DEPARTMENT OF BOTANY
Phytochemistry is the study of phytochemicals
Phytochemicals are certain non-nutritive plant chemicals which
have some disease preventive properties.
Ruta used for different medicinal purposes and possesses
Ruta contains many secondary metabolites such as
furocoumarins, furoquinolines and acridone alkaloids, mainly
present in the leaves.
This project is continuation work of the project “Antibacterial
activity of Ruta graveolens” done by Ms.Surumi A.B. in the
previous year, 2012.
To investigate on the phytochemicals responsible for the
antibacterial activity of Ruta graveolens.
Class : Dicotyledon
Sub class : Polypetalae
Series : Disciflorae
Cohort : Geraniales
Family : Rutaceae
Genus : Ruta
Species : graveolens
The plant material used for the study is the aerial parts
of Ruta graveolens.
The leaves were shade dried at room temperature (32-
35oc) to constant weight over a period of 60 days.
100 gm of plant part were coarsely powdered using a
mixy and put through a sieve in order to separate the
fine from the coarse particles.
The powder was then transferred into closed container
and kept in low temperature for future purposes.
Methods of Phytochemical Screening
Detection of alkaloids
Extracts were dissolved individually in dilute hydrochloric acid and filtered.
1. Mayer’s test: Filtrates were treated with Mayer’s reagent (Potassium Mercuric
iodide). Formation of yellow coloured precipitate indicates the presence
2. Wagner’s test: Extracts when treated with Wagner’s reagent (iodine in potassium
iodide). Formation of brown /reddish precipitate indicates the presence of alkaloids.
3. Hager’s test: Filtrates were treated with Hager’s reagent (saturated picric acid
solution). Presence of Alkaloids confirmed by the formation of yellow coloured
Detection of amino acids and proteins
1. Xanthoproteic Test: The Extracts were treated with few drops of concentrate nitric
acid. Formation of yellow colour indicates the presence of protein.
2. Ninhydrin Test: To the extracts, 0.25%w/v ninhydrin reagent was added and
boiled for few minutes. Formation of blue colour indicates the presence of amino
3. Biuret Test: To 2 ml of the test solution add 2 ml of 10 %sodium hydroxide: mix
well. Add few drops of 0.1%copper sulphate solution. Formation of violet or pink
colour confirms the presence of protein.
Detection of Anthraquinone
1. Borutrager’s Test: To 1 ml of the extract , add 1 ml of 10 % ferric chloride and .5
ml of concentrate hydrochloric acid. Boil in a water bath for few minutes. Filter it
and the filtrate is treated with 1 ml of Diethyl ether and concentrate ammonia.
Appearance of pink or deep red colour.
Detection of carbohydrates
Extracts were dissolved individually in 5 ml distilled water and filtered. The
filtered were used to test for the presence of carbohydrates.
1. Molish’s Test: Filtrates were treated with2 drops of alcoholic α- naphthol solution
in a test tube. Formation of the violet ring at the junction indicates the presence of
2. Benedict’s Test: Filtrates were treated with Benedict’s reagent and heated gently.
Orange red precipitate indicates the presence of reducing sugars.
3. Fehling’s Test: Filtrates were hydrolysed with dilute HCl, neutralized with alkali
and heated with Fehling’s A&B solutions. Formation of red precipitate indicates
the presence of reducing sugars.
Detection of Cardiac glycosides
1. Legal’s test: Extracts were treated with sodium nitropruside and pyridine
and sodium hydroxide. Formation of pink to red colour indicates the presence
of cardiac glycosides.
Detection of flavanoids
1. Alkaline reagent test: The Extracts were treated with few drops of sodium
hydroxide solution. Formation of intense yellow colour, which becomes colour
less on addition of dilute , indicates the presence of flavanoids.
2. Lead acetate Test: The Extracts were treated with few drops of Lead acetate
solution. Formation of yellow colour precipitate indicates the presence of
3. Ferric chloride test: 1 ml of the extract was treated with 1 ml of ferric
chloride.Formation of brown colour precipitates indicates the presence of
Detection of glycosides
Extracts were hydrolysed with dilute HCl and then subjected to test for glycosides.
1. Modified Borntrager’s Test: Extracts were treated with Ferric Chloride solution
and immersed in boiling water for about 5 minutes. The mixture was cooled and
with extracted with equal volume of benzene.The benzene layer was separated
and treated with ammonia solution. Formation of rose – pink colour in the
ammonical layer indicates the presence of anthranol glycosides.
2. Sulphuric acid test: 1 ml of concentrated sulphuric acid was added to 1 ml of test
solution and was allowed to stand for 2 minutes, the formation of reddish colour
indicates the presence of glycosides.
3. Keller Killan’s Test: 5 ml of the extract is treated with 2 ml of glacial acetic acid
containing one drop of ferric chloride solution and 1 ml concentrated sulphuric
acid. A brown ring at the interface indicates the presence of deoxy sugar. A
characteristics feature of cardenolides.
Detection of phenols
1. Ferric chloride Test: Extracts were treated with 3-4 drops of ferric chloride
solution. Formation of bluish black colour indicates the presence of phenols.
2. Lead tetra acetate Test: 1 ml of the Extracts were treated with few ml of lead
acetate solution. A precipitate production shows the presence of phenolic
Detection of phlobatanin.
1. 1ml of the extract was boiled with 1% hydrochloric acid. Formation of red
precipitate indicates the presence of Phlobatanin.
Detection of Saponins
1. Foam test: o.5gm of extract was shaken with 2 ml of water. It foam produced
persists for ten minutes it indicates the presence of saponins.
Detection of Tannins
1. Gelatin Test: To the extract, 1% gelatine solution containing sodium chloride was
added. Formation of white precipitate indicates the presence of Tannins.
2. Modified Prussian blue test: To 1 ml of extract, add 1 ml .008M potassium ferric
cyanide and 1 ml of .oo2 M ferric chloride in 0.01 M HCl. Presence of blue colour
indicates the presence of tannins.
3. Lead acetate Test: To the extract few drops of Aqueous basic lead acetate solution
were added, reddish brown bulky precipitate indicates the presence of tannin.
4. Ferric chloride Test: To the extract, few drops of 1%natural ferric chloride
solution was added formation of blackish blue colour indicates the presence of
Detection of Terpenoids
1. Salkowski’s test :5ml of each extract was made mixed in 2 ml of
chloroform and concentrate sulphuric acid was carefully added to
form a layer. A reddish brown coloration of the interface was formed
to show positive result for presence of terpenoids.
2. Copper acetate Test: Extract were dissolved in water and treated
with 3-4 drops of copper acetate solution. Formation of emerald
green indicates the presence of terpenoids.
Table2 : powder analysis with chemical agents
Sl.No Reagents Colour observed
1. Powder Brown
2. Powder + Conc.HCl Brown
3. Powder + Conc.H2So4 Brown
4. Powder +5%NaOH Brown
5. Powder +5%FeCl3 Brown
DETECTION OF ALKALOIDS
1. Mayer’s Test 2. Wagner’s test
A B A B
Detection of Terpenoids.
Phytochemical activity of Ruta graveolens is tested by
different solvents like Water, Acetone, Methanol, Ethanol,
Petroleum ether and Chloroform.
13 phytochemicals were tested during the experiments.
Out of 13 phytochemicals, 12 phytoconstituents were present
in the extraction.
phlobatanin were absent , where the flavanoids were present
in all the extracts.
Identify the phytochemicals which are responsible for the
antibacterial activity in the plant Ruta graveolens.