azadirachta indica---a continuing source of exiting …
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AZADIRACHTA INDICAAZADIRACHTA INDICA------A CONTINUING A CONTINUING SOURCE OF EXITING NEW CHEMISTRY SOURCE OF EXITING NEW CHEMISTRY
AND POTENTIAL USESAND POTENTIAL USES
BINA S. SIDDIQUIBINA S. SIDDIQUI, SYED TARIQ ALI, , SYED TARIQ ALI, M.RASHEED, S.K.ALI, S.FAIZI, S.N.H. NAQVI M.RASHEED, S.K.ALI, S.FAIZI, S.N.H. NAQVI
AND R.M.TARIQAND R.M.TARIQ
INTERNATIONAL CENTER FOR CHEMICAL SCIENCESH. E. J. Research Institute of Chemistry
Dr. Panjwani Center for Molecular Medicine and Drug ResearchUniversity of Karachi, , Karachi-75270, PAKISTAN
• CHEMISTRY
• BIOSYNTHETIC RATIONALE
• BIOACTIVITY DIRECTED ISOLATION STUDIES ON NEEM FRUITS
• SYNTHETIC ANALOGUES
• STRUCTURE/ACTIVITY(INSECTICIDAL)RELATIONSHIP
• PRODUCTS DEVELOPED
• PRESENT STATUS/FUTURE STRATEGIES
MeOOCAcO OH O
O
TigO
O
OHOHO H
MeOOCO
H H
PARTS STUDIED PARTS STUDIED • WHOLE FRUITS (Fresh and dried)• SEEDS (De-pulped, fresh and dried)• COATINGS • KERNELS• STEM BARK• TWIGS• HEART WOOD• ROOT BARK• FLOWERS
MAJOR COMPOUNDS ISOLATED FROM NEEM(TERPENOIDS)
TRITERPENOIDS
* Protolimonoids* Monortriterpenoids* Dinortriterpenoids* Trinortriterpenoids* Tetranortriterpenoids* Pentanortriterpenoids* Hexanortriterpenoids* Heptanortriterpenoids* Octanortriterpenoids* Nonanortriterpenoids
HOOH
O
HO
O OAc
OO
COOMe
O
O
OO
OH
O OHOAc
COOH
HOH2CAcO OH
MeO
O
TigO
COOH
OHO
O
Azadirol (C-30)Apo-protolimonoid
Melianol (C-29)
OH
Limocinol (C-30)(Protolimonoid)
Azadirolic acid (C-28) Limbonin (C-27)
OAcO
OO
OH
30
Diepoxyazadirol
OO CH3
O
O
OH
OHHO
OO CH3
O
O
OAc
OHAcO
Azadirol Diacetyl azadirol
J. Nat. Prod.,54, 408-415 (1991)
C OH
OH
O
HR
C OH
OH
OC CH 3
OHR
CH 2
O
HO
C CH 3
OH
O
O
H 3 C
R
OH
O
O
HH
HH
:
..
+
-
OOH O
H
R
O
C
O
ON N
C
O
O
O
+
+
-
O
O OAc
Nimocin (C-26)
Mahmoodin (C-26)
O OAc
O
O
O
HOH2CH2CO
O OAc
O
O
HO
OH
AcO
O
O OBz
Azadirone (C-26)
O OAc
O
OH
O
OH
AcO
Nimolicinolide (C-26) Isonimolicinolide (C-26
COOMeO
O
OR
OCOOMe
R1
R2
O
O
O
COOMe
O
R
R1
R R1O H,OHH,OH O
MargosinolideIsomargosinolide
R2R1RH O H,OHH H,OH OAc H,OH OAc O H,OH
6-Deacetylnimbinolide 6-Deacetyl-isonimbinolide
IsonimbinolideNimbinolide
O OHOAc
OOH
O
O
O
OR
OCOOMe
HAc
RNimbinene
Nimolicinolic acid (C-24)
Nimbocin (C-24)
AcO OCinOH
O
OTig6-Deacetylnimbinene (C-25)
O
O
R
OAc
R3
O OHOAc
O
α-Nimolactone α-methylDesfuranoazadiradione (C-22)
β-methylβ-Nimolactone(C-21)
DITERPENES
• ABIETANES• PODOCARPANES• MISCELLANEOUS
ABIETANESOH
O OO O
OH
HO O
HO
OH
OH
O
CH2OH
O O
OH
OH
Nimosone(Stem bark)
Margocin(Root bark)
Margocilin(Root bark)
Nimbocidin(Root bark)
Margocilin(Root bark)
Nimbocidin(Root bark)
PODOCARPANES
OHCH3
O O
CH3
O O
CH3
OH
HO O
OH
HO O
CH2CH2OHCH3
O
CH3
O
OH
CH2CH2OH
OH
OH
Nimbinone(Stem bark)
Nimbisonol(Stem bark)
Demethylnimbinol(Stem bark)
Azadirin A(Root bark)
Azadirin B(Root bark)
Nimbione(Stem bark)
MISCELLANEOUS DITERPENOIDS
OH
O
OH
O
OOH
O
CH 3
OCOCH 3
O
OH
O
OCH 3
Margosone(Stem bark)
Azadricin(Root bark)
Nimbocinin(Root bark) Nimbilicin
(Root bark)
Azadirilin(Root bark)
OH
TABLEVARIATIONS OBSERVED IN THE CONSTITUENTS OF DIFFERENT PARTS OF THE NEEMDISTRIBUTION OF TERPENOIDS IN VARIOUS PARTS
----+--Nonanortriterpenoids
----+++Octanortriterpenoids
-+(1)---+-Hexanortriterpenoids
+
_
(1)
_
+
_
+
_
+
_
+
+
+
_
Tetranortriterpenoids
Pentanortriterpenoids
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
+
_
+
+
+
_
_
_
_
+
_
+
Apo-tirucallanes/Apo-euphanes
DEGRADED LIMONOIDS
Mononortriterpenoids
Dinortriterpenoids
Trinorterpenoids
____+++Tirucallanes/Euphanes
PROTOLIMONOIDS
FlowersRoot bark
Stem bark
TwigsCoatingsSeedsLeaves
DISTRIBUTION OF TERPENOIDS IN VARIOUS PARTS
Leaves Seeds Coatings Twigs Stem Root Flowersbark bark
DITERPENOIDS
Abietanes - - - - + + -
Podocarpanes - - - - + + -
Miscellaneous - - - - + + -
HO
HO
HO
O
OH
H
D1
Butyrospermol (D)
E
O OH
O
HO OH
O
OHO
HO OH
O
O
HO
H
H+
O OH
O
O
OHO
O OH
O
O
OO
O OH
O
O
J
G3
I
OH
O OH
O
G2
O
O
OH
OOCRx
G4
..
-
OOCRx
G6
HO OH
OO
HO
HO OH HO OH
O
H
F
O
H
+.. ..
F1 G
HO OH
O
O
O
OH
OH
HO OH
O
O
OH
OHCO
H
+H
ROOCO OH O
O
O
CO
RO
O
O OHOHO HR
R
K
Azadirachtin
HO OHO
O
OOHC
OHH
HO OH
O
O
OH
OHCOH
H OH
K1 K2
L
O OH
O
OH
O
OH
N
HO O
O
O
OHCHO
+H
CHO OH
O
OH
OH
OHC
HO O
O
O
OHCOOR
Q
R
O
O
OH
OCHO
HO
O
O
O
OH
OCOOR
ROO
P
ON1
• STUDIES ON THE FRUIT COATINGS
MOTIVES OF ESEARCH
• Lack of systematic studies on fruit coatings.• Comparative effectiveness of extract of fruit
coatings over that of seeds against house-insects.• No azadirachtin in fruit coatings.
MeOOC
O
OAcO
TigOOH
O OO
MeOOC OH
OHH
Azadirachtin
Neem Fruits50 kg
FFC (RB-b)Fresh Fruit Coats, 23 kgLC50, 290 ppm(Anopheles stephensi)LC50, 319 ppm(Aedes aegypti)
EtOH (x5), RTFFS (RB-a)Fresh Fruit Seeds, 27 kgLC50, 784 ppm(Anopheles stephensi)LC50, 446 ppm(Aedes aegypti)
Shaken with EtOAc and water
EtOAc Fraction LC50, 165 ppm(An. stephensi)
Water Fraction
4% aqueous Na2CO3Partitioned into acidic and neutral
(RB-b 'A') LC50, 300 ppm
Acidic Fraction Neutral Fraction(RB-b 'N') LC50, 43 ppm
Bioassay directed extraction and isolation scheme
Neutral Fraction
(i) Washed with H2O(ii) Dried over Na2SO4 (anhyd.)(iii) Shaken with PE
Petrol-ether PE 'S' soluble FractionLC50, 159 ppm
Petrol-ether PE 'I' insoluble FractionLC50, 154 ppm
Shaken with Ether
Ether soluble(RB-b 'ES')NFCN, 189 gLC50, 106 ppm
Ether insoluble(RB-b 'EI')Azadiradione (2), 82 gLC50, 15 ppm
VLC (x3)
Shaken with 90% Aq. MeOH-PE (1:1)
Aq. MeOH Phase PE soluble Phase
Aq. Phase EtOAc Phase
(i) Saline(ii) Shaken with EtOAc
13 Fractions ('A' to 'M')
VLC Fractions
Fr. 'A'300 mg
LC50 100 ppm
Fr. 'B'2.5 g
LC50 200 ppm
Fr. 'C'6.5 g
LC50 150 ppm
n-pentadecane (3)n-Hexadecane (4)n-Heptadecane (5)n-Octadecane (6)n-Nonadecane (7)n-Eicosane (8)n-Heneicosane (9)n-Docosane (10)n-Tricosane (11)n-Tetracosane (12)n-Pentacosane (13)n-Hexacosane (14)n-Heptacosane (15)n-Octacosane (16)n-Nonacosane (17)n-Hentriacontane (18)
Butylated Hydroxy-toluene (19)4,4,5,8-Tetramethyl-3,4-dihydrocoumarin (20)3,4-Dihydro-4,4,7,8-tetra-methyl-coumarin-6-ol (21)α-Hexyl-cinnam-aldehyde (22)Methyl trans,trans-Farnesoate (23)Galoxolide (24)Methyl 14-methyl-penta-decanoate (25)Ethyl palmitate (26)Ethyl oleate (27)
Methyl 14-methyl-pentadecanoate (25)Ethyl palmitate (26)Ethyl oleate (27)Dihydromyrcenol (28)Asarone (29)
(continued)100% PE 99:1 to 92:8
PE-EA91:9 to 70:30
PE-EA
PE-EA (50:50)
Fr. 'F'
Fr. 'H'5.38 g
LC50 215 ppm
PE-EA (40:60)
PE-EA (30:70)
Fr. 'G'combined
hot EAincold PE
AldrichFCCPE
solublePE
insoluble Fr.(H-1 to H-23)
2, 30 andDeoxygedunin
29 mgLC50 150 ppm
Prep. TLC / hplc
Limocin C
and Limocin D 16.5 mg
Azadiradionolide (4.8 mg)
Fr. 'I'89.2 g
LC50 107 ppm
Fr. 'K'100.7 g
LC50 212 ppm
Fr. 'J'93.4 g
LC50 136 ppm
Fr. 'L'1.53 g
LC50 66 ppm
EA (100%) eluate
Fr. 'M'182 g
LC50 182 ppm
PE-EA (30:70)
PE-EA (20:80)
PE-EA (10:90)
MeOH (100%)eluate
PE-EA(70:30)
Fr. 'D'
PE-EA(60:40)
Fr. 'E'
Fractions 'D' through 'M'
CrystalsfromcoldMeOH
Epoxazadiradione 18 g
LC50 18 ppm
Azadirone 26 mg
LC50 10 ppm
Pencilcolumn
2 & 30(1:1) ratio
(Fr. H-3 to H-5)
Chromatographed on Eyla FCCinto 16 fractions (LFC-1 to LFC-16)
Fr. 'LFC 1-2'159 mg
LC50 36 ppm
Fr. 'LFC 3-6'181.9 mg
LC50 23.5 ppm111.2 mg
LC50 19.2 ppm
Fr. 'LFC 7-8'
combined on the basis of tlcRepeatedPrep. TLC
LFC-618 (38 mg)
LFC-608 (29 mg)
LFC-611 (27 mg)
Prep.TLC / hplc
Prep.TLC
Prep.TLC
Meliacinolactol 16 mg
Tetraacetyl-meliacinolactol
4 mg
Gedunin 22 mg
LC50 120 ppm
α-Nimolactone 12 mg
LC50 60 ppmβ-Nimolactone
16 mgLC50 45ppm
Meliacinin 14.2 mg, LC50 13 ppmDesfuranoazadiradione 20.2 mg, LC50 100 pmAzadironic acid 7.3 mg, LC50, 4.5 ppmAzadironic acid methyl ester 3.2 mg, LC50 2.8 ppmLimocin A andLimocin B 10.2 mg, LC50 19 ppm
CHCl3 (100%) CHCl3 -MeOH(99:1)
CHCl3 -MeOH(95:5 to 96:4)
Fr. 'L', 1.53 g, LC50 66 ppm
291.3 mg chromatographed on Aldrich FCC into 22 fractions (LFC-601 to 622)
(CHCl3, 100%) CHCl3 -MeOH (99.1 : 0.99) to CHCl3-MeOH (95.5 : 4.5)
O
OO
OO
CH3
O23
645 7
8
916
15
1719
18
11
14
12
13
10
30
28
20 21
22
23
1
29
2'1'
O
O
O
CH3O
H
H O CH3
O23 64
5 78
9 1615
1719
18
1114
1213
10
30
28
20
21
22
23
1
29
2425
2'1'
LC 50 13ppm
• Azadiradionolide Meliacinin
Azadironic acid Meliacinolactol
O
COOR
H
H O CH3
O23 64
5 7
8
91615
1719
18
11
14
1213
10
30
28
20
21
22
23
1
29 37 R = H37a R = Me
2'1'
LC50 4.5ppm
O
OH
OH
O
CH2OH
O CO
CH
CH
H3CO OH
H
2' 3'
4'
5'
1'
6' 7'
8'
9'
123 4 5 6 7
8
919
10
11 1213
14 15
1617
18
2829
2022
23
21
24
30
Acetylmeliacinolactol Limocin C and D
O
R
R'
O
H
O CO
CH3
(33) R = H, R' = OCH2CH3
(34) R = OCH2CH3, R' = H2"1"
2"1"
HH
O
OAc
OAc
O
CH2OAc
O CO
CH
CH
H3CO OAc
H
2' 3'
4'
5'
1'
6' 7'
8'
9'
123 4 5 6 7
8
919
10
11 1213
14 15
1617
18
2829
2022
23
21
24
30
O
O OAc
R
R=O, Azadiradione LC50 15 ppm
R=O, 14,15-Epoxyazadiradione LC50 18 ppm
R=H2, Azadirone LC50 10 ppm
Limonoids of azadirone class
R
OAcO
O
R=CH2, 18α Me, Desfuranazadiradione LC50 37 ppm
R=O, 18α Me, α-Nimolactone LC50 60 ppm
R=O, 18β Me, β-Nimolactone LC50 45 ppm
Enneanortriterpenoid
14 15
18
Previously Reported Limonoids
O
O OAc
R1
R2
R1=H, R2=OMe, Limocin A &
R1=OMe, R2=H, Limocin B (39)LC50 19 ppm
Limonoids with acetal ring
14,15-Deoxygedunin LC50 150 ppm
Gedunin LC50 120 ppm
Limonoids of gedunin class
O
O
O OAcO
O14
15
Previously Reported Limonoids
O
O OH
O
O OR
O
R=Ac, Azadiradione (2)LC50 15 ppm
R=H, Nimbocinol (2a)LC50 30 ppm
17β-Hydroxynimbocinol (2b) LC50 15 ppm
Analogues of azadiradione
(MeOH, K2CO3)
OHO
STRUCTURE-ACTIVITYRELATIONSHIP
O
O OAc
R1
R2
R1=H, R2=OMe, Limocin A &R1=OMe, R2=H, Limocin B
LC50 19 ppm
O
O OAc
O
Nimbinin or Nimolin or14,15-Epoxyazadiradione
LC50 18 ppm
O
O OAc
O
Nimolicin or Azadiradione LC50 15 ppm
O
O OAc
Azadirone LC50 10 ppm
O
oxo
epoxy
acetal
O
O Ac
O
O
O
O OAc
O
Azadiradione LC50 15 ppm
Epoxyazadiradione )LC50 18 ppm
O
O
O OAcO
O
O
O
O OAc
O
Gedunin LC50 120 ppm
Deoxygedunin LC50 150 ppm
Lactone
Lactone
O OAc
O
Desfuranazadiradione (34)LC50 37 ppm
O
O OAc
O
Azadiradione (2)LC50 15 ppm
O
OAcO
O18
α-nimolactone (41: 28α-Me)LC50 60 ppm
β-nimolactone (42: 28β-Me)LC50 45 ppm
Desfuran
O
O OAc
Azadirone (2)LC50 15 ppm
OAcO
COOR
H
H
O
O
O OAc
O
Deoxygedunin (40)LC50 150 ppm
Azadironic acid (37: R = H)LC50 13 ppm
Methyl ester of 37 (37a: R = Me) LC50 2.8 ppm
21 23
O
O
O
CH3O
H OAc
Meliacinin (38)LC50 4.5 ppm
2125
Furan open
Furan open
O
O OR
O
R=Ac, Azadiradione (2)LC50 15 ppm
R=H, Nimbocinol (2a)LC50 30 ppm
O
O OR
OOH
R=H, 17-Hydroxynimbocinol LC50 15 ppm
PRODUCTS DEVELOPED
• BIOPESTICIDE BIOSAL(AGRICULTURE)
• BIOPESTICIDE NIMOSAL (HORTICULTURE)
• BIOINSECTICIDE BIOAMAN• (HOUSEHOLD)• BIOFERTIZER NEEM PLUS
-CUM-PESTICIDE• GERMICIDAL NIMECTIN
CREAM/SPRAY (FOR LIVE STOCK)• MOSQUITO REPELLANTS.
BIOSAL
(FOR USE IN AGRICULTURAL CROPS)
• Biosal is a emulsifiable concentrate prepared by solvent extraction of neem seeds and formulating the product in a manner to give water miscible pesticide.
• Found effective in protecting crops like cotton (SUCKING PESTS), rice, vegetables including brinjal, tomato, pumpkins, cucumber, chillies, onion, ladyfinger, cabbage and peas etc as well as of fruit trees (Mango, Coconut, Palm, Guava, Ziziphus) .
.
COTTON FIELDS:START OF TREATMENT WITH BIOSAL
COTTON FIELDS TREATED WITH BIOSAL
COTTON FIELDS TREATED WITH BIOSAL
COTTON FIELDS TREATED WITH BIOSAL
BIOSAL(FOR USE IN AGRICULTURAL CROPS)
NIMOSAL
(FOR USE IN HORTICULTURE)• Nimosal is aza powder-based emulsified concentrate containing a specific
concentration of neem oil.• It is miscible in water.• 1-2 % dilution with water is found effective hence saves environment from
pollution.• Controls sucking pests, (Jassids, Thrips, Aphids), leaf folders, fruit borer, mealy
bug and other pests of ornamental plants (Hibiscus, Croton, Rose, Jasminumetc.).
NIMOSAL(FOR USE IN HORTICULTURE)
OBSERVATIONS AND ADVANTAGES
Observations:• Best results are obtained on prophylactic (cure before infestation) and
regular use of these pesticides. • Treatment by 1% concentration (10 ml Biosal / Nimosal +990 mL water)
proves effective in low infestation. • 1 Litre (Nimosal / Biosal) is enough for 1 acre area. • Pronounced effect is noted after 72 hrs. of treatment.
Advantages:• Resistance does not develop against these pesticides upto many
generations.• No toxic residues are left as these are biodegrdable.• Only negligible quantities of solvents is left in the final sprays hence these
are pollution free products and environmental friendly.• IGR, anti-feedent and repellent effects are more obvious.
BIO-AMAN(For HOUSE HOLD PEST CONTROL)
Bio-aman is an oil-based bioinsecticide ready to use formulation as aerosol.Effectively controls cockroaches, mosquitoes and flies and other household insects.Being based on botanicals it is safe for human beings.
NEEM PLUS(For Lush green, healthy plants)
• Neem plus is a processed neem seeds product.• Enhances the growth of plants and fruit yields and protects the plants from pests
and nematodes.• Like other neem products it is environment friendly.• It is spread is in the beds (basal treatment).
NEEM PLUS(For Lush green, healthy plants)
AFTE TREATMENTBEFORE TREATMENT
Banana Tree
TREATMENT WITH NEEM PLUS
NIMECTIN(For USE IN LIVE STOCK AND PETS)
Nimectin is a neem-based germicide.
It has been found very effective for the treatment of goats, cows, buffaloes and sheep suffering from skin diseases like mange caused by ectoparasites, ticks and flies etc.
NIMECTIN(For USE IN LIVE STOCK AND PETS)
AFTE TREATMENTBEFORE TREATMENT
BIOSAL-A(MOSQUTOES REPELLENTS)
Biosal-A has been formulated in three different forms:
• Biosal-A liquid vaporizer.
• Biosal-A lotion.
• Biosal-A coil.• The function of all of them is to repel mosquitoes.
PRESENT STATUS/FUTURE STRATEGIES
• Demonstration of use of these products has been successfully demonstrated in fields of different crops to farmers for three years.
• They are continually trained through Farmer Field Schools (FFS) and Training of Farmers Schools (ToFS).
• Interaction with private partners is in process.• The will and support of Government is strongly
needed.• More awareness programs are to be undertaken.
In the name of Allah the Most Merciful
the Most Benevolent