1. By
Saptarshi Das
M.Pharm, 2nd year
Bengal School Of Technology.
UNDER SUPERVISIONS OF Prof. ( Dr. ) HIMANGSHU SEKHAR
MAJI & Asst. Prof. Mr. BISWANATH GHOSH OF
BENGAL SCHOOL OF TECHNOLOGY .

2. Introduction..
 The World Health Organization (WHO) has compiled a list
of a 20,000 medicinal plants used in different part of the
world. Eighty Percent of world population really on
traditional system for primary health care.
 Plants have been a rich source of medicines because they
produce a host of bioactive molecules evolved as chemical
defenses against prediction or infection. Medicinal activity
of plants are generally due to the secondary metabolites
which occur in the tree form of steroids, diterpene,
triterpane, flavones, alkaloids etc as well as in the form of
ether, ester and glycoside.

3. Objective..
 The work deals with the extraction, detection of the
active compounds from seeds of Annona squamosa.
 Phytochemical screening of the seed extracts.
 Isolation of cardiac glycosides from the ethanolic
extract of seeds of Annona squamosa, and evaluation
its possible chemical structure.

4. Taxonomical Classification..
 Kingdom- Plantae
 SubKingdom - Tracheobionta
 Superdivision - Spermatophyta
 Division - Magnoliophyta
 Class - Magnoliopsida
 Subclass - Magnoliidae
 Order - Magnoliales
 Family - Annonaceae
 Genus - Annona L.
 Species - Annona squamosa L.
 Local name - Ata, Maden, Mandargom.
 Distribution : Through out south west Bengal.

5. Description..
 Annona squamosa, the sugar-apple, sweetsop, or sugar-pineapple,
is a species in the Annonaceae family that is native to the tropical
Americas and widely grown for its fragrant, juicy, and flavorful
fruit, which contains more vitamin C than an orange. Sugar-apple
is the most widely cultivated of tropical fruits in the family, which
includes cherimoya (A. cherimola), sugar-apple (A. squamosa),
and paw paw (Asimina triloba). Sugar-apple is grown in lowland
tropical climates worldwide.

6.  The seeds are toxic, and have been used as an insecticide and
to treat head lice (although the preparation is an eye-irritant
and can cause blindness). Seeds are, however, high in oil,
which can be used in soap manufacture or, if treated to
remove the toxic alkaloids, as a cooking oil.
 Leaves, unripe fruits, and extracts of bark and root, all rather
astringent, have been used in traditional medicine to treat
fevers, rheumatism, diarrhea, dysentery, and other ailments.
The aromatic leaves are occasionally used in perfumes, and
fibers from the bark are used to make cords and ropes .

7. Uses& Benefits of Custard Apple
 The bark of custard apple tree can be used to stop diarrhea in children and
adults. In addition, the plant is effective to treat diabetes.
 Its fruit is used to make a hair tonic in some parts of India.
 The plant bears some amazing medicinal qualities, like serving as an
insecticide, antiovulatory and abortifacient.
 The grounded seeds can be applied on hair, to get rid of lice. However,
make sure that it does not come in contact with ice or else, it can irritate the
eye, leading to blindness.
 Custard apple can treat burning sensation, as it is an effective coolant.
 It is used to produce sugar wine apple and is the perfect plant for indoors.
 The crushed leaves of the tree are used to treat hysteria (fearful state of
mind) and fainting spells.
 The treatment of ulcer, wound, dysentery and other ailments is also done
by its concentrated leaf extract (in which the leaf is boiled and its essence is
extracted).

8. Plant Chemicals ..
Annotemoyin-2
Squamocenin
Squamocin
Squamocin -A
Squamocin -B
Squamostatin-D
Squamostatin-E
Cherimolin-1
Cherimolin-2
Motrilin
Reticulatain-2
from the ethyl alcohol extract of A.
squamosa L. Squamocenin is a new
Acetogenin, Annotemoyin and
Reticulatain-2.

9. Pharmacological Activity ..
From the literature review we have some important pharmacological
activities of Annona squamosa seed, they are as following..
 Anti diabetic Activity
 Antifertility activity
 Antimicrobial activity
 Antitumor Activity
 Antioxidant Activity
 Larvicidal and Chemosterilant Activity
 Anti-platelet activity
 Antifertility activity
 Antiviral activity
 Antihelmintic activity

10. Methodology at a glance…
Plant material
Collection &
Authentication
Soxhletion Distillation
Thin layer
Chromatography
Column
Chromatography
High
Performance
Liquid
Chromatography
Infrared
Spectroscopy
Mass
Spectroscopy
Proton Nuclear
Magnetic
Resonance
Conclude with
probable
Structure
UV
Spectroscopy

11. Results..
Petroleum Ether Extract yield :-
Wt. of paper container = 4.80 gm.
Wt. of crude drug (seed) with paper
container = 72.04 gm.
Therefore, wt. of crude (seed) = 67.24 gm.
Now, wt. of the product after extraction =
5.28 gm.
Percentage yield = ( Wt. of the extracted
product / Wt. of the crude taken ) × 100
= (5.28 / 67.24) × 100
= 78.52 %
Ethanol Extract yield :-
Wt. of paper container = 4.68 gm.
Wt. of crude drug (seed) with paper
container = 74.06 gm.
Therefore, wt. of crude (seed) = 69.38 gm.
Now, wt. of the product after extraction =
6.24 gm.
Percentage yield = ( Wt. of the extracted
product / Wt. of the crude taken ) × 100
= (6.24 / 69.38) × 100
= 89.93 %

12. Phytochemical Screening …

13. Result(s) of Thin layer chromatography
• TLC of Ethanolic extract
• Rf factor of ethanolic extract ,
4.8 cm / 5.3 cm = 0.90
• Mobile phase used = ethyl acetate :
methanol : water ( 100 : 13.5 : 10 )
• TLC of petroleum ether extract
• Rf factor of petroleum ether extract ,
4.7 cm / 5.8 cm = 0.81
• Mobile phase used = ethyl acetate :
methanol : water ( 81 : 11 :8 )

14. Result(s) of HPLC

15. Result(s) of FTIR
Transmittance at 3628 cm-1,is
due to –OH stretching.
Transmittance at 2921 cm-1,is
due to –CH stretching.
Transmittance at 1737 cm-1, is
due to ˃C=O stretching. Or
presence of a ketone group.
The compound, can be
concluded to contain an alkyl
group ( -CH3 ) group, hydroxyl
( O-H ) group and a carboxyl
( C=O ) group or a ketone
group.

16. Result(s) of Mass Spectrometry

17. Result(s) of 1H NMR Chemical shift value at 0.8,
is due to a primary proton.
Chemical shift value at 1.38
is due to a secondary proton,
Chemical shift value at 1.5 is
due to a tertiary proton,
Chemical shift value at 3.6 -
3.5 is due to ester / ether
linkage,
Chemical shift value at 2 -
2.7 is due to carboxyl group,
Chemical shift value at 6 - 7
is due to an aromatic ring,
Chemical shift value at 4.6 –
5.9, is due to vinylic carbon.
( C=C-H )
Chemical shift value at 7.26
& 6.98, is due to presence of
two pyrone ring

18. Conclusion
This report describes their isolation and characterization was obtained
as colourless plate (C2H5OH), and positive to Liberman–Burchard
reaction. The UV ( 295 nm ) and IR absorption spectra suggested the
presence of 2-pyrone ring ( 1635 cm– 1 ) and ketone group (1712 cm–
1). In the 1H. NMR spectrum, signals at 7.26, and 6.98) were
characteristic of the 2-pyrone ring may of bufadienolide . All the
information above showed that compound was may be a
bufadienolide. From the UV spectrometry scan, HPLC data curve
peak, 1H NMR spectra, IR spectra, the possible structure of the
compounds is as follows, which similar to a cardiac glycoside, further
test and elaboration will continued..

19. Probable structure of the isolated compound is..

20. Reference (s)..
 Gupta Veena and sing AK (2005) Proceeding of seminar,’Herbs in health care
information up date’Ramkrishna Mission AshramaNarendrapur;p-50.
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medicinal plants of the Scrophulariaceae and Acanthaceae. Int. J. Pharmacognosy
1996; 34: 243-248.
 Vohar SB, Ishwar Kumar and Naquvi SAH. Phytochemical, Pharmacological,
antibacterial and anti-ovulatory studies on Annona Squamosa. Planta Med. 1975;
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 Kaleem M, Asif M, Ahmed QU, Bano B. Antidiabetic and antioxidant activity
ofAnnona squamosa extract in streptozotocin induced diabetic rats. Singapore
Med J. 2006; 47(8): 670-675.

21. Continued..
 CHEN Yong, XU Shasha, CHEN Jianwei, Wang Yu, XU Huiqing, FAN Naibing,
LI Xiang.“Antitumor activity of Annona squamosa seed extract containing
annonaceous acetogenins compounds.”
 Li Qiao , Yong-Fu Huang , Jia-Qing Cao , Yu-Zhi Zhou , Xiu-Lan Qi & Yue-Hu
Pei (2008) One new bufadienolide from Chinese drug “Chan'Su”, Journal of
Asian Natural Products Research, 10:3, 224-227, DOI:
10.1080/10286020701603146
 IUPAC, Compendium of Chemical Terminology, 2nd ed. (the "Gold Book")
(1997). Online corrected version: (2006–) "Retention factor, k in column
chromatography“
 Harwood, Laurence M.; Moody, Christopher J. (13 Jun 1989). Experimental
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