CHAPTER I

INTRODUCTION

Annona squamosa locally known as the Atis which is a member of

the Annonaceae family that bears edible fruits called sugar apples. Studies have
showed analgesic, anti-inflammatory, anti-microbial, anti-diabetic, cytotoxic,
antioxidant, antilipimic, anti-ulcer, molluscicidal, genotoxic, vasorelaxant,
hepatoprotective, larvicidal, anthelmintic properties.
Ficus septica locally known as the labnog tree which is a member of the
Moraceae family is known to have diuretic, sudorific, antiherpetic, antirheumatic
properties. Studies have shown that its leaves yielded phenanthroindolizidine Noxide, ficuseptine-A together with 18 known compounds. Some of the
compounds showed possible cytotoxic activity against two human cancer cell
lines.
Medicinal plants play an important role in terms of healthcare system in
some portion of the world. There are several medicinal plants which are widely
used in the traditional systems of medicine for the prevention and cure of
diseases like cancer. Several plant derived compounds have been found to play
significant role in the development of clinically useful anti-cancer agents.
According to the World Health Organization, there were an estimated 4
million new cancer cases and 8.2 million cancer-related deaths in 2012
worldwide. Cancer is the name given to a collection of related diseases. In all
types of cancer, some of the bodys cells begin to divide without stopping and

spread into surrounding tissues. Cancer can start almost everywhere in the
human body, which is made up of trillion cells. Many cancers form solid tumors,
which are masses of tissue. (NCI, 2015). Tumors can grow and interfere with the
digestive, nervous, and circulatory systems and they can release hormones that
alter body function. When a tumor successfully spreads to other parts of the body
and grows, invading and destroying other healthy tissues, it is said to have
metastasized. The process itself is called metastasis, and the result is a serious
condition that is very difficult to treat. (Peter Crosta, 2008)

Background of the Study

Study showed that cancer is the fifth leading cause of morbidity and
mortality worldwide. There are a lot of therapies that cancer patients can undergo
but not all can acquire it due to its expensiveness. People know that medicinal
herbs and plants have been used in folk medicine for millennia. For years, Ficus
septica and Annona squamosa are claimed to have properties that can cure
common diseases. The said information aroused the researchers interest to
investigate and to conduct a further study to confirm its feasibility as an
alternative cure for cancer and tumors.

Statement of the Problem

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This study aimed to determine and identify the mitotic index and toxicity of
the ethanolic leaf extracts Annona squamosa and Ficus septica . In particular, the
researcher conducted a phytochemical screening to determine the active
compounds present in each leaves, the brine shrimp lethality assay also being
performed to further manifest its cytotoxic activity, the antioxidant potency of the
ethanolic leaf extracts using DPPH Radical Scavenging Assay and to test the
Allium cepa through distinguishing and discerning chromosomal aberration.
Specifically, the study sought to answer the following questions:
1. What are the phytochemical substances found in Annona squamosa and
Ficus septica?
2. Are the ethanolic extracts of Annona squamosa and Ficus septica
exhibited cytotoxic activities when tested using the brine shrimp lethality
test?
3. Are the ethanolic extracts of Annona squamosa and Ficus septica has the
antioxidant potential when tested using the DPPH Radical Scavenging
Assay?
4. Is there significant mean difference of the mitotic phases of the Allium
cepa roots treated with the Annona squamosa and Ficus septica ethanolic
extracts as compared to the control?

Significance of the Study

The results of this study may provide strong evidence about the
uniqueness of the chemical characteristics of Annona squamosa and Ficus
septica ethanolic leaf extracts. Positive results may stimulate the use of natural
medication particularly in cancers and tumors. It may also equip baseline
information to the locality in their way of finding possible alternative source of
medicine for nursing common illness.

Scope and Limitations of the Study

This study only focuses on the determination of the following components:

phytochemicals, cytotoxic activity, antioxidant activity and the chromosomal
aberration in the ethanolic leaf extracts of Annona squamosa and Ficus septic
leaves. These components are exhibited in terms of phytochemical analysis,
brine shrimp lethality test, Free Radical Scavenging Activity and Allium cepa
chromosomal aberration assay.

Operational Definition of Terms

Annona squamosa is a small, well-branched tree or shrub from the family

Annonaceae that bears edible fruits called sugar apples.
Brine shrimp lethality assay is a simple, high throughput cytotoxicity test of
bioactive chemicals. It is based on the killing ability of test compounds on a
simple zoological organism-brine shrimp.

Cancer - A group of diseases involving abnormal cell growth with the potential to
invade or spread to other parts of the body. A potentially deadly form of
uncontrolled cell division.
Chromosomal Aberration A missing, extra, or irregular portion of chromosomal
DNA. It can be atypical number of chromosomes or a structural
abnormality in one or more chromosomes.
Cytotoxic is the quality of being toxic to cell.
DPPH is a common abbreviation for an organic 1-picrylhydrazyl. It is a dark
colored crystanine powder composed of stable free-radical molecules.
Ficus septica is a shrub or tree of the family moraceae living at low altitudes.
Mitotic Index Is defined as the ratio between the numbers of cells in a
population undergoing mitosis to the number of cells not undergoing
mitosis.
Phytochemical are chemical compounds that occur naturally in plants.
Tumor - An abnormal growth of body tissue. Tumors can be cancerous
(malignant) or noncancerous (benign).

CHAPTER II
REVIEW OF RELATED LITERATURE

This chapter represents the review of related literature, which is all

directed towards the accomplishments of the aims and purposes of this research.
These literature and research works are reviewed to serve as bases of the data
gathered for this study.

Geographical location of Sta. Filomena

Figure 1. Map of Sta. Filomena (https://www.google.com.ph/maps/place)

Sta. Filomena is a barangay located in Iligan City, Philippines. It is where

the researcher collected the samples of Annona squamosa and Ficus septica
leaves.

Annona squamosa is a small, well-branched tree or shrub from the family

Annonaceae that bears edible fruits called sugar apples. It tolerates a tropical
lowland climate better than its relatives helping make it the most widely cultivated
of these species. (Morton, 1987)
Ficus septica is a shrub or tree of the Moraceae family living at low
altitudes. It lives on the edge of vegetation, often in degraded environments.
(Shanahan et al., 2001)

Physical Description
Annona squamosa is a small, semi-deciduous, much branched shrub or
small tree (9.8 to 26 ft tall) with a broad, open crown or irregularly spreading
branches and a short trunk short, not buttressed at base. Its fruit has delicious
whitish pulp, and is popular in tropical markets.
Branches have light brown bark and visible leaf scars; inner bark light
yellow and slightly bitter; twigs become brown with light brown dots.
Thin, simple, alternate leaves occur singly, 5 centimeters to 17
centimeters long and 2 centimeters to 6 centimeters wide; rounded at the base
and pointed at the tip (oblong-lanceolate). Pale green on both surfaces and
mostly hairless with slight hairs on the underside when young. The sides
sometimes are slightly unequal and the leaf edges are without teeth,
inconspicuously hairy when young. Leaf stalks are 0.4 centimeters to 2.2
centimeters long, green, sparsely pubescent.

Flowers are in solitary or in short lateral clusters of 2-4 about 2.5

centimeters long, greenish-yellow flowers on a hairy, slender 2 centimeters long
stalk. Three green outer petals, purplish at the base, oblong and three inner
petals reduced to minute scales or absent. Very numerous stamens; crowded,
white, less than 1.6 centimeters long; ovary light green. Styles white, crowded on
the raised axis. Each pistil forms a separate tubercle which matures into the
aggregate fruit. Flowering occurs in spring-early summer and flowers are
pollinated by nitidulid beetles.
Aggregate and soft fruits form from the numerous and loosely united pistils
of a flower which become enlarged and mature into fruits which are distinct from
fruits of other species of genus. The round or heart-shaped greenish yellow,
ripened aggregate fruit is pendulous on a thickened stalk with many round
protuberances and covered with a powdery bloom. Fruits are formed of loosely
cohering or almost free carpets.
The pulp is white tinged yellow, edible and sweetly aromatic. Each carpel
containing an oblong, shiny and smooth, dark brown to black, long seeds.
(Wikipedia, 2015)

Ficus septica is an erect, small tree, growing 3 to 8 meters high, smooth,

with more or less hairy young shoots. Leaves are smooth and shining, not all
roughened, oblong-ovate to elliptic-ovate, 10-20 centimeters long, with tip
tapering to a rather sharp point, and the base pointed. Receptacles are axillary,

solitary, depressed-globose or turbinate, obscurely ridged or angled, 1.5 to 2

centimeters in diameter, and shortly peduncled. (Stuartxchange, n.d.)
Figs grow often in pairs but can be solitary or in groups of up to four. Figs
are depressed-globose to ellipsoid, the apex is flat or concave. Seven to twelve
ribs toward to ostiole. At maturity, whitish to yellowish dots appear on the fig. The
individuals from Philippines have their stems covered by short hairs while those
found in Taiwan are glabrous. (Wikipedia, 2015)

Distribution
Annona squamosa is cultured throughout the Philippines; occasionally
instinctive. It is also native to the tropical Americas and West Indies, but the spoton origin is unknown. It is now the most widely cultured of all the species of
Annona, being grown for its fruit throughout the tropics and warmer subtropics,
such as Indonesia, Thailand, and Taiwan; it was acquainted to southern Asia
before 1590. It is naturalized as far north as southern Florida in the United States
and as south as Bahia in Brazil, and is an invasive species in some areas.

Ficus septica is distributed in thickets at low and medium altitudes

throughout the Philippines. Also occurs in China, from Northeast India to
Australia and throughout Malesia.

Development

Using histological techniques and microscopy, Annona squamosa seeds

were studied with regard to their anatomical characteristics. In general, they
presented the typical anatomical patterns of seeds of the Annonaceae family. The
multilayered testa was comprised of exo-, meso- and endotestas, with the
presence of longitudinal and oblique fibers. The embryo was small and straight
with moderately developed embryonic axis, rudimentary plumule and flat and thin
cotyledons, wherein one could observe the procambium. In particular, the
perichalaza was observed in the medium longitudinal plane of the endotesta with
more than two rows of vascular tissue. The tegmen or inner integument was
collapsed. The micropylar plug was tapered, woody and composed of transverse
fibers; a fracture line was also observed. The ruminate endosperm interfered with
both the inner and external integuments. Oleiferous idioblasts were observed at
the outer edges of the ruminations. (Martinez et al., 2013)
Ficus septica Cotyledons are almost orbicular, about 3 mm diam. First pair
of leaves toothed. At the tenth leaf stage: leaf blade broadly elliptic, apex acute,
base obtuse, margins sinuate on the upper half of the leaf blade, teeth obscure,
upper surface glabrous; oil dots not visible; flat glands sometimes visible on the
underside of the leaf blade at the junction of the main lateral veins and the
midrib; petiole with a few scattered hairs; stipules sheathing the terminal bud,
glabrous, about 10-15 mm long. (Burman, 1768)

Life Cycle

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Sugar apple seeds have a relatively long life, having kept well for 3 to 4
years. They germinate better a week after removal from the fruit than when
perfectly fresh. Germination may take 30 days or more but can be hastened by
soaking for 3 days or by scarifying. The percentage of germination is said to be
better in unsoaked seeds. While the tree is generally grown from seed,
vegetative propagation is practiced where the crop is important and early fruiting
is a distinct advantage.
Sugar apple trees are spaced at 10 x 10 ft (3x3 m) in order to elevate
atmospheric humidity and improve pollination. Commercial fertilizer containing
3% N, 10 % P and 10% K significantly increases flowering, fruit set and yield.
Judicious pruning to improve shape and strength of tree must be done only in
spring when the sap is rising; otherwise pruning may kill the tree. Irrigation during
the dry season and once during ripening will increase fruit size. (Morton, 1987)

Figure 2. Annona squamosa (Atis) Seedlings

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Figure 3. Annona squamosa (Atis) Full grown tree

Ficus septicas obligate pollination mutualism is similarly specialized. Life

cycles of pollinating fig wasps and their Ficus host plants are completely
interdependent. Volatile signals attract female fig wasps to a receptive fig of a
host where they gain access to the unisexual flowers of the enclosed
inflorenscence through a narrow bract-covered opening, called the ostiole.
Females oviposit in pistillate flowers and deposit pollen in the process. In most
cases, pollinators do not have a second chance at reproduction as they cannot
leave the fig upon entering (Moore et al. 2003). In monoecious figs, flowers either
set seed, form galls that nourish fig wasp offspring, or produce pollen that
eclosing wasps later transport to other receptive figs. In dioecious species,
reproductive functions are separated between plants bearing either seedproducing inflorescences (female figs) or inflorescences producing wasps and
pollen (male figs).
Cotyledons almost orbicular, about 3 mm diameter. First pair of leaves
toothed. At the tenth leaf stage: leaf blade broadly elliptic, apexacute,
base obtuse, margins sinuate on the upper half of the leaf blade, teeth obscure,
12

upper surface glabrous; oil dots not visible; flat glands sometimes visible on the
underside of the leaf blade at the junction of the main lateral veins and
the midrib; petiole with a few scattered
hairs; stipules sheathing the terminal bud, glabrous, about 10-15 mm long. (Moe
and Weiblen, 2012)

Figure 4. Ficus septica (Labnog) Seedlings and full grown tree

Tumors and Cancers

The terms tumor and cancer are sometimes used interchangeably which
can be misleading. A tumor is not necessarily a cancer. The word tumor simply
refers to a mass. For example, a collection of fluid would meet the definition of a
tumor. A cancer is a particularly threatening type of tumor. It is helpful to keep

13

these distinctions clear when discussing a possible cancer diagnosis. (Johns

Hopkins University, n.d.)
According to Canadian Cancer Society, malignant tumors are cancerous.
Cancer can start in any one of the millions of cells in our bodies. Cancer cells
have a larger nucleus that looks different from a normal cells nucleus, and
cancer cells behave, grow and function quite differently from normal cells.
Malignant tumors vary in size and shape. They grow in an uncontrolled,
abnormal way and can grow into (invade) nearby tissues, blood vessels or
lymphatic vessels. They can interfere with body functions and become lifethreatening. Cancer cells can break off and spread to distant locations in the
body (metastasize). Cancer that spreads from its original location (the primary
tumor) to a new part of the body is called metastatic cancer. Malignant tumors
can also come back (recur) after they are removed.
The most common way to name cancers is to do so according to the place
in the body they start, such as breast cancer or prostate cancer. Cancers of the
blood are called leukemia, while cancer of the plasma cells is called multiple
myeloma and cancers of the lymphatic system are called lymphoma. Some
cancers have been named after the person who first described them (for
example, Hodgkin lymphoma or Wilms tumor). Another way to name some
benign or malignant tumors is after the type of cell or tissue it develops from
(tissue of origin). Most benign tumors and some malignant tumors have the suffix
"oma" at the end of their name. When a malignant tumor has the same name as
a benign tumor, the word "carcinoma" or "sarcoma" is added to the end to identify

14

it as cancer. For example, a benign tumor of fatty tissue is called a lipoma,

whereas a malignant tumor of fatty tissue is called a liposarcoma.

Bioactive compounds in Annona squamosa

Annona squamosa leaves yield an alkaloid, chloroplatinate. Anonaine, an
alkaloid, is found in the bark, leaves and seeds. Seed yields an alkaloid, neutral
resin, fixed oil, contains yellow, non-drying oil and an irritant which kills lice. Fruit
peel extracts yielded alkaloids, proteins, carbohydrates, flavonoids, glycosides,
saponins, and tannins. Roots yielded two oxoaporphine alkaloids: liriodenine and
oxoanalobine. (Stuartxchange, n.d.)
According to Vijayalaksham et al., (2011) Forty-four compounds were identified in
the ethyl acetate fraction of Annona squamosa leaf extract by GC-MS analysis.
The prevailing compounds were sodium benzoate (27.50%), 4, 4- TertButylcalix(4)arene (12.34 %),4, 4- Dimethylcholesterol (10.30%),
Butyloctylpthalate (9.67%), stigmasterol acetate (2.92%), isoamylacetyate
(2.29%). The present investigation clearly indicates the highest percentage of
sodium benzoate in ethylacetate fraction of Annona squamosa leaf extract.
Sodium benzoate was used as an antifungal agent, antibacterial agent, as CNS
stimulant and as a diagnostic aid in liver function tests.

15

Bioactive compounds in Ficus septica

Phytochemical study isolated isoflavones: ficusin A and ficusin B from the
root bark. Two indolizidine alkaloids: a novel ficuseptine and antofine.
Phytochemical screening of an ethanol extract yielded alkaloids, quarternary
base, tannins, 2-deoxysugars and benzopyrone nucleus. Methanol extract of
stems yielded eight new alkaloids, namely, ficuseptines B-D (1-3), 10R, 13aRtylophorine N-oxide, 10R, 13aR-tylocrebrine N-oxide, 10S, 13aR-tylocrebrine Noxide, 10S, 13aR-isotylocrebrine N-oxide, and 10S, 13aS-isotylocrebrine Noxide, together with six known phenanthroindolizidine alkaloids.

Annona squamosa Health Benefits

Annona squamosa leaves, fruit and seeds are vermicidal and insecticidal.
The unripe fruit is astringent, used for diarrhea and dysentery and dyspepsia.
The bark is astringent and tonic. Roots make a drastic purgative. Leaves are
emmenagogue, febrifuge and tonic. Studies have showed analgesic, antiinflammatory, anti-microbial, anti-diabetic, cytotoxic, antioxidant, antilipimic, antiulcer, molluscicidal, genotoxic, vasorelaxant, hepatoprotective, larvicidal,
anthelmintic, insecticidal properties. .(Stuartxchange, n.d.)

High in Vitamin C
A medium-size sweetsop fruit weighs about 5.5 ounces and contains 56.3
milligrams of vitamin C -- this is roughly 75 percent of the daily recommendation
of 75 milligrams for women and 62 percent of the recommended daily intake of

16

90 milligrams for men. Vitamin C helps maintain healthy bones and muscles, as
well as helps blood vessels stay supple and strong. Although the value of vitamin
C to fight colds remains unconfirmed, the July 2012 issue of "American Family
Physician" points out that vitamin C does seem to shorten the duration of a cold
in adults and children.

Rich in Fiber
Sweetsop is rich in fiber and carbohydrates. One 5.5-ounce serving, or
roughly 3-inch fruit, provides you with 6.8 grams of fiber, which is about 18
percent of the daily recommendation for an adult. With 36.6 grams of
carbohydrates per piece of fruit, sweetsop also is an excellent source of energy.
In comparison, a medium-size apple, or about 6.4 ounces has only 25 grams of
carbs and, even with the skin, just 4.4 grams of fiber. Fiber is an essential
nutrient that helps lower cholesterol, normalizes bowel movements and helps
control blood sugar levels. (Dray, 2015)

Antioxidant Properties
In a 2011 study published in the "Journal of Pharmacy Research,"
researchers studied Annona squamosa to see if it contains any compounds that
could be useful for medical treatments or nutritional support. Results from testtube research shows that sweetsop has free radical scavenging activity, making it
an effective antioxidant.

17

Anecdotal Use
Sweetsop is used extensively in India and tropical American countries to
treat a number of conditions. Most of its uses have been passed down through
generations, though studies are needed to confirm whether sweetsop really
works in treating these conditions. For example, sweetsop is used in India to
treat wounds and ulcers, as well as an anti-diarrheal tonic. In tropical American
countries, sweetsop has different medicinal uses, such as being a treatment for
colds, digestive problems and high fever.

Antidiabetic
Beneficial effects of Annona squamosa extract in streptozotocin-induced
diabetic rats: Study results showed that Annona squamosa extract has an
antihyperglycemic effect and alleviated liver and renal damage associated with
STZ-induced diabetes mellitus in rats. Study of aqueous leaf extracts were
investigated on STZ-nicotinamide induced diabetic rats. The diabetic groups
treated with aqueous leaf extract were compared with standard glibenclamide.
Study evaluated the antidiabetic activity of a hydroalcoholic extract of
Annona squamosa in experimentally induced diabetic rat model. Extract of
leaves showed significant reduction in blood glucose after glucose loading, with
activity comparable to glibenclamide.

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Anti-Inflammatory / Cyclic Peptides

Study yielded two new cyclic peptides, cyclosquamosin H and I,
together with six known cyclic peptides, squamin A, squamin B, cyclosquamosin
A, D E and cherimolacyclopeptide B from the seeds. Compound 7 showed an
inhibitory effect on the production of pro-inflammatory cytokines.

Hepatoprotective / DEN-induced Hepatotoxicity

Study on diethylnitrosamine (DEN)-induced liver injury in Swiss albino
mice showed hepatoprotective effect, with improvement in biochemical
parameters and confirmation by histopathological studies.
Study showed the extracts of Annona squamosa were not able to
completely revert the hepatic injury induced by isoniazid + rifampin, but it could
limit the effect of the drugs on the liver. The effect compared with standard drug
silymarin.

Antibacterial
Study screened the ethanol crude extract of the fruit of Annona squamosa
for antimicrobial activity against some pathogenic microorganisms. It showed
inhibitory activity against S aureus and S pneumoniae. Results conclude the
plant extract may serve as a valuable source of compounds with therapeutic
antibiotic potentials.

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Antithyroid Activity / Quercetin

Extract of the seeds of Annona squamosa was evaluated for it
ameliorative effect in the regulation of hyperthyroidism in a mouse model.
Phytochemical study revealed the presence of quercetin in the seed extract and
the results of the effects of quercetin suggest an involvement of this
phytochemical in the mediation of antithyroidal activity of A squamosa seed
extract.

Antigenotoxic Activity
Study showed both aqueous and ethanolic bark extracts of Annona
squamosa showed antigenotoxic effect. The bark extract demonstrated more
prominent antigenotoxic effect in DBMA induced genotoxicity in Syrian hamsters.

Molluscicidal Activity
Study on molluscicidal activity of leaves, bark and seed of Annona
squamosa against snail Lymnaea acuminata was studied. Highest activity was
observed in the seed extracts. The acetogenins from the seed were more toxic
than synthetic pesticides.

Anti-Head Lice Activity

Study identified the active compounds against head lice from the hexane
extract of Annona squamosa seeds. The two major compounds were oleic acid
and triglyceride with one oleate ester. The triglyceride with one oleate ester and

20

the crude hexane extract diluted with coconut oil 1:1 were found to kill all tested
head lice.

Antimicrobial / Phytochemicals
Phytochemical screening yielded phenols, tannins, alkaloids, saponins,
flavanoids, reducing sugars and oil. The methanol extract showed maximum
antibacterial activity against E. coli. Seed extract showed maximum antifungal
activity against T. rubrum.

Anti-Ulcer
Phytochemical investigation of twigs isolated twelve known compounds.
Three of the compounds, (+)-O-methylarmepavine (2), N- methylcorydaldine (3),
isocorydine (6), showed promising anti-secretory activity, comparable to standard
drug omeprazole. An ethanol extract and its chloroform and hexane fractions
exhibited gastroprotection via inhibition of H+K+-ATPase (proton pump) activity
and simultaneously strengthening mucosal defense mechanisms.

Toxicity
The seeds are acrid and poisonous. Bark, leaves and seeds contain the
alkaloid, anonaine. Six other aporphine alkaloids have been isolated from the
leaves and stems: corydine, roemerine, norcorydine, norisocarydine, isocorydine
and glaucine. Aporphine, norlaureline and dienone may be present also.
Powdered seeds, also pounded dried fruits serve as fish poison and insecticides

21

in India. A paste of the seed powder has been applied to the head to kill lice but
must be kept away from the eyes as it is highly irritant and can cause blindness.
If applied to the uterus, it induces abortion. Heat-extracted oil from the seeds has
been employed against agricultural pests. Studies have shown the ether extract
of the seeds to have no residual toxicity after 2 days. High concentrations are
potent for 2 days and weaken steadily, all activity being lost after 8 days. In
Mexico, the leaves are rubbed on floors and put in hen's nests to repel lice.
(Stuartxchange, n.d.)

Ficus septica Health Benefits

Ficus septica have diuretic, sudorific, antiherpetic, antirheumatic
properties. In the Philippines, Ficus septica are used for diarrhea, cough, malaria
and stomach problems.

Mucarinic Receptor Activity

Malaysian study of 224 plant extracts from 50 plant families for muscarinic
receptor binding activity showed the greatest inhibition, and with other extracts
that exhibited significant muscarinic properties were suggested to be worthy of
further investigation.

Anti-inflammatory
Study examined the molecular mechanisms for the anti-inflammatory
activity of phenanthroindolizidine alkaloids isolated from the leaves of Ficus

22

septica. Study suggests that it exerts its anti-inflammatory effects by inhibiting

expression of the proinflammatory factors and related signaling pathways.

Antimicrobial / Antifungal / Antiprotozoal

Study of ethanol extracts of Ficus septica and S foetida showed
antibacterial activities, inhibiting the growth of S aureus and E coli. Antifungal
assay showed inhibition of Candia albicans. Antiprotozoal assay showed activity
against T vaginalis and Entamoeba histolytica. Results suggest the extracts can
be used to produce alternative forms of antimicrobials.

Antioxidant / Hepatoprotective
Study evaluated the antioxidant properties of leaf extract of Ficus septica
by measuring malondialdehyde (MDA) levels, as by-products of lipid
peroxidation, in the liver of ICR mice. Extract treated mice had lower MDA levels
with various signs of histological cellular repair. Results suggested
hepatoprotection.

Antiangiogenic Activity
Study evaluating the potential of leaves of various plants, including Ficus
septica for angiosuppressive activity. All the extracts tested except for P.
laevigata can reduce CAM (chorio-allantoic membrane) vascular density pointing
to an antiangiogenic activity. (Stuartxchange, n.d.)

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Free Radicals
Free radicals are atoms or groups of atoms with an odd (unpaired)
number of electrons and can be formed when oxygen interacts with certain
molecules. Once formed these highly reactive radicals can start a chain reaction,
like dominoes. Their chief danger comes from the damage they can do when
they react with important cellular components such as DNA, or the cell
membrane. Cells may function poorly or die if this occurs. To prevent free radical
damage the body has a defense system of antioxidants. (Antioxidants and Free
Radicals, n.d.)
A notable example of a free radical is the hydroxyl radical (HO), a
molecule that has one unpaired electron on the oxygen atom. Two other
examples are triplet oxygen and triplet carbine (:CH2) which have two unpaired
electrons. In contrast, the hydroxyl anion (HO-) is not a radical, since the
unpaired electron is resolved by the addition of an electron; singlet oxygen and
singlet carbine are not radicals as the two electrons are paired.
Free radicals may be created in a number of ways, including synthesis
with very dilute or rarefied reagents, reactions at very low temperature, or
breakup of larger molecules. The latter can be affected by any process that puts
enough energy into the parent molecule, such as ionizing radiation, heat,
electrical discharges, electrolysis, and chemical reactions. Indeed, radicals are
intermediate stages in many chemical reactions. (Wikipedia, 2015)

24

Some conditions cause by free radicals include, deterioration of the eye

lens, which contributes to blindness, inflammation of the joints (arthritis), damage
to nerve cells in the brain, which contributes to the conditions such as
Parkinsons disease, acceleration of the aging process, increased risk of
coronary heart disease, since free radicals encourage low-density lipoprotein
(LDL) cholesterol to stick to artery walls, certain cancers, triggered by damage
cellDNA. (Anioxidants, n.d.))
A diet in antioxidants may reduce the risk of many diseases, including
heart disease and certain cancers. Antioxidants scavenge free radicals from the
body cells, and prevent to reduce the damage caused by oxidation.

DPPH Radical Scavenging Assay

DPPH is a common abbreviation for an organic chemical compound 2, 2diphenyl-1picrylhydrazyl. It is a dark-colored crystalline powder composed of
stabl free-radical molecules. DPPH has two major applications, both in laboratory
research: one is a monitor of chemical reactions involving radicals, most notably
it is a common antioxidant assay, and another is a standard of the position and
intensity of electron paramagnetic resonance signals. (Sharma and Bhat, 2009)

Allium cepa
Is known as garden onion or bulb onion. Its family is a large and
diverse one containing over 500 species (Simoons, et al. 1998). Alliun cepa is a
biological material or great importance for mutagenicity screening of many

25

substances. The Allium cepa Test or Allium Test is widely employed because of
its simplicity, the fast growth of its roots, and the response of its genetic material
to the presence of substances with cytotoxic and genotoxic potential (Smackakincl et al., 1996; Evadri et al., 2000). The test is routinely used around the world,
at laboratories working with toxicological genetics. It provides the assessment of
the degree of pollution of environments and of the toxicity caused by industrial,
agricultural and domestic effluents, by determining the reduction in the mitotic
index (MI) and the formation of chromosomal aberrations. (Fisjek , 1993)

Mitotic Phases
Mitosis simply refers to a type of cell division in which one cell (the
mother) divides to produce two new cells (the daughters) that are genetically
identical to itself. However, in the context of the cell cycle, mitosis also has a
narrower definition: it refers to just one part of the overall division process, the
part in which the DNA of the nucleus is split into two equal sets of chromosomes.
The great majority of the cell divisions that happen in your body, or in the bodies
of plants, animals, fungi, and other eukaryotic organisms, involve mitosis. During
development and growth, mitosis populates an organisms body with cells, and
throughout an organisms life, it replaces old, worn-out cells with new ones. For
single-celled eukaryotes like yeast, mitotic divisions are actually a form of
reproduction, adding new individuals to the population. (Khan Academy, 2015)
Mitosis consists of four basic phases: prophase, metaphase, anaphase,
and telophase. Some textbooks list five, breaking prophase into an early phase

26

(simply called prophase) and a late phase (called prometaphase). These phases
occur in strict sequential order, and cytokinesis - the process of dividing the cell
contents to make two new cells - starts in anaphase or telophase.

Figure 5. Cell Cycle of Mitosis (http://biology.tutorvista.com/cell/cell-cycle.html)

Mitosis begins with prophase, during which chromosomes recruit
condensin (large protein complexes that play a central role in chromosome
assembly and segregation during mitosis and meiosis) and begin to undergo a
condensation process that will continue until metaphase. In most species,
cohesin (a protein complex that regulates the separation of sister chromatids
during cell division, either mitosis or meiosis) is largely removed from the arms of
the sister chromatids during prophase, allowing the individual sister chromatids to
27

be resolved. Cohesin is retained, however, at the most constricted part of the

chromosome, the centromere. During prophase, the spindle also begins to form
as the two pairs of centrioles move to opposite poles and microtubules begin to
polymerize from the duplicated centrosomes.
Prometaphase begins with the abrupt fragmentation of the nuclear
envelope into many small vesicles that will eventually be divided between the
future daughter cells. The breakdown of the nuclear membrane is an essential
step for spindle assembly. Because the centrosomes are located outside the
nucleus in animal cells, the microtubules of the developing spindle do not have
access to the chromosomes until the nuclear membrane breaks apart.
Prometaphase is an extremely dynamic part of the cell cycle. Microtubules
rapidly assemble and disassemble as they grow out of the centrosomes, seeking
out attachment sites at chromosome kinetochores, which are complex platelike
structures that assemble during prometaphase on one face of each sister
chromatid at its centromere. As prometaphase ensues, chromosomes are pulled
and tugged in opposite directions by microtubules growing out from both poles of
the spindle, until the pole-directed forces are finally balanced. Sister chromatids
do not break apart during this tug-of-war because they are firmly attached to
each other by the cohesin remaining at their centromeres. At the end of
prometaphase, chromosomes have a bi-orientation, meaning that the
kinetochores on sister chromatids are connected by microtubules to opposite
poles of the spindle.

28

After, chromosomes assume their most compacted state during

metaphase, when the centromeres of all the cell's chromosomes line up at the
equator of the spindle. Metaphase is particularly useful in cytogenetics, because
chromosomes can be most easily visualized at this stage. Furthermore, cells can
be experimentally arrested at metaphase with mitotic poisons such as colchicine.
Video microscopy shows that chromosomes temporarily stop moving during
metaphase. A complex checkpoint mechanism determines whether the spindle is
properly assembled, and for the most part, only cells with correctly assembled
spindles enter anaphase.
The progression of cells from metaphase into anaphase is marked by the
abrupt separation of sister chromatids. A major reason for chromatid separation
is the precipitous degradation of the cohesin molecules joining the sister
chromatids by the protease separase. Two separate classes of movements occur
during anaphase. During the first part of anaphase, the kinetochore microtubules
shorten, and the chromosomes move toward the spindle poles. During the
second part of anaphase, the spindle poles separate as the non-kinetochore
microtubules move past each other. These latter movements are currently
thought to be catalyzed by motor proteins that connect microtubules with
opposite polarity and then "walk" toward the end of the microtubules.
Mitosis ends with telophase, or the stage at which the chromosomes
reach the poles. The nuclear membrane then reforms, and the chromosomes
begin to decondense into their interphase conformations. Telophase is followed
by cytokinesis, or the division of the cytoplasm into two daughter cells. The
29

daughter cells that result from this process have identical genetic compositions.
(Nature Education, 2014)

30

CHAPTER III
METHODOLOGY

Materials

Beaker

Water bath

Test tubes

Spatula

Test tube holder

Dropper

Chemicals Used

HCl

Concentrated

Glacial acetic acid

Mayers reagent

Sulphuric Acid

Potassium Hydroxide

Wagners reagent

Chloroform

Aqueous copper(II)

Dragendroffs reagent

Ammonia

sulfate

Hagers reagent

Ferric Chloride

HNO3

Sodium Hydroxide

H2SO4

Casein

Lead Acetate

-naphthol

Egg albumin

1% Gelatin Solution

Phenylhydrazine

Tyrosine

containing NaCl

Hydrochloride

Acetic Acid

Acetic Anhydride

Sodium acetate

31

Collection of Samples
Handpicked leaf samples of Annona squamosa and Ficus septica was
collected at Barangay Sta. Filomena, Iligan City.

Preparation of Samples
Leaves of A. squamosa and F. septica were washed, air-dried until crisp,
powdered prior to extraction with 95% ethanol at room temperature for 72 hours.
The process was repeated 3 times and the combined ethanol fractions were
evaporated under reduced pressure using a rotary evaporator at 55C.
Phytochemical Screening
Phytochemical examinations were carried out for all the extracts as per
the standard methods.

Detection of Alkaloids - Extracts were dissolved individually in dilute

Hydrochloric acid and filtered.
a) Mayers Test Filtrates were treated with Mayers reagent (Potassium
Mercuric Iodide). Formation of a yellow colored precipitate indicates the presence
of alkaloids.
b) Wagners Test: Filtrates were treated with Wagners reagent (Iodine in
Potassium Iodide). Formation of brown/reddish precipitate indicates the presence
of alkaloids.

32

c) Dragendroffs Test Filtrates were treated with Dragendroffs reagent (solution

of Potassium Bismuth Iodide). Formation of red precipitate indicates the
presence of alkaloids.
d) Hagers Test Filtrates were treated with Hagers reagent (saturated picric
acid solution). Presence of alkaloids confirmed by the formation of yellow colored
precipitate.

Detection of Flavonoids
a) Alkaline Reagent Test Extracts were treated with few drops of sodium
hydroxide solution. Formation of intense yellow color, which becomes colorless
on addition of dilute acid, indicates the presence of flavonoids.
b) Lead acetate Test Extracts were treated with few drops of lead acetate
solution. Formation of yellow color precipitate indicates the presence of
flavonoids.

Detection of Tannins
Gelatin Test To the extract, 1% gelatin solution containing sodium chloride was
added. Formation of white precipitate indicates the presence of tannins.

Detection of Saponins
a) Froth Test Extracts were diluted with distilled water to 20ml and this was
shaken in a graduated cylinder for 15 minutes. Formation of 1 cm layer of foam
indicates the presence of saponins.

33

Detection of Steroids
a) Libermann Burchard Reaction: 2 ml extract was mixed with chloroform. To
this 1-2 ml acetic anhydride and 2 drops concentrated sulphuric acid were added
from the side of test tube. First red, then blue and finally green color appears.

Detection of Anthraquinone
a) Borntragers Test To 3 ml extract dilute sulphuric acid was added, boiled and
filtered. To the cold filtrate equal volume benzene or chloroform was added. The
organic layer was separated and ammonia was added. Ammonical layer turns
pink or red.

Detection of Glycosides: Extracts were hydrolysed with dil. HCl, and then
subjected to test for glycosides.
a) Modified Borntragers Test Extracts were treated with Ferric Chloride solution
and immersed in boiling water for about 5 minutes. The mixture was cooled and
extracted with equal volumes of benzene. The benzene layer was separated and
treated with ammonia solution. Formation of rose-pink color in the ammonical
layer indicates the presence of anthranol glycosides.
b) Foam Test 0.5 gm of extract was shaken with 2 ml of water. If foam
produced persists for ten minutes it indicates the presence of saponins.

34

Brine Shrimp Lethality Test/ Cytotoxic Activity Test

Plant test extract preparation
50 mg of the plant extract was dissolved in 5 mL of methanol. 0.5 mL of
the above Solution A was diluted to 10 mL with methanol (Solution B). Pipette
100 L of Solution B, 50 L of Solution A and 500 L of Solution A were
separated into vials, respectively labelled 1, 2 and 3. A control vial was prepared
containing 1 mL of methanol. The solutions were dried in each of the above vials
under nitrogen and then prepared five replicates for each dose level.

Hatching the shrimp

A shallow rectangular dish (22 cm x 32 cm; a soap dish will do) was filled
with the artificial sea water. A plastic divider was placed, punched with several 2
mm holes, in the dish to divide into two unequal compartments. The minute
brown brine shrimp eggs were sprinkled (ca. 50 mg) into the larger compartment.
The larger compartment was covered to keep away from light; the smaller
compartment was left open. The smaller open compartmentwas illuminated then
after 48 hours, pipette the hatched brownish orange nauplii from the illuminated
compartment of the dish.
Note: The brine shrimp egg shells are left in the darkened side of the dish.

35

Bioassay
Counting the nauplii
Pipette nauplii and was counted macroscopically in the stem of the
pipette, and was held against a well lighted background.

Concentration of sample vials 1, 2 and 3

Each sample vials diluted to 5 mL with artificial seawater made a final
concentration of 10,100 and 1000 g/mL respectively.

With a 9 in. pipette, ten nauplii was transfered into each sample vial
labelled 1, 2 and 3 and control vial. Artificial sea water was added to each vial,
samples and control, to make a total volume of 5 mL. A drop of yeast suspension
(3 mg/ 5 mL of sea water) was added as food in each vial. The vials were kept
under illumination. The survivors were counted first after 6 hours then after 24
hours; The 24 hour count was used to record the number of deaths then
determined the percent deaths for each dose level and for the control vials.

In cases where control deaths occurred, the data was corrected using
Abbotts formula:
% deaths = (death in test vial-death in control vial) x 100
death in control vial

Free Radical Scavenging Activity

36

Spray reagents:
Solution A 1 % iron(III) chloride aqueous solution
Solution B 1 % potassium ferricyanide aqueous solution
Equal volumes of Solution A and b were mixed before use. This spray
reagent can only detect phenolic antioxidants.

Thin-layer plates were prepared and then take an equivalent of 10 g plant

material from the stock plant extract prepared and were evaporated to incipient
dryness over a steam bath; and were cooled to room temperature. The residue
was extracted with just enough diethyl ether and drops of methanol were added
so as to break down any emulsified material. Spots of 2 L were applied of the
appropriate plant sample solutions and 2 L each of the standard solutions of
BHA and BHT (0.05% in ethyl alcohol) to a thin layer of silica gel
chromatographic plate. The chromatogram was developed in the dark in a
developing chamber that has been equilibrated with n-hexane:2-butanone:n-butyl
ether (34:7:6) for 30 minutes. When the solvent reaches the solvent front, the
plate was removed from the tank and allow the chromatogram to stand in a
stream of cold air until most of the solvent has evaporated then immediately
spray the chromatogram with the spray reagent. And the sizes were compared
and intensities of the sample blue spots with that of the standard.

Alternative spray reagents for chromatograms C1 and C2:

37

Solution C consists of a 2% solution of -ocimene in hexane.

Solution D1 consists of a 2% solution of iron (II) thiocyanate in 30%
perchloric acid.
Solution D2 consists of a 2% solution of 2.4-dinitrophenylhydrazine in
30% perchloric acid.

Alternatively develop two chromatograms, C1 and C2, containing the

same plant sample and standards. The developed chromatograms was sprayed,
C1 and C2, with Solution C, and then were allowed standing at room temperature
for about 1 hour or heat at 50C for 10-15 minutes. Chromatogram C1 was
sprayed with Solution D1. White spots in a pink background indicate positive
results. The results were immediately documented because the color is very
unstable. The chromatogram C2 was sprayed with Solution D2. The antioxidants
are revealed as white spots on a yellow to brown background. The spots
remained visible for days on inert supports.

Determination of the Eukaryotic Cell Inhibition: Antimitotic Assay

Onion Collection
16 pieces of the same size Onion Bulbs were bought from the market and
were then separated into two variables: Four onion bulbs for the negative control
(distilled water) and to the respective treatments with the concentrations of 20%,
40% and 60%.

38

Preparation of Treatments
A 20%, 40%, and 60% concentration of Annona squamosa and Ficus
septica extracts already mixed with distilled water was prepared separately and a
100% concentration of Annona squamosa and Ficus septica extracts and was
placed in a 15 ml glass.

Cell Counting and Determination of Mitotic Index

The assay was carried out with four sample concentrations. A 100 ml of
distilled water as the negative control followed by a 20%, 40% and 60% extracts
of Annona squamosa and Ficus septica with distilled water (treatment). 4 onions
were exposed to each concentration. In each beaker containing the solution, an
onion bulb was placed on the top of it and the roots were allowed to grow for 4
days. The test was carried out at a room temperature and the onions were kept
away from direct sunlight. 5 root tips at a length of 10 mm was cut off for the cell
counting of different mitotic phases and was placed in a test tube with 2 ml acetic
acid and hydrochloric acid solution and heated for 3 minutes at 50C. Then the
roots were placed on a slide and the terminal root tips which measure 1-2 mm
are cut off for further preparation. Two drops of Benedicts Reagent was also
added and mixed with the root tips. Then the cover slip was placed on the root
cells and absorbed stain for 4 minutes. The cells were squashed by placing the
clean absorbent fabric on the cover glass and press it slightly with the hand.
Then, cell counts of different mitotic phases were counted under a compound
microscope.

39

40

CHAPTER IV
RESULTS AND DISCUSSIONS

Annona squamosa (Atis)

Kingdom: Plantae
Phylum: Spermatophyta
Class: Dicotyledonae
Order: Magnoliales
Family: Annonaceae
Genus: Annona
Species: A. squamosa

Ficus septica (Labnog)

Kingdom: Plantae
Phylum: Tracheophyta
Class: Magnoliopsida
Order: Rosales
Family: Moraceae
Genus: Ficus
Species: F. septica

Qualitative Determination of Phytochemicals

Phytochemical Components Tested

Phytochemical Components
Flavanoids
Alkaloids
Tannins
Saponins
Steroids
Anthraquinone
Cyanogenic glucoside

Annona squamosa
+++
+++
+++
+++
+++
-

Ficus septica
+++
+++
+++
+++
-

Legend:
(+) slight turbidity
(++) definite turbidity
(+++) heavy precipitation
(-) no detection

Findings exhibited that Annona squamosa contains Flavanoids, Alkaloids,

Tannins, Saponins and Steroids while Ficus septica contains Flavanoids,
Alkaloids, Tannins and Saponins only.
Alkaloids are an amazing group of phytoprinciples. Some alkaloids, such
as nicotine, are used in pesticides, and others are used as chemical reagents.
The primary use of alkaloids, however, is in medicine, because they can act
quickly on specific areas of the nervous system. Alkaloids have potent anticancer
activity against various cancers (Mohan et al., 2011).
Saponins are glucosides with foaming characteristics. Studies have shown
that saponins have antitumor and anti-mutagenic activities and can lower the risk
of human cancers, by preventing cancer cells from growing. Saponins seem to

react with the cholesterol rich membranes of cancer cells, thereby limiting their
growth and viability. Studies have also shown that saponins can cause apoptosis
of leukemia cells by inducing mitotic arrest (Top Cultures, N.D.).
Flavonoids are plant-based compounds with powerful antioxidant
properties found in many fruits and vegetables like blueberries and grapes. They
serve a variety of functions such as protecting blood vessel walls in people who
have heart disease or diabetes, alleviating allergies, protecting brain health
against

dementia

bioflavonoids

and

even

another

preventing
word

for

some
the

cancers.
same

Flavonoids
compounds

or

have medicinal properties that include the ability to defend against cancer,
viruses, not to mention anti-microbial, antihistamine and anti-inflammatory
characteristics (Mother Nature Network, 2015).
Steroids on the other hand were developed as medical treatments and
they come in two varieties. Anabolic steroids are the kind you hear about the
most. They behave like male sex hormones, and doctors prescribe them for
treating problems like late puberty as well as significant muscle loss in patients
with cancer and AIDS (Kristen Philipkoski, 2012).
Table 1. Cytotoxic Activities of A. squamosa and F. septica leaf extracts within 6
and 24 hours
Sample

LC50 (6 hours)

Annona squamosa
Ficus septica

137.08 ppm
105.76 ppm

LC50 (24
hours)
192.34 ppm
126.59 ppm

Interpretation
Active (Toxic)
Active (Toxic)

As presented in Table 1, the leaf extracts of both Annona squamosa and

Ficus septica exhibited cytotoxic activity. This implies that both were found to be
toxic.

Table 2. Mitotic activities in Allium cepa root cells treated with F. septica
Formul-

Prophase

Metaphase

Anaphase

Telophase

Total

Total

Mitotic

ation

Cells

Cells

Cells

cells

dividing

cells

Index

Control
T1 (20%)
T2 (40%)
T3 (60%)
T4

128
30
36
30
11

102
124
29
86
26

116
21
44
37
11

174
153
124
115
64

cells
520
328
233
268
112

counted
1276
862
746
954
621

40.75
38.05
31.23
28.09
18.03

(100%)

Table 3. Mitotic activities in Allium cepa root cells treated with A. squamosa
Formul-

Prophase

Metaphase

Anaphase

Telophase

Total

Total

Mitotic

ation

Cells

Cells

Cells

cells

dividing

cells

Index

Control

143

93

98

152

cells
486

counted
1216

39.96

T1 (20%)

25

68

22

102

217

915

23.71

T2 (40%)
T3 (60%)

9
15

37
13

13
52

56
74

115
154

601
845

19.13
18.22

T4

17

25

12

39

93

711

13.08

(100%)

Graph 1. Mitotic Activities of A. squamosa and F. septica leaf extracts

45

M
I
T
O
T
I
C
I
N
D
E
X

40
35
30
25
Ficus septica

20

Annona squamosa

15
10
5
0
Control

T1 (20%)

T2 (40%)

T3 (60%)

T4 (100%)

FORMULATI

Tables 2 and 3 exhibited the data on the mitotic activities observed in

onion root cells treated with Annona squamosa and Ficus septica leaf extracts.
The mitotic index values of each leaf extracts was obtained by dividing the total
dividing cells to the total cells counted and was multiplied to 100.
The mitotic index values were lowered in the treated onion root cells when
compared with the control, and the mitotic index values were observed to be
decreasing with increasing concentrations of the Annona squamosa and Ficus
septica leaf extract.
The decreased mitotic index values in the treated onion roots is an
indication of the presence of cytotoxic substances in the Annona squamosa and
Ficus septica leaf extracts, which caused inhibition of mitotic activities.

Table 4. Chromosomal aberrations an Allium cepa root cells treated with F.

septica leaf extract.
Formul-

Total

Total

Small

Bridge

Laggard

Vagrant

Total

Aber-

ation

cells

dividing

and big

and two

Chromo-

Chromo

aber-

ration

counted

Cells

frag-

frag-

some

-some

rant

inci-

1276
862
746
954
621

520
328
233
268
112

ments
0
0
0
2
2

ments
0
0
1
1
5

0
0
2
3
2

0
0
0
2
1

cells
0
0
3
8
10

dence
0
0
1.28
2.98
8.92

Control
T1 (20%)
T2 (40%)
T3 (60%)
T4
(100%)

Table 5. Chromosomal aberrations an Allium cepa root cells treated with A.

squamosa leaf extract.
Formul-

Total

Total

Small

Bridge

Laggard

Vagrant

Total

Aber-

ation

cells

dividing

and big

and two

Chromo-

Chromo

aber-

ration

counted

Cells

frag-

frag-

some

-some

rant

inci-

1216
915
601
845
711

486
217
115
154
93

ments
0
0
0
1
2

ments
0
0
0
1
3

0
0
0
2
1

0
0
0
1
1

cells
0
0
0
5
7

dence
0
0
0
3.24
7.52

Control
T1 (20%)
T2 (40%)
T3 (60%)
T4
(100%)

Tables 4 and 5 exhibited that chromosome aberrations observed includes

small and big fragments, bridge and two fragments, laggard chromosome,
vagrant chromosome and chromosome breakages. The aberration incidence of
each leaf extracts was obtained by dividing the total aberrant cells to the total
dividing cells and was multiplied to 100.
The number of aberrant cells was also observed to be increasing with the
concentration of the Annona squamosa and Ficus septica leaf extracts. The

observation of aberrant cells in the treated onion root tip indicates genotoxic
effects of the leaf extracts.

Table 6. Antioxidant activity of A. squamosa using the DPPH method

ppm

Microgra

Vol.

Conc.

Vol. of

Final

New

Ave.

Control

extract

used

Of

DPPH,

vol.,

conc.,

abs

at 517

extract

mL

DPPH,

mL

mL

ppm

reading

nm

mM

%FRSAC

at 517
nm

10
20
30
40
50

30
60
90
120
150

3
3
3
3
3

0.1
0.1
0.1
0.1
0.1

2
2
2
2
2

5
5
5
5
5

6
12
18
24
30

0.057
0.046
0.039
0.033
0.026

0.381
0.381
0.381
0.381
0.381

85.04
87.93
89.76
91.34
93.18

Table 7. Antioxidant activity of F. septica using the DPPH method

ppm

Microgra

Vol.

Conc.

Vol. of

Final

New

Ave.

Control

extract

used

Of

DPPH,

vol.,

conc.,

abs

at 517

extract

mL

DPPH,

mL

mL

ppm

reading

nm

mM

%FRSAC

at 517
nm

10
20
30
40
50

30
60
90
120
150

3
3
3
3
3

0.1
0.1
0.1
0.1
0.1

2
2
2
2
2

5
5
5
5
5

6
12
18
24
30

0.368
0.349
0.34
0.333
0.329

0.381
0.381
0.381
0.381
0.381

3.41
8.40
10.76
12.60
13.65

Table 8. Antioxidant activity of Vit. C using the DPPH method (Positive control)
ppm

Microgra

Vol.

Conc.

Vol. of

Final

New

Ave.

Control

extract

used

Of

DPPH,

vol.,

conc.,

abs

at 517

extract

mL

DPPH,

mL

mL

ppm

reading

nm

mM

%FRSAC

at 517
nm

10

30

0.1

0.313

0.334

6.29

20
30
40
50

60
90
120
150

3
3
3
3

0.1
0.1
0.1
0.1

2
2
2
2

5
5
5
5

12
18
24
30

0.295
0.253
0.209
0.009

0.334
0.334
0.334
0.334

11.68
24.25
37.57
97.31

The antioxidant activity was determined using the DPPH method that
stands for Diphenyl Picrylhydrazyl. There are assorted methods in determining
the antioxidant activity however; scavenging of DPPH free radical is the common
basis for antioxidant assay. Antioxidants play an important role in human health,
scientist have said that antioxidants can lessen the issue of chronic diseases
such as cancer and heart disease. Plants sourced antioxidants like Vitamin C
that has the potential to reduce disease risk. (Tailor Chandra, 2014)

The percentage of Free Radical Scavenging Activity was also observed to

be increasing along with the concentration of the Annona squamosa and Ficus
septica leaf extracts. The observation of the increased percentage of FRSAC
indicates stronger antioxidant activity.

CHAPTER V
CONCLUSION AND RECOMMENDATIONS

Conclusion
Phytochemical Findings exhibited that Annona squamosa contains
Flavanoids, Alkaloids, Tannins, Saponins and Steroids while Ficus septica
contains Flavanoids, Alkaloids, Tannins and Saponins only which means that
both leaf extracts have useful bioactive compounds. The brine shrimp lethality
test results also implies that both were found to be toxic and could be preceded
for further studies like anti-microbial and anti-viral. Both leaf extracts also
exhibited decreased mitotic index by the use of Allium cepa chromosome
aberration assay which is an indication of the presence of cytotoxic substances
and which also caused inhibition of mitotic activities. The Free Radical
Scavenging Activity exhibits that the Annona squamosa and Ficus septica have
antioxidant activity using the DPPH method.
Therefore, both Annona squamosa and Ficus septica leaf extracts
exhibited an active substance and is toxic. But among the two leaf extracts,
results showed that Annona squamosa leaf extract have stronger cytotoxic
activity compared to the Ficus septica leaf extract. Thus, it could be a promising

substance in the form of medicine for possible treatment for anti-tumor and anticancer.

Recommendations
The researcher cited the following recommendations for further related
studies:
1. Investigate the possibility or potency to produce alternative form of
antimicrobials and anti diabetic in the leaf extracts.
2. The results suggest that more specific bioassays should be performed on
those plant extracts in order to confirm the conclusions.

BIBLIOGRAPHY

Online Journals
Burman, N.L. (1768). Ficus septica. Australian Tropical Rainforest Plants.
Retrieved from: http://keys.trin.org.au/key-server/data/0e0f0504-0103430d-8004-060d07080d04/media/Html/taxon/Ficus_septica.htm
Dray, T. (2015). What are the Health Benefits of Sweetsop?. Livestrong.com.
Retrieved from: http://www.livestrong.com/article/549365-what-are-thehealth-benefits-of-sweetsop/
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APPENDIX

Phytochemical screening

Boiling of water

Chemicals used

Ficus septica (Labnog)

Annona squamosa (Atis)

Allium cepa test

Preparation of Onion root tip

Preparation of Onion root tip

Squashing of onion root tips

Visualization using the light microspe