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Chemical composition of the leaves of Azadirachta indica Linn (Neem)

Article · January 2014

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 Chemical composition of the leaves of Azadirachta Indica Linn
(Neem)
Prashanth G.K1, G.M. Krishnaiah2

Department of Chemistry, Sir M. Visvesvaraya Institute of Technology, Hunasamaranahalli,

Bangalore-562 157, India.

Abstract:

The aim of this study was to carry out the phytochemical screening and GC-MS
analysis of the leaves of Azadirachta Indica Linn. Phytochemical screening of the
aqueous and ethanolic extracts of the leaves revealed the presence of alkaloids,
reducing sugars, saponins etc, in them. GC-MS of the ethanolic extract revealed the
presence of many compounds in the leaves of Azadirachta Indica Linn.

Keywords: Azadirachta indica Linn, Phytochemical screening, GC-MS

INTRODUCTION

Meliaceae or the Mahogany family is a flowering plant family of

mostly trees and shrubs. They are characterized by alternate usually pinnate
leaves without stipules and by syncarpous apparently bisexual flowers borne in
panicles, cymes, spikes, or clusters. Most species are evergreen, but some
are deciduous, either in the dry season or in winter. The family includes about
50 genera and 550 species. Azadirachta indica Linn is a tropical evergreen tree
native to India and is also found in other southeast countries. It is a tree in
the mahogany family Meliaceae. Azadirachta indica is locally known as Neem.
It is a tree in the mahogany family of Meliaceae. It is one of two species in the
genus of Azadirachta. It is native to India, Bangladesh, Thailand, Nepal and
Pakistan. It grows well in tropical and sub-tropical regions. In India, Neem is
known as “the village pharmacy” because of its healing versatility. The tree has
been used in Ayurvedic medicine due to its medicinal properties. Neem is also
Prashanth,Krishnaiah Page 21
 called ‘arista’ in Sanskrit- a word that means ‘perfect, complete and
imperishable’. Fruit, seeds, oil, leaves, roots, bark and almost every part of the
tree is bitter and contain compounds with proven antiviral, antiretroviral, anti-
inflammatory, anti-ulcer and antifungal, antibacterial, anti plasmodial,
antiseptic, antipyretic and anti diabetic properties [1-3]. The various parts of this
tree have many uses that aptly give Neem its name in Sanskrit-“sarva roga
nivarini”, meaning “the curer of all ailments”.
India has encouraged scientific investigations of Neem tree as a part of
its program to revitalize Indian tradition and also to increase commercial
interest on Neem [4]. In India, this tree is called “Divine tree”, “Wonder tree”,
“heal all”, “Materia Medica”, “Free tree of India”, Nature’s drugstore”, “Village
Pharmacy”, “Panacea for all diseases” [5-7]. The Neem oil is isolated from its
fruits and seed [2, 8-10]. Neem is the most important medicinal plant that has
been declared as the “Tree of the 21st century” by the United Nations.
Thus as the plant possesses immense medicinal properties, the aim of the
present study was to identify the phyto-components present in the aqueous and
etanolic extracts of the leaves of Neem by qualitative photochemical testing and
to identify the compounds present in the ethanolic extract of the leaves by Gas
chromatography-Mass spectrum (GC-MS) analysis.

EXPERIMENTAL DETAILS

Plant collection
Neem leaves were collected in Hunasamaranahalli, Bengaluru. It was ensured
that the plant was healthy and uninfected. Leaves were washed under running
tap water to remove any traces of soil particles and other dirt. Then washed with
distilled water, air dried and cut in to small pieces and dried for 15 days in
shade. Then the leaves were ground and sieved to get fine powder.

Prashanth,Krishnaiah Page 22
 Preparation of aqueous and ethanolic extracts
All the chemicals and reagents used in this study were of analytical grade.
The powdered leaves (20g) were extracted successively in double distilled water and
absolute ethanol at 50-600 C for 18 hours using Soxhlet apparatus. The solvents used
were recovered under pressure until dry extracts were obtained.

Phytochemical screening
Detailed phytochemical examinations were carried out for both the extracts as
per the standard methods [11-13].
Tests for Alkaloids
To the extract, dilute hydrochloric acid was added, shaken well and
filtered. With the filtrate, the following tests were performed.
Mayer’s reagent test
To 3 ml of filtrate, few drops of Mayer’s reagent were added along sides
of tube. Formation of creamy precipitate indicates the presence of alkaloids.
Wagner’s test
To 2 ml of filtrate, few drops of Wagner’s reagent were added in a test
tube. Formation of reddish brown precipitate indicates the presence of alkaloids.
Hager’s test
To 2 ml of filtrate, few drops of Hager’s reagent were added in a test
tube. Formation of yellow color precipitate indicates the presence of alkaloids.
Tests for Carbohydrates

Molisch test

2 ml of aqueous extract was treated with 2 drops of alcoholic α-naphthol

solution in a test tube and then 1 ml of concentrated sulphuric acid was added
carefully along the sides of the test tube. Formation of violet ring at the junction
indicates the presence of carbohydrates.

Prashanth,Krishnaiah Page 23
 Barfoed’s test
1 ml of extract and Barfoed’s reagent were mixed in a test tube and
heated on water bath for 2 minutes. Red color due to formation of cupric oxide
indicates the presence of monosaccharide.
Tests for Reducing Sugars
Fehling’s test
To 1 ml of aqueous extract, 1 ml of Fehling’s A and 1 ml of Fehling’s B
solutions were added in a test tube and heated on a water bath for 10 minutes.
Formation of red precipitate indicates the presence of reducing sugar.
Benedict’s test
Equal volume of Benedict’s reagent and extract were mixed in a test
tube and heated on a water bath for 5-10 minutes. Solution appears green,
yellow or red depending on the amount of reducing sugar present in the test
solution which indicates the presence of reducing sugar.
Tests for Flavonoids
Alkaline reagent test
The extract was treated with few drops of sodium hydroxide solution
separately in a test tube. Formation of intense yellow color, which becomes
colorless on addition of few drops of dilute acid indicates the presence of
flavonoids.
Lead Acetate Test
The extract was treated with few drops of lead acetate solution.
Formation of yellow precipitate indicates the presence of flavonoids.
Tests for Glycosides
Borntrager’s test
To 3 ml of test solution, dilute sulphuric acid was added, boiled for 5
minutes and filtered. To the cold filtrate, equal volume of benzene or
chloroform was added and it was shaken well. The organic solvent layer was

Prashanth,Krishnaiah Page 24
 separated and ammonia was added to it. Formation of pink to red color in
ammonical layer indicates the presence of anthraquinone glycosides.
Legal’s test
1 ml of test solution was dissolved in pyridine. 1 ml of sodium
nitropruside solution was added and made alkaline using 10% sodium hydroxide
solution. Formation of pink to blood red color indicates the presence of cardiac
glycosides.
Keller-Killiani test
To 2 ml of test solution, 3 ml of glacial acetic acid and 1 drop of 5%
ferric chloride were added in a test tube. Carefully 0.5 ml of concentrated
sulphuric acid was added by the sides of the test tube. Formation of blue color
in the acetic acid layer indicates the presence of cardiac glycosides.
Tests for Tannin and Phenolic compounds
Ferric chloride test
A small amount of extract was dissolved in distilled water. To this
solution 2 ml of 5% ferric chloride solution was added. Formation of blue, green
or violet color indicates presence of phenolic compounds.
Lead Acetate Test
A small amount of extract was dissolved in distilled water. To this
solution few drops of lead acetate solution were added. Formation of white
precipitate indicates the presence of phenolic compounds.
Dilute iodine solution test
To 2-3 ml of extract, few drops of dilute iodine solution were added.
Formation of transient red color indicates the presence of phenolic compounds.
Test for Saponin
Froth test
The extract was diluted with distilled water and shaken in a graduated
cylinder for 15 minutes. The formation of layer of foam indicates the presence

Prashanth,Krishnaiah Page 25
 of saponins.
Tests for Protein and Amino acids
Ninhydrin test
3 ml of the test solution was heated with 3 drops of 5% Ninhydrin
solution on a water bath for 10 minutes. Formation of blue color indicates the
presence of amino acids.
Biuret test
The extract was treated with 1 ml of 10% sodium hydroxide solution in a
test tube and heated. A drop of 0.7% copper sulphate solution was added to the
above mixture. The formation of violet or pink color indicates the presence of
proteins.
Tests for Triterpenoids and Steroids:
Salkowski’s test
The extract was treated with chloroform and filtered. The filtrate was
added with few drops of concentrated sulphuric acid, shaken and allowed to
stand. If the lower layer turns red, sterol is present. Presence of golden yellow
layer at the bottom indicates the presence of triterpenes.
Libermann-Burchard’s test
The extract was treated with chloroform. To this solution few drops of
acetic anhydride were added, boiled and cooled. Concentrated sulphuric acid
was added through the sides of the test tube. Formation of brown ring at the
junction of two layers, if upper layer turns green, indicates the presence of
steroids and formation of deep red color indicates the presence of triterpenoids.
GC-MS analysis of the ethanolic extract of Neem leaves
The chemical composition of ethanolic extract of the leaves was analyzed by
GC-MS. The analysis was carried out on Jeol spectrometer (Model: Accu TOF
GCV).
RESULTS AND DISCUSSIONS

Prashanth,Krishnaiah Page 26
 Results of qualitative phytochemical analysis of the aqueous and ethanolic
extracts of Neem leaves
The results of qualitative phytochemical analysis of aqueous leaf extract
(ALE) and ethanolic leaf extract (ELE) of Neem are given in Table 1. Results
indicate the presence of many phyto-components in both the extracts. The
results of our present studies are almost in agreement with the results published
by the other research groups [14-16].

Sl. Constituent Test Result Sl. Constituent Test Result

No. ALE ELE No. ALE ELE
1 Alkaloids Mayer’s reagent + + Glycoside Bomtrager’s test - +
test
Wagner’s + + Killer-Killiani test + +
reagent test
Hager’s reagent + + 6 Tannins Ferric chloride test + +
test and
2 Carbohydrat Molish’s test + + Phenolic Lead acetate test + +
es compounds
Barfoed’s test + + Dilute iodine + +
solution test
3 Reducing Fehling’s test + + 7 Saponins Froth test + +
sugars Benedicts test + + 8 Proteins Biuret test - -
4 Flavanoids Alkaline + + and Amino Ninhydrin test - -
reagent test acids
Lead acetate + + 9 Triterpenoi Salwonski test - -
test ds and
5 Glycoside Legal’s test - + Steroids Libermann and - -
Burchard’s test

Table 1 Results of Qualitative Phytochemical Screening

+ is present - is absent

GC-MS analysis

A typical GC-MS of the ethanolic extract of the leaves of Neem is

presented in Fig. 1. Mass spectra of the ethanolic extracts of Neem leaves are
depicted in Fig. 2 (a-b). The fragmentation patterns of the mass spectra were
compared with those of the known compounds stored in the National Institute of
Standards and Technology (NIST) research library. It revealed the presence of
(Acetyloxy) acetic acid (C4H6O4), Hydroxy pivalic acid (C5H10O3), Phytol
(C20H40O), 4-Cycloocten-1-ol, 8,8’-(iminodi-2,1-phenylene) bis- (C28H35NO2),

Prashanth,Krishnaiah Page 27
 1,3-Diphenyl-2-azafluorene (C24H17N), Lup-20 (29)-2n-3-ol, acetate, (3β)-
(C32H52O2), Germanicol (C30H50O).

Fig. 1 GC-MS of the ethanolic extract of the leaves of Neem

Prashanth,Krishnaiah Page 28
 Fig. 2 (a-b) Mass spectra of the ethanolic extract of the leaves of
Neem

Prashanth,Krishnaiah Page 29
 CONCLUSION

Thus in our present studies, the phytochemical screening of the aqueous

and ethanolic extracts of Neem leaves revealed the presence of many phyto-
components. The GC-MS analysis of ethanolic extract showed a number of
components. This study may be useful to explore the pharmacological and
biosynthetic activity of Neem plant further.

ACKNOWLEDGEMENT

The authors thank the Management and the Principal of Sir MVIT for the
support and encouragement extended towards this work. The authors
acknowledge SAIF, IITB for GC-MS analysis.

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