PHYTOCHEMICAL ANALYSIS OF

RUTA GRAVEOLENS

BYRUGMA RAVISBAE10170744

DEPARTMENT OF BOTANYMAHARAJAS COLLEGE

INTRODUCTION Phytochemistry is the study of phytochemicalsPhytochemicals are certain non-nutritive plant chemicals which

have some disease preventive properties. Ruta used for different medicinal purposes and possesses

antibacterial activity.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.

AIMTo investigate on the phytochemicals responsible for the

antibacterial activity of Ruta graveolens.

MATERIALsSystematic position Class : DicotyledonSub class : PolypetalaeSeries : DiscifloraeCohort : GeranialesFamily : RutaceaeGenus : RutaSpecies : 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 of alkaloids.

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 precipitate.

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 acids.

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

4. 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 carbohydrates.

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 flavanoids.

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 flavanoids.

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 compounds.

Detection of phlobatanin.

3. 1ml of the extract was boiled with 1% hydrochloric acid. Formation of red precipitate indicates the presence of Phlobatanin.

Detection of Saponins

4. 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 tannins.

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.

RESULTS

Sl. No

Chemical Compounds

EXTRACTS

Water Acetone Ethanol Methanol Petroleum ether

Chloroform

1

Alkaloids + + + + + +

2

Amino acids &proteins

+ + + - + +

3

Anthraquinones

+ + + - + + 4

Carbohydrates

+ + - - - -

5

Cardiac glycosides

+ + - - - + 6

Flavanoids

+ + + + + +

7

Glycosides

+ + + - - + 8

Phenol

+ + - + - - 9

Phlobatanin

- - - - - -

10

Saponins

+ - - - - -

11

Steroids

- + + - - +

12

Tannin

+ + + + - + 13

Terpenoids

+ + + - + +

Table 1: Phytochemical analysis of Ruta graveolens

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 ALKALOIDS

3.Hager’s test

A B

Detection of aminoacids and proteins

1. Biuret Test

A B

Detection of carbohydrates1.Molish’s Test 2. Benedict’s Test

A B A B

Detection of Cardiac glycosides

1. Legal’s test

A B

Detection of flavonoids

1. Alkaline reagent test 2. Lead acetate Test

A B A B

Detection of flavonoids

3. Ferric chloride test

A B

Detection of glycosides1. Sulphuric acid test

A B

Detection of phenols

1.Ferric chloride Test 2. Lead tetra acetate Test

A B A B

 Detection of Steroids

1.Salkowski’s Test 2. Libermann Burchard’s Test

AB A B

Detection of Tannins

1.Gelatin Test 2.FerricchlorideTest

A B A B

Detection of Tannins

3.Lead acetate Test

A B

Detection of Terpenoids.

1.Salkowski’s Test

A B

CONCLUSIONPhytochemical 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.

FUTURE PROSPECTSIdentify the phytochemicals which are responsible for the

antibacterial activity in the plant Ruta graveolens.

THANKS