neem applications
TRANSCRIPT
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%
Limit: not less than 80.0%
Limit: not less than 60.0%
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 -‑)
1H NMR SPECTRUM OF AIN –P-02-019
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 +)
1H NMR SPECTRUM OF AIN –P-03-019
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 +)
1H NMR SPECTRUM OF AIN –P-01-018
MASS SPECTRUM OF AIN –P-01-018
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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