Neem Applications

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

COMPOUNDSA 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 Godand

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

Order

Suborder

Family

Subfamily Tribe

Genus Species

Rutales Rutineae Meliaceae (Mahogany family) Melioideae Melioideae Azadirachta indica

The neem tree has been described as A. indica as early as 1830 by

De Jussieu and its taxonomic position is as follows[1].

Vernacular Names

English - Margosa tree

Hindi - Nim, Nimba

Sanskrit - Arista

Marathi - Kadunimb, Limba, Nim

Tamil - Vemmu, Veppu

Telgu - Vemu

Gujrati - Limbado, Limado

Botanical name : Azadirachta

indica

Natural order : Meliaceae

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

O

H3C

O

CH2

CH3

OR1

OR2

CH3 O

CH3 CH3

COOMe

(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 SourceBiological activity

1 Nimbidin Seed oil

Anti-inflammatory

Antiarthritis

Antipyretic

Hypoglycaemic

Antigastric ulcer

Spermicidal

Antifungal

Antibacterial

2 Sodium nimbidate Seed Antiinflamatory

3 Nimbin Seed oil Spermicidal

4 Nimbonlide Seed oilAntibacterial

Antimalarial

5 Gedunin Seed oilAntifungal

Antimalarial

6 azadirachtin Seed Antimalarial

7 Mahmoodin Seed oil Antibacterial

8

Gallic acid

Epicatechin

And catechin

Bark

Anti-inflammatory

And

immunomodulator

9

Margolone

Margolonone

Isomargolonone

Bark Antibacterial

10Cyclic trisulphide

& cyclic tetrasulphideLeaf Antifungal

11Polysaccharides Gla,

GlbBark Anti-tumour

12Polysaccharides Glla,

GlllaBark Anti-inflammatory

13 NB-ll peptidoglycan Bark Immunomodulatory

PHARMACOLOGICAL ACTIONS OF NEEM

Part Medicinal use

LeafLeprosy, 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.

FruitRelieves piles. Intestinal worms, urinary disorder, epistaxis, phlegm, eye

problem, diabetes, wounds and leprosy.

TwigRelieves cough, asthma, piles, phantom tumor, intestinal worms,

spermatorrhoea, obstinate 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 and fruit

together Blood morbidity, biliary afflictions, itching, skin ulcers, burning sensation

and leprosy

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 toxicity is manifested

Reference

Lethal toxicity Mice, guinea pigs 77

Reduced heart rate and increased pulse rate

Guinea pigs 70

CNS-depressant Mice 69

Hapatonephropathy Hisex Chick 71

Genotoxicity Mice 72

Antifertility Mice 62, 77

Decreased sperm count and motility

Rat 73

Antiandrogenic Rat 74

Hypoglycaemia Rat, rabbit 56, 58

MATERIAL AND METHODS

SOURCE OF NEEM EXTRACT

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].

Aqueous extract of Neem leaves was procured from SAMI LABS

LIMITED. Batch No. A50387

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 EXTRACTISOLATION 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-02-019Stationary 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

SequenceSyringe size 100 µlNumber of tracks 4Application position Y 15.0 mmBand 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 5Table5: HPTLC sample application

Development

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

(5: 2: 2: 2)

Spraying reagent: Ninhydrin spraying reagent

No. Appl.

PositionAppl.

volumeVial Sample ID

1 20.0 mm 5.0 µl 1Neem

Extract2

2 40.0 mm 5.0 µl 1Neem

Extract2

3 60.0 mm 3.0µl 2 Proline2

4 80.0 mm 3.0µl 2 Proline2

Detection Instrument CAMAG TLC Scanner 3

"ScannerNumber of tracks 4Position of first track X 20.0 mmDistance between tracks 20.0 mmScan start pos. Y 15.0 mmScan end pos. Y 92.0 mm

Measurement Wavelength 480Lamp 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 elutionTime

(in min)

Flow

(ml/min)%A %B

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 PARAMETERSTABLE7: 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/mlLimit: between 0.40g/ml-

0.70g/ml

Physical parameters Observations limits

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

0.50g/ml

Sieve test (passes through)-20 mesh-40 mesh-80 mesh

100.0%

99.91%

99.27%

Limit: not less than 100.0%



Content of bitter principle by

gravimetery4.92%w/w Limit: not less than 4.0%

PHYTOCHEMICAL SCREENING OF NEEM EXTRACTTABLE8: VARIOUS CHEMICAL CONSTITUENTS IN EXTRACT

S.No Chemical constituentsNeem oil Extract

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 PARAMETERSTotal 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

Total Sugars

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)

Total Sugars

=13.74%

100 x 1 x x W100 x 100 x A

x100P x 10 x 100 x 5 x x WA

TBS

SBT

100 x 2 x 100 x 100 x 0.1264

100 x 99 x 10 x 5 x 40 x 0.175

Total amino acid content by spectrophotometric method

Observation

Table10: Sample v/s U.V absorbance (Total Amino acid content)

Calculations

0.1mM L-Leucine concentration =13.117mg/ltr

0.1mM L-Proline concentration =11.513mg/ltr

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

Table11: Volume v/s amount of Standards

Volume of L-

Leucine taken

Amount of L-

Leucine

Volume of L-

Proline Taken

Amount of L-

Proline

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

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

y = 22.612x

R2 = 0.9976

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0 0.005 0.01 0.015 0.02 0.025 0.03

5.0

1000096.0

Plot: Amount of L-Proline taken v/s Absorbance

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

%age of Total amino acid content=7.030%

y = 5.1469x

R2 = 0.9913

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0 0.005 0.01 0.015 0.02 0.025

%10.508.0

100*00408.0

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 spectrum146.1(M +), 168.1(M + + Na +), 100.0(M + - COO -‑)

1H NMR SPECTRUM OF AIN –P-01-019

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 -‑)



STRUCTURE (PROLINE)


Proton spectrum

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

Mass Spectrum

189.1(M + + Na +)



STRUCTURE (PHENYLALANINE)


Proton spectrum

3.37(CH), 1.93(CH3)

Mass

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



STRUCTURE (ALANINE)

Characterization of AIN-P-01-002Elemental analysisNitrogen- 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

1H NMR SPECTRUM OF AIN-P-01-002

13C NMR SPECTRUM OF AIN-P-01-002

MASS SPECTRUM OF AIN-P-01-002

STRUCTURE(2-HYDROXYPROPIONATE)

HPTLC ANALYSIS

TABLE12: HPTLC PEAK TABLE

Track PeakStart Rf

Start Height

Max Rf

Max Height

Height %

End Rf

End Height

AreaArea %

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

%age of L-Proline present in extract

spot per µg 0.36 =3054.65

1838.5 x 0.6

3.6%10

100 x 0.36

HPLC ANALYSISHPLC CHROMATOGRAM OF STANDARD

HPLC CHROMATOGRAM OF EXTRACT

TABLE13: HPLC PEAK TABLE

Amino acids Mol. Wt

Amount of

Std

in µg

Area Std Sample area

% of amino

acid

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

Amino acids Mol. WtAmount of Std

in µgArea Std Sample area

% of amino

acid

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

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