ISOLATION AND CHARACTERIZATION OF THERAPEUTICALLY

RELEVANT COMPOUNDS FROM AQUEOUS EXTRACT OF LEAVES OF

NEEM (AZADIRACHTA INDICA) AND DEVELOPMENT OF QUALITATIVE AND
QUANTITATIVE METHOD FOR ISOLATED COMPOUNDS
A dissertation submitted to
Manipal University, Manipal
In partial fulfillment of the degree of

Master of Pharmacy in
Pharmacognosy

Avinash Kumar Garg

070605017
Under the guidance of
Dr. C.S. Shreedhara Dr. Aniruddha Datta
Professor and head Senior Maneger
Department Pharmcognosy Natural Product Research and
MCOPS Development
Manipal Panacea Biotech Ltd, Lalru,
Karnataka Punjab
 Dedicated
to
My Almighty God
and
Beloved Parents
INTRODUCTION
 Azadirachta indica A.Juss and Melia azadirachta - closely related species
of Meliaceae
 A.indica A.Juss - Indian neem (Margosa tree) or Indian lilac
 M. azadirachta - Persian lilac
 Arishtha -Sanskrit name of neem
 Neem- considered as Sarbaroganibarini and Village dispensary - in
India[1]
 Neem seed - abundant in Limonoids
 about 300 active principles- diterpenes, limonoids (tetranortriterpenoids),
sulphur compounds, flavonoids, amino acids and carbohydrates
 over 160, about 80% of which are bitter limonoid
 Azadirachtin - in the neem seed kernel ( 0.1% to 0.5% by weight)
 Other limonoids - azadirone, azadiradione, epoxyazadirone and salannin,
azadiradione, epoxyazadiradione, melianone
 Flavonoids - kaempferol, quercetin, yricetin, isorhamnetin and their
glycosides
 Proteins
 Heteropolysaccharides- Polysaccharides Gla and Glb
 Peptidoglycan - compound NB-ll
 Fatty acids - oleic and linoleic acids
Literature
Review
The neem tree has been described as A. indica as early as 1830 by
De Jussieu and its taxonomic position is as follows[1].

 Rutales
Order
 Rutineae
Suborder  Meliaceae (Mahogany family)
Family  Melioideae
Subfamily
 Melioideae
Tribe  Azadirachta
Genus  indica
Species
 Botanical name : Azadirachta indica
Natural order : Meliaceae

Vernacular Names
English - Margosa tree
Hindi - Nim, Nimba
Sanskrit - Arista
Marathi - Kadunimb, Limba, Nim
Tamil - Vemmu, Veppu
Telgu - Vemu
Gujrati - Limbado, Limado
DISTRIBUTION
 Southern tip of Kerala to the Himalayan hills
 Tropical to sub tropical
 Semi dried to wet tropical regions
 Sea level to about 700 meter elevation
 Cultivated in India and African countries
 In India- Uttar Pradesh, Bihar, West Bengal, Orissa, Delhi,

Maharashtra, Gujarat, Andhra Pradesh, Tamil Nadu

GENERAL DESCRIPTION

 Flowers from January to may

 Ripening time of fruits from may to august.
 Tree is an evergreen, or deciduous, fast-growing
 Height up to 25 meters
 The trunk is relatively short, straight and may reach a girth of 1.5-3.5 m.
 The bark -thick, fissured, gray outer bark and a reddish brown inner one.
 Leaves -unpaired, pinnate , 20 to 40 cm long , medium to dark green [3].
 Average Fruit Yield -About 205 Kg/Tree

 The weight of the seed kernel accounts for about 10% of that of the whole
fruit[3] [5].

 Growes at an altitude up to 1500m,

 Temperature range is about 9.5 - 37 °C.
 distributed throughout southeast Asia, East and sub-Sahelian Africa, Fuji,
Mauritius, parts of Central America, the Cambean and Puerto Rico.
ACTIVE COMPOUNDS FROM NEEM
1. Azadirone group
2. Amoorastatin group
3. Vepinin group
4. Vilasinin group
5. Gedunin group
6. Nimbin group
7. Nimbolinin group
8. Salannin group
9. Azadirachtin group
AZADIRONE GROUP
GEDUNIN AND VILASNIN GROUP
NIMBIN GROUP
NIMBOLININ GROUP
SALANNIN GROUP
H3C

COOMe H3C
CH2
1
OR
CH3 CH3

2 O
OR
H3C O

(22) R1-Tig R2-Ac Salannin

AZADIRACHTIN AND ITS ANALOGUES
OTHER TERPENOIDS
ORGANIC SULPHURIC COMPOUNDS
POLYSACCHARIDES
BIOLOGICAL ACTIVITIES OF SOME NEEM
COMPOUNDS
S.No. Compound Source Biological activity

Anti-inflammatory

Antiarthritis

Antipyretic

Hypoglycaemic
1 Nimbidin Seed oil
Antigastric ulcer

Spermicidal

Antifungal

Antibacterial

2 Sodium nimbidate Seed Antiinflamatory

3 Nimbin Seed oil Spermicidal

Antibacterial
4 Nimbonlide Seed oil
Antimalarial

Antifungal
5 Gedunin Seed oil
Antimalarial
6 azadirachtin Seed Antimalarial

7 Mahmoodin Seed oil Antibacterial

Gallic acid
Anti-inflammatory
8 Epicatechin Bark
And immunomodulator
And catechin

Margolone

9 Margolonone Bark Antibacterial

Isomargolonone

Cyclic trisulphide
10 Leaf Antifungal
& cyclic tetrasulphide

11 Polysaccharides Gla, Glb Bark Anti-tumour

12 Polysaccharides Glla, Gllla Bark Anti-inflammatory

13 NB-ll peptidoglycan Bark Immunomodulatory

PHARMACOLOGICAL ACTIONS OF NEEM
Part Medicinal use

Leaf Leprosy, eye problems, epistaxis, intestinal worms, anorexia, biliousness, skin ulcers.

Bark Analgesic, alternative and curative of fever.

Flower Bile suppression, elimination of intestinal worms and phlegm.

Relieves piles. Intestinal worms, urinary disorder, epistaxis, phlegm, eye problem, diabetes,
Fruit
wounds and leprosy.

Relieves cough, asthma, piles, phantom tumor, intestinal worms, spermatorrhoea, obstinate
Twig
urinary disorder, diabetes.

Gum Effective against skin diseases like ring worms scabies, wounds and ulcers.

Seed pulp Leprosy and intestinal worms.

Oil Leprosy and intestinal worms.

Root, bark, leaf, flower
Blood morbidity, biliary afflictions, itching, skin ulcers, burning sensation and leprosy
and fruit together
BIOLOGICAL ACTIONS OF AQUEOUS EXTRACT
OF NEEM LEAVES

 Immunostimulant activity[53,54].
 Hypoglycaemic activity[55,56,57,59,60].
 Antiulcer effect[61,47]
 Antifertility effect[62,63,42]
 Antiviral activity[64,38]
 Anticarcinogenic activity[65,66,67]
 Hepatoprotective activity[44,68].
 Anti-inflammatory effect[40].
 Spermicidal effect[48].
 Antipsoriatic effect[43].
 Antihypertensive effect[50].

 Primary Chemopreventive effect[46].

 Growth, Pigments and Photosynthesis inducing effect[45].
 Restorative effect[49].
 Anti-coccidial effect[41].
 Allelopathic effect[39].
 Biosorbent effect[51].
SAFETY EVALUATION OF AQUEOUS
EXTRACT OF NEEM LEAVES
Toxic/adverse effect Animal in which Reference
toxicity is manifested

Lethal toxicity Mice, guinea pigs 77

Reduced heart rate and Guinea pigs 70

increased pulse rate

CNS-depressant Mice 69

Hapatonephropathy Hisex Chick 71

Genotoxicity Mice 72

Antifertility Mice 62, 77

Decreased sperm count

Rat 73
and motility

Antiandrogenic Rat 74

Hypoglycaemia Rat, rabbit 56, 58

 MATERIAL
AND METHODS
SOURCE OF NEEM EXTRACT

Aqueous extract of Neem leaves was procured from SAMI LABS LIMITED. Batch
No. A50387
Description of aqueous extract of neem leaves
 Extract- purified aqueous extract of Azadirachta indica leaves.
 Objective - finding markers in a Polyherbal Formulation
 Active against HIV and sexually transmitted disease pathogens in vitro
 Phase ll clinical trial with the Polyherbal tablets in women with (AVDS)
 In total, 137 women (97 %) reported complete (n=62, 44%) and partial (n= 75,
53%) relief
 71 (74 %) women -confirmed to be cured of AVDS.
 Cure rate was 77% for C. albicans and 68% for bacterial vaginosis[52].
INSTRUMENTS
 Electronic weighing balance- Mettler
 Rota vapor- Buchi, Heidolph
 Sonicator- Toshcon
 TLC chamber- Camag
 UV- VIS Spectrophotometer- Perkin Elmer
 HPTLC- Camag Linomat 5
 HPLC- PICO-TAG (Waters)
 Infrared spectrophotometer- Perkin Elmer
 NMR spectrophotometer- Brucker
 pH meter Cyberscan
CHROMATOGRAPHIC METHODS

 Column chromatography
 Column filtration
 Thin layer chromatography
 High performance liquid chromatography (HPLC)
 High performance liquid chromatography (HPTLC)
 Preparative TLC
 PHYSICAL ANALYSIS
 Water extractable matter [79] 

 Alcohol extractable matter[70]

 Loss on drying[78] (Ph. Eur. method 2.8.17)
 Ash content

Method II[78] (Ph. Eur. method 2.4.16)

 Apparent densities[78] (Ph. Eur. method 2.9.15
 Content of bitter principles by gravimetery
PHYTOCHEMICAL ANALYSIS

 Preliminary phytochemical screening

 Total sugar content

 Total Free Amino Acid content

ISOLATION OF MARKERS FROM EXTRACT
ISOLATION OF AIN- P- 01- 019
ISOLATION OF AIN- P- 02- 019 AND AIN- P - 03 - 019
ISOLATION OF AIN - P- 01 - 018
ISOLATION OF AIN-P-01-002
HPTLC ANALYSIS OF AIN-P-02-019
Stationary phase
Plate size (X x Y) 10 x 10 cm
Material HPTLC plates silica gel 60 F 254
Manufacturer E. MERCK KGaA
Pre-washing No
Modification No

Sample application - CAMAG Linomat 5

Instrument CAMAG Linomat 5

Linomat 5 application parameters

Spray gas Inert gas
Sample solvent type Methanol
Dosage speed 150 nl/s
Sequence
Syringe size 100 µl
Number of tracks 4
Application position Y 15.0 mm
Band length 6.0 mm

Standard preparation
0.2g per litr concentration of L-Proline was prepared by dissolving 200mg
per litr of methanol

Sample preparation
2g per litr concentration of aqueous extract of neem leaves was prepared
by dissolving 1g in 500 ml of methanol.
Sample application- CAMAG Linomat 5
Table5: HPTLC sample application

No. Appl. Position Appl. volume Vial Sample ID

1 20.0 mm 5.0 µl 1 Neem Extract2

2 40.0 mm 5.0 µl 1 Neem Extract2

3 60.0 mm 3.0µl 2 Proline2

4 80.0 mm 3.0µl 2 Proline2

Development
Mobile phase: isopropyl alcohol: glacial acetic acid: toluene: water
(5: 2: 2: 2)
Spraying reagent: Ninhydrin spraying reagent
Detection
Instrument CAMAG TLC Scanner 3 "Scanner
Number of tracks 4
Position of first track X 20.0 mm
Distance between tracks 20.0 mm
Scan start pos. Y 15.0 mm
Scan end pos. Y 92.0 mm

Measurement
Wavelength 480
Lamp W
HPTLC PLATE SHOWING DIFFERENT TRACKS
HPLC ANALYSIS
HPLC conditions
Column Pico Tag silica based column, 3.9mm x 15cm
(Waters).
Column temp 500C
Detection Wavelength 254nm
Injection Volume 20µl
Mobile Phase Eluent A- Sodium Acetate Trihydrate sol.
containing 60% acetonitrile solution
Eluent B- 60% acetonitrile in water
Sample preparation
 1mg/ml concentration of extract was prepared in water
 10µl of this solution was taken in sample tubes and tubes were put in
reaction vial
 The sample was then dried till 65millitorr
Redrying
 20µl of redrying solution(2:2:1 solution of Water:ethanol:triethylamine) is
added to sample
 it was dried till the vacuum reached 65millitorr.

Derivatisation
 20µl of Derivatisation solution (7:1:1:1 solution of
ethanol:water:PITC:triethylamine) is added to sample and again it was
vacuum dried.
 The samples were run after diluting them in PICO.TAG Sample diluent( a
solution of Dissodium Hydrogen Phosphate with 5% acetonitrile and pH
7.4).
Standard Preparation
 5µl of amino acid std in tubes was taken, dried, redried and derivatised.

 Std concentration of all amino acids was- 2.5µM/ml. It was diluted to 200µl
and 20µl was injected.
Running of the Samples
The sample as obtained in dry condition in the sample tube was run after
diluting them in sample diluent to 100µl. About 20 µl of this solution was
loaded onto the column through injection.
Gradient elution
Table6: HPLC gradient elution

Time Flow
%A %B
(in min) (ml/min)

0 1.0 100 0

18 1.0 65 35

18.5 1.0 0.0 100

22.0 1.5 0.0 100

22.5 1.5 100 0.0

24.0 1.0 100 0.0

24.5 0.1 100 0.0

RESULTS
 PHYSICAL PARAMETERS
TABLE7: VARIOUS PHYSICAL PARAMETERS OF EXTRACT

Physical parameters Observations limits

Description Pale reddish brown powder with characteristic odour, hygroscopic

Water solubles 98.15% Limit: not less than 70.0%

Alcohol solubles 61.44% Limit: not less than 60.0%

Loss on drying 2.92% Limit: not less than 6.0 %

Ash content 14.68% Limit: not less than 15.0%

Tapped bulk density 0.70g/ml Limit: between 0.40g/ml- 0.70g/ml

Physical parameters Observations limits

Loose bulk density 0.50g/ml Limit: between 0.20g/ml -0.50g/ml

Sieve test (passes through)

-20 mesh 100.0% Limit: not less than 100.0%
-40 mesh 99.91% Limit: not less than 80.0%
-80 mesh
99.27% Limit: not less than 60.0%

Content of bitter principle by

4.92%w/w Limit: not less than 4.0%
gravimetery
PHYTOCHEMICAL SCREENING OF NEEM
EXTRACT
TABLE8: VARIOUS CHEMICAL CONSTITUENTS IN
EXTRACT
Neem oil Extract
S.No Chemical constituents

1. Carbohydrates Present

2. Reducing sugars Present

3. Hexoses Present

4. Amino acids Present

5. Phenolic compounds Present

6. Saponins glycosides Present

7. Coumarin glycosides Present

8. Flavonoids Present
CHEMICAL PARAMETERS
Total sugar content by spectrophotometric method
 Observation
Table9: Sample v/s U.V absorbance(Total Sugar content)
S.No. Sample Absorbance

1. Blank 0.0000

2. Standard 0.1268

3. Standard 0.1265

4. Standard 0.1263

5. Standard 0.1263

6. Standard 0.1263

7. Test solution 0.1755

Calulations

ABT x WS x 5 x 100 x 10 x P x100

Total Sugars 
ABS x 100 x 100 x WT x 1 x 100

Where
ABT = Absorbance of the test solution.
ABS = Mean of the absorbance of the standard solution.
WS = Weight of D - Glucose standard solution.
WT = Weight of the test sample taken (in mg).
P = Potency of D-Glucose standard in % w/w (99% in this case)

0.175 x 40 x 5 x 10 x 99 x 100

Total Sugars 0.1264 x 100 x 100 x 2 x 100

=13.74%
Total amino acid content by spectrophotometric method
Observation
Table10: Sample v/s U.V absorbance (Total Amino acid content)

S.No 440nm 570nm

Sample Absorbance Sample Absorbance

1. 0.0ml 0.0 0.0ml 0.0

2. 0.5ml 0.0319 0.5ml 0.1521

3. 1.0ml 0.0662 1.0ml 0.2867

4. 1.5ml 0.0847 1.5ml 0.4402

5. 2.0ml 0.1171 1.5ml 0.5800

6. Extract 0.0701 Extract 0.4365

Calculations
0.1mM L-Leucine concentration =13.117mg/ltr
0.1mM L-Proline concentration =11.513mg/ltr
Table11: Volume v/s amount of Standards
Volume of L-Leucine Amount of L-Leucine Volume of L-Proline Amount of L-Proline
taken Taken

0.5ml 0.006mg 0.5ml 0.0057

1.0ml 0.013mg 1.0ml 0.0115

1.5ml 0.019mg 1.5ml 0.0172

2.0ml 0.026mg 2.0ml 0.0230

Plot: Amount of L-Leucine taken v/s Absorbance
0.7
y = 22.612x
0.6 R2 = 0.9976

0.5

0.4

0.3

0.2

0.1

0
0 0.005 0.01 0.015 0.02 0.025 0.03

For neem extract y=0.4365

x=0.019mg/2ml
= 0.0096mg/ml
% age of amino acids (other than L-Proline and L- Hydroxy proline) present
in Extract
0.0096 100

0.5
Plot: Amount of L-Proline taken v/s Absorbance
0.14
y = 5.1469x
0.12
R2 = 0.9913
0.1

0.08

0.06

0.04

0.02

0
0 0.005 0.01 0.015 0.02 0.025

For neem extract y=0.0421

x=0.00817mg/2ml
= 0.00408mg/ml
%age of amino acids (L-Proline and L- Hydroxy proline) present in
Extract
 0.00408 *100
0.08
 5.10%
%age of Total amino acid content=7.030%
 
TLC finger Print profile

Instrument / apparatus: Pre-coated TLC plate, TLC chamber, TLC dipping

system

Mobile phase: isopropyl alcohol: glacial acetic acid: toluene: water

(5: 2: 2: 2)

Spraying reagent: Ninhydrin spraying reagent (freshly prepared as follow)

-30mg of Ninhydrin was dissolved in 10ml n-Butanol followed by 0.3ml of
98% acetic acid.

Derivatisation: Sprayed with Ninhydrin reagent and heated at 105oC for 5-

10min.
TLC PLATE BEFORE HEATING AND AFTER HEATING
TLC FINGER PRINT PROFILE
Characterization of AIN –P-01-019
 
Proton spectrum
3.29(CH), 2.46(CH2), 2.10(CH2)
 
Mass spectrum
146.1(M +), 168.1(M + + Na +), 100.0(M + - COO -‑)
 
 
H NMR SPECTRUM OF AIN –P-01-019
1
MASS SPECTRUM OF AIN –P-01-019
Characterization of AIN –P-02-019

Proton spectrum

2.0(NH), 3.62(CH), 2.70(CH2), 1.95; 1.70(CH2),

Mass

116.1(M + + 1), 138.1(M + + Na +), 70.2(M + - COO -‑)

 
H NMR SPECTRUM OF AIN –P-02-019
1
MASS SPECTRUM OF AIN –P-02-019
STRUCTURE (PROLINE)
Characterization of AIN –P-03-019

Proton spectrum

8.53(NH2), 4.914 (CH), 3.43; 3.17(CH2)

Mass Spectrum

189.1(M + + Na +)
H NMR SPECTRUM OF AIN –P-03-019
1
MASS SPECTRUM OF AIN –P-03-019
STRUCTURE (PHENYLALANINE)
Characterization of AIN –P-01-018

Proton spectrum

3.37(CH), 1.93(CH3)

Mass

89.2(M +), 111.4(M + + Na +)

H NMR SPECTRUM OF AIN –P-01-018
1
MASS SPECTRUM OF AIN –P-01-018
STRUCTURE (ALANINE)
Characterization of AIN-P-01-002
Elemental analysis
Nitrogen- 0%
Carbon- 26.65%
Hydrogen- 5.8%
Oxygen- 21.77%
 
IR (KBr,Vmax cm-1):
3360.00, 1606.90, 1122.79.
 
1H- NMR (DMSO, 400MHz,  ppm):

1.212(H1), 3.394(H2),
 
13C-NMR (DMSO, 400MHz, ppm):

20.38(C-3), 67.64(C-2), 176.06(C-1).

 
MS (M+): 127.00(M+ - C3H5O3), 219.20(M+)
IR SPECTRUM OF AIN-P-01-002
H NMR SPECTRUM OF AIN-P-01-002
1
13
C NMR SPECTRUM OF AIN-P-01-002
MASS SPECTRUM OF AIN-P-01-002
STRUCTURE(2-HYDROXYPROPIONATE)
HPTLC ANALYSIS
 TABLE12: HPTLC PEAK TABLE

Max
Start End
Track Peak Start Rf Max Rf Heigh Height % End Rf Area Area %
Height Height
t

1 1 0.17 6.7 0.23 54.9 22.72 0.26 39.7 1878 18.2

2 1 0.17 7.1 0.23 51 22.68 0.25 43.2 1799 17.9

3 1 0.15 4.5 0.23 81.6 100 0.29 20.4 3094 100

4 1 0.15 5.5 0.23 79.5 81.8 0.28 17.6 3016 90.47

Sample mean area= ½ (1878.0 + 1799.2) =1838.5
Standard mean area= ½ (3094 + 3016.0) = 3054.95

Amount of L-Proline present per spot of extract

0.6 x 1838.5

3054.65
= 0.36 µg per spot

%age of L-Proline present in extract

0.36 x 100

10
 3.6%
HPLC ANALYSIS
HPLC CHROMATOGRAM OF STANDARD
HPLC CHROMATOGRAM OF EXTRACT
 TABLE13: HPLC PEAK TABLE

Amount of Std % of amino acid

Amino acids Mol. Wt Area Std Sample area
in µg in sample

Asp+Asn 133.11 166.39 675711 58156 0.7159

Glu+Gln 147.13 183.91 619609 94673 1.4050

Ser 105.1 87.44 685204 0 0

Gly 75.07 93.83 707569 0 0

His 155.16 129.09 623389 0 0

Arg 174.2 144.93 692240 0 0

Thr 119.12 99.11 665851 0 0

Ala 89.1 74.13 695271 67081 0.5372

Pro 115.13 143.91 644197 36174 3.9700

Tyr 181.19 226.48 734030 0 0

 Amount of Std % of amino acid
Amino acids Mol. Wt Area Std Sample area
in µg in sample

Val 117.15 97.46 677080 0 0

Met 149.21 124.14 650971 0 0

Cys 121.16 75.73 585753 0 0

Ile 131.18 163.98 670392 0 0

Leu 131.18 163.98 592859 0 0

Phe 165.19 137.43 431370 400208 9.5550

Lys 146.19 121.63 1101406 0 0

Amino acids present in extract and their percentages
Asp 0.719%
Glu 1.405%
Ala 0.537%
Pro 3.970%
Phe 9.555%
References
 REFERENCES
1. Biswas, K., Chattopadhyay, I., Banerjee, R.K. and Bandyopadhyay, U., 2002.
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1336-1343.
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from Melia azadirachta. Tetrahedron, 27(16), 3927-39.
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azadiradione. Tetrahedron, 53(41), 14131-14140.
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Several antifeedants from neem Oil, Azadirachta indica A Juss. against
Reticulitermes aperatus Kolbe (Isoptera , Zhinotermitidae). Biosci. Biotech.
Biochem., 56,1835- 1838.
14. Lee, S. M. and Olsen, J. I., Schweizer, M. P., Klocke, J. A., 1988. 7-Deacetyl-17b-
hydroxyazadiradione, a new limonoid insect growth inhibitor from Azadirachta
indica. Phytochemistry, 27(9), 2773-2775.
15. Jeyabalan, D., Murugan, K., 1997. Effect of neem Limonoids on feeding and
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