A. squamosa seeds had been used in south of China as a folk medicine to treat “malignant sore” (cancer)

1. CUSTARD APPLE SEEDS: BIOACTIVE COMPOUNDS,
BIOLOGICALACTIVITY AND ITS APPLICATION IN
FOOD INDUSTRY
ADVISER:
Dr. Rakhi Singh
SUKH VEER SINGH
PhD I year
FST

2. 1. Introduction
2. Bioactive compounds
3. Biological activity
a. Antitumor activity
b. Medicinal property
c. Toxic property
5. Application
6. Conclusion
Content

3.  Custard apple (Annona squamosa L.) is an edible tropical
fruit, and is also called sugar apple or sweetsop.
 A. squamosa seeds had been used in south of China as a
folk medicine to treat “malignant sore” (cancer)
(Guangdong Food and Drug Administration, 2004)
 Annonaceous acetogenins are a group of compounds
isolated only from the Annonaceae family (Bermejo et
al., 2005)
Introduction

4.  These compounds are characterized by a terminal γ-methyl
γ-lactone and by a long aliphatic chain bearing
tetrahydrofuranic (THF) and tetrahydropyranic rings,
epoxy rings and (or) double bonds.
Previous study showed that the total content of acetogenins
(ACGs) in the seeds of A. squamosa was higher than in
four other species (A. glabra, A. muricata, A. reticulate
and A. bullata) (Yangetal.,2009b)

5.  Seeds of A. squamosa contain active compounds such as alkaloids,
cyclohexapeptides, and acetogenins (Chen Y, et al 2102).
 Isolation of six cytotoxic acetogenins from the seeds of A. squamosa
(Chen, Chen, & Li, 2011b, 2012; Yang et al., 2009a) has also been
reported and the structure activity relationships of diverse ACGs
(Yang, Zhang, Li, Chen, & Cai, 2009c).
 Annosquacins A–D (1–4) are adjacent bistetrahydrofuran
annonaceous acetogenins.
 Annosquatin A (5) and annosquatin B (6) are nonadjacent
bistetrahydrofuran annonaceous acetogenins and the first examples in
which the tetrahydrofuran ring system is located between C-9 and C-
20.
Bioactive compounds

6. Amorphous and waxy:
• Dieposabadelin
• Squamocenin
• Lepirenin
• Dotistenin
Others:
• Corepoxylone,
• Diepomuricanins A and B,
• Dieporeticenine,
• Tripoxyrollin,
• Bullatencin,
• Glabrencin B,
• Reticulatains-1, -2,
• Uvariamicins I, II, III,
• Erythrosolamin,
• Annotemoyin-1 and -2, and
• Solamin
Acetogenins compound

7.  The ACGs have antitumor activities through the depletion of ATP levels via
inhibiting mitochondrial complex I and NADH oxidase (Laprevote et al.
1992).
 Owing to the implicated structures of ACGs and the absence of a structural
model of the inhibitory binding site of complex I, it is an intricate task to
predict the inhibitory efficacy of a new ACGs (Bermejo et al. 2005).
 In order to find ACGs with stronger antitumour activity, three new
acetogenins were isolated from the supercritical fluid extraction (SFE) of
the seeds of sugar apple, named:
Dieporeticenin B (1),
Squamocin P (2) and
Annosquatin III (3)
Antitumor annonaceous acetogenins

8. Cont…
Five known acetogenins:
• Diepomuricanin A (4) (Laprevote et al. 1992)
• Diepomuricanin B (5) (Sahpaz et al. 1997)
• Dieporeticenin (6) (Tam et al. 1994)
• Coronin (7) (Gleye et al. 2001)
• EpoxyrollinA (8) (Konno & Makabe 2003) were also isolated.
 Six of the isolated compounds (1, 4–8) are epoxy-ACGs, the key
metabolites in the biosynthesis of THF ACGs (Bermejo et al. 2005).

9. Air-dried seeds of A. squamosa (45 kg) were powdered
Extraction
Supercritical fluid CO2 extraction (SFE)
SFE extraction(3.3 kg) passed through silica gel (200–300 mesh)
All fractions were collected
TLC analysis to obtain twelve fractions (Fr 1-Fr 12)
Fr 3 (178.8 g) was separated by silica gel
Further purified on HPLC
Yeild 1 (50 mg) with five known acetogenins
Extraction and isolation

10. • Three multidrug-resistant (MDR)
(cancer cell lines)
 The cytotoxic assay was performed in MTT method in 96-
well plates (Sladowski et al. 1993).
 All IC50 values of isolated compounds were summarised in
table.
 All isolated compounds showed potential cytotoxicities
against MDR cancer cell lines.
SMMC 7721/T
MCF-7/ADR
A549/T
In vitro cytotoxic activity

11. Dieporeticenin B
Squamocin P
Annosquatin III
Diepomuricanin A
Diepomuricanin B
Dieporeticenin
Coronin
EpoxyrollinA
Cytotoxic IC50 values of compounds 1-8 against multidrug
resistant cancer cell lines

12.  ACGs exhibit a broad range of biological properties, such as
antioxidant, cytotoxic, immunosuppressive, pesticidal, antiparasitic
and antimicrobial activities (Alali, Rogers, Zhang, & McLaughlin,
1999; Lima et al.,2010; Yazbak, Sinha, & Keinan, 1998)
 Numerous annonaceous acetogenins have been shown to be
responsible for the medicinal effects:
1. Anti-HIV activity
2. Antidiabetic
properties
3. Wound healing
properties. (Pandey
and Barve 2011)
Medicinal properties

13. Acetogenins namely
Annotemoyin-1, Annotemoyin-
2, Cholesteryl glucopyranoside
and Squamocin have been
previously isolated from the
seeds of Annona squamosa and
have shown remarkable
antimicrobial and cytotoxic
activities (Rahman et al. 2005).
Antimicrobial compounds and other action

14.  Investigation of phytochemicals was through standard qualitative
screening procedures while antioxidant activities of the extract were
evaluated through the radical scavenging effect of DPPH, H2O2 and
the FRAP assay.
 Methanol was the most effective solvent in capturing phytochemicals
including tannins, alkaloids, flavonoids, fixed oils and phenols.
 Seed extracts of A. squamosa exhibited significant scavenging effects
on DPPH and H2O2 radicals.
 It was also observed that the seed extracts contained a high level of
flavonoids and phenols that are indicated for their antioxidant
potential.
Antioxidant activity

15.  Allen, an American Botanist, mentions personal knowledge of a case
of blindness resulting from “the juice of the crushed custard apple
seeds” coming in contact with the eyes (Allen PH, 1943).
 Others have mentioned that the crushed seeds on contact with the eye
can cause conjunctival irritation and ulcers in the eye (Alali FQ, et al
1999).
 Testing of toxic extracts of Annona seeds on rabbit eyes caused
conjunctival inflammation and corneal epithelial delayed damage
(Sookvanichsilp N, et al 1994)
Toxic properties

16.  Essential oils are aromatic and volatile liquids are extracted from
plants.
 Food industry primarily uses essential oils as flavorings, they
represent an interesting source of natural antimicrobials for food
preservation
 Foodborne diseases are a growing public health problem worldwide,
calling for more effective preservation strategies (Hyldgaard et al.,
2012).
 Application of essential oils as food preservatives requires detailed
knowledge about their properties, such as the minimum inhibitory
concentration (MIC), the range of target organisms, the mode of
action, and the effect of food matrix components on their
antimicrobial properties (Hyldgaard et al., 2012).
Application in food

17. Characterization of antimicrobial compounds in Annona
squamosa seed.
Extraction of oil from custard apple seed and its fatty acid
profiling.
Extraction of gum from custard apple seed and its utilization
Extraction and isolation of essential oil and its antimicrobial
activity against Bacillus subtilis and Staphylococcus
aureus.
Research objectives:

18.  In conclusion, two THF ACGs and six precursors of THF ACGs were
isolated from the seeds of A. squamosa.
 Cytotoxicity evaluation of compounds showed squamocin P (2) and
annosquatin III (3) significant activities against MDR (SMMC
7721/T, MCF-7/ADR, A549/T) cancer cells.
 While, precursors (1, 4–8) showed no obvious cytotoxic activities
against these MDR tumour cells, this result is the same as previous
researches (Takada et al. 2000).
 The study revealed that the seeds of A. squamosa contain a
considerable high level of phenolic flavonoid compound that was
believed to be the major contributor to their antioxidant activities.
Conclusion

19.  There are several medicines available in the market to treat the
various types of cancer but no drug is found to be fully effective and
safe.
 The major problem in the cancer chemotherapy is the toxicity of the
established drugs.
 However plants and plant derived products have proved effective and
safe in the treatment and management of cancers.
 These days most of the research work on cancer drugs is targeted on
plants and plants derived natural products.
 Many natural products and their analogues have been identified as
potent anti-cancer agents and day by day the anti-cancer property of
various plants is being identified.

20. • Chen Y, Chen JW, Wang Y, Xu SS, Li X. Six cytotoxic annonaceous acetogenins
from Annona squamosa seeds. Food Chem. 2012;135:960–6. [PubMed]
• (Alali, Rogers, Zhang, & McLaughlin, 1999; Lima et al.,2010; Yazbak, Sinha, &
Keinan, 1998) Goniotriocin and (2,4-cis- and -trans)-xylomaticinones, bioactive
annonaceous acetogenins from Goniothalamus giganteus Journal of Natural
Products, 62 (1999), pp. 31-34
• Food Chem. 2012;135:960–6. Wu YC, Hung YC, Chang FR, Cosentino M, Wang
HK, Lee KH. Identification of ent-16 beta, 17- dihydroxykauran-19-oic acid as
an anti-HIV principle and isolation of the new diterpenoids annosquamosins A
and B from Annona squamosa. J Nat Prod. 1996;59:635–7.
• Gupta RK, Kesari AN, Watal G, Murthy PS, Chandra R, Tandon V. Nutritional and
hypoglycemic effect of fruit pulp of Annona squamosa in normal healthy and
alloxan-induced diabetic rabbits. AnnNutr Metab. 2005;49:407–13
• Allen PH. Poisonous and injurious plants of Panama. Am J Trop Med Hyg.
1943;23:76.
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22. LET YOUR FOOD BE YOUR MEDICINE