylum (Dennst.)
Alstone (Vitaceae) found in Western Ghats of Kerala, India Res. J. Pharmaceutical Sci. 2(10): 1-6.
III. Prashant Tiwari, Bimlesh Kumar, Mandeep Kaur, Gurpreet Kaur, Harleen Kaur. 2011. Phytochemical screening and
Extraction: A Review Internationale Pharmaceutica Sciencia 1(1): 98-106.
IV. Google images.