Annona squamosa as botanical pesticide; Annonaceous acetogenins; annonin/squamocin; Advancement in botanical research; Structure activity relationship of squamocin/annonin; Chemistry and mode of action of squamocin/annonin

1. Term Paper Presentation
AC 602
Advancement in botanical research
with reference to Annona squamosa
Indian Agricultural Research Institute, New Delhi
Prithusayak Mondal
Roll No. 10064

2. Introduction
• Importance of botanical pesticides
• Current botanicals (Neem, rotenone, pyrethrum, nicotine &
essential oils etc.)
• Potential new botanical pesticide – Squamocin (Anonin)
from the plant Annona squamosa
Indian Agricultural Research Institute, New Delhi

3. Plant Profile
Scientific Name: Annona squamosa Linn.
Kingdom Plantae – Plants
Subkingdom Tracheobionta – Vascular plants
Superdivision Spermatophyta – Seed plants
Division Magnoliophyta – Flowering plants
Class Magnoliopsida – Dicotyledons
Subclass Magnoliidae
Order Magnoliales
Family Annonaceae – Custard-apple family
Genus Annona
Species squamosa – Sugar apple
Indian Agricultural Research Institute, New Delhi

4. Properties
Indian Agricultural Research Institute, New Delhi
Plant parts Use
Bark Prevention of diarrhoea
Root Treatment of dysentry
Leaf Cold remedy, urine purification, treatment of hysteria
Fruit Haematinic, cooling, sedative, stimulant, expectorant &
maturant tonic; treatment of anaemia & burning sensation;
making of ice cream & milk beverages
Seed Abortifacient & pesticidal activity
Antifeedant, insecticidal, antidiabetic, anti-tumour,
anticancer, antibacterial & antiviral activity (Pandey et al.,
2011)

5. Chemical constituents
• The plant is reported to contain glycoside, alkaloids,
saponins, flavonoids, tannins, carbohydrates, proteins,
phenolic compounds, phytosterols, amino acids (Pandey
et al., 2011)
• The diterpenoid alkaloid atisine is the main component of
the root
• Annonaceous acetogenins – Botanical pesticides
Indian Agricultural Research Institute, New Delhi

6. Preparation of Extract
• Shade drying of leaves & homogenization to
coarse powder
• Sohhlet extraction using following solvents:
1) Petroleum ether (40-60°C), 2) Chloroform,
3) Ethylacetate, 4) Acetone, 5) Methanol
• Concentration of extracts using rotary evaporator
at 40-50C & drying
Percentage practical yield of petroleum ether (40-60oC), chloroform,
ethyl acetate, acetone and methanolic extracts were found to be
3.85, 2.33, 2.39, 1.2, 7.07 % w/w respectively
Indian Agricultural Research Institute, New Delhi
Agarwal et al., 2012

7. Preliminary phytochemical
screening
Indian Agricultural Research Institute, New Delhi

8. HPTLC Study
Indian Agricultural Research Institute, New Delhi

9. Annonaceous Acetogenins
• Acetogenins have gained much attention recently,
because of its wide range of bioactive spectra
• Series of C-35/C-37 natural products derived from C-32/
C-34 fatty acids that are combined with a 2-propanol unit
• Characterized by a long aliphatic chain bearing a terminal
methyl-substituted α,β-unsaturated γ-lactone ring (or
ketolactone ring), with 1,2 or 3 THF rings located along
the hydrocarbon chain and a number of oxygenated
moities (hydroxyls, acetoxyls, ketones, epoxides) &/or
double bonds
Indian Agricultural Research Institute, New Delhi

10. Acetogenins Extraction
Ground Annona
squamosa seeds
AcOEt
Indian Agricultural Research Institute, New Delhi
(2 kg)
1) Petroleum ether
2) Partition between petrolem ether & aquousmethanol
Methanol layer
Residue
Petroleum ether
Crude acetogenin
fraction
layer
Silica gel chromatography
CHCl3/ AcOEt=2:1 to
AcOEt/ MeOH= 20:1
Araya et al., 2004
Fr. 1 2 3 4 5 6 7 8 9 10

11. Structures of Acetogenins
R1=R2=R4=H, R3=OH: Squamocin
R1=R2=R3=H, R4=OH: Squamocin-C
R1=OH, R2=R3=R4=H: Squamocin-G
R1=R2=R3=R4=H: Squamocin-L
R1=R4=H,R2=R3=OH: Squamocin-O1,-O2
R 4
R 3 O H
O O
O
O H
R 2 R 1
O
2 4 1 5
3 7
R1=R2=H, R3=OH: Squamocin-D
R1=R3=H, R2=OH: Squamocin-F
R1=OH, R2=R3=H: Squamocin-H
R1=R2=R3=H,: Squamocin-M
R3
O O
O
OH
R2 R1
O
OH
24 15
37
Indian Agricultural Research Institute, New Delhi

12. O O
Squamocin-N
Squamosten-A
O
OH
O
24 15
37
OH
O
O
O H
O H
O H
O
O H
2 3 1 5
3 7
Structures of Acetogenins …
Indian Agricultural Research Institute, New Delhi

13. R=OH: Squamocin-B
R=H: Squamocin-J
R=OH: Squamocin-E
R=H: Squamocin-K
O O
O H
2 2 1 3
Squamocin-I
O H
O
O
3 5
O O
2 2 1 3
O
O
3 5
R O H
O H
O O
2 2 1 3
O
O
3 5
O H
O H
R
Structures of Acetogenins …
Indian Agricultural Research Institute, New Delhi

14. Structures of Acetogenins …
24
O O
R1=OH: Squamostatin-C
R1=H: Squamostatin-E
O
O
OH OH OH
R1
R2
19
12
37
R1=H, R2=OH: Squamostatin-A
R1=OH, R2=H: Squamostatin-B
R1=R2=H: Squamostatin-D
24
O O
O
O
OH OH OH
R1
19
12
37
H
O
O
O
15
18
Squamostanal-A
Indian Agricultural Research Institute, New Delhi

15. Structural Features
The fundamental structural features of acetogenins are:
(1) Having hydrocarbon chain, of C35 or C37 in length
(2) 1～3 tetrahydrofuran rings are present
(3) One γ-lactone is present at an end of hydrocarbon
chain
(4) 2～8 hydroxyl groups (rarely carbonyl or acetoxy
group) are present
Indian Agricultural Research Institute, New Delhi

16. Classification of Annonaceous
Acetogenins
Indian Agricultural Research Institute, New Delhi

17. Structure Activity Relationship
(1) Generally, intensity of the bioactivity is (A) type (adjacent bis-
THF) > (B) type (non adjacent bis-THF) > (C) type (mono-THF) >
(D) type (non-THF)
(2) γ-Lactone is crucial for activity
(3) If all other structural features are identical, C35 acetogenins
are more potent than the C37 acetogenins
(4) Thirteen carbons space between the OH-flanked THF and γ-
lactone is optimum for activity
(5) Three hydroxyl groups, two flanking the THF ring(s) and
another somewhere in the long hydrocarbon chain provide both
the optimal position and polarity needed for the most potent
activity, and for tetra-hydroxylated acetogenins the activity drops
drastically
Indian Agricultural Research Institute, New Delhi
Alali et al., 1999

18. Structure Activity Relationship …
(6) Neither the 4-OH group nor the 10-OH group is essential
for activity
(7) A ketone instead of a hydroxyl functional group decreases
the activity
(8) Derivatives (acetates, chloride etc.) decrease the activity
(9) Ketolactone acetogenins are usually less active and more
selective than their parent compounds
(10) The THP ring compounds are as active as the THF
compounds and have the same mechanism of action
Indian Agricultural Research Institute, New Delhi
Alali et al., 1999

19. Commercial Product
ANOSOM®
It is a biological insecticide based on botanical extract of Annona squamosa
(Custard Apple / Seethaphal). ANOSOM® contains squamocin (annonin) as
active ingredient and is formulated as 1% Emulsifiable Concentrate (10,000
ppm). ANOSOM® is non phytotoxic and not toxic to higher animals.
Mode of Action
Annonaceous acetogenins present in ANOSOM® have pesticidal and/or
insect antifeedant properties.
Target Pests
Helicoverpa arimigera, Helicoverpa zea, Spodoptera litura, Spodoptera
exigua, Earias spp. , Achaea janata, Nephotettix virescens, Bunch caterpillar,
Green Leaf Hopper, Leaf Folder, Army worm, Cut Worm.
Indian Agricultural Research Institute, New Delhi

20. Commercial Product …
Crops
ANOSOM® is suitable for application on cereals, millets, pulses, oilseeds,
fibre crops, sugar crops, forage crops, plantation crops, vegetables, fruits,
spices, flowers, medicinal crops, aromatic crops, orchards and
ornamentals.
Method of Application
Foliar application : Mix ANOSOM® @ 2ml/ L of water and spray on crop
canopy. The spray volume depends upon the crop canopy. It is
recommended that BioPesticides are sprayed in early hours of morning or
late afternoon for better results. UV radiation in peak sunny hours of the
day may reduce the bio efficacy of BioPesticides.
Shelf Life
It is stable for a period of 24 months from the date of manufacturing.
Indian Agricultural Research Institute, New Delhi

21. Mode of Action
• They are potent inhibitors of NADH : ubiquinone oxidoreductase, which
is an essential enzyme in complex I of the electron transport system
(ETS) which eventually leads to oxidative phosphorylation in
mitochondria (Lewis et al., 1993).
• They act directly at the ubiquinone catalytic site(s) within complex 1
and in microbial glucose dehydrogenase (Friedrich et al., 1994).
• They also inhibit the ubiquinone-linked NADH oxidase that is peculiar
to the plasma membranes of cancerous cells and functions to permit
cytosolic phosphorylation (substrate level phosphorylation) by
restoration of NAD levels.
Thus, the end result of both of these mechanisms is ATP
deprivation (Morre et al., 1995).
Indian Agricultural Research Institute, New Delhi

22. Indian Agricultural Research Institute, New Delhi