CHAPTER 1

INTRODUCTION
Medicinal plants play a vital role in the society. We usually
saw those plants as ornamentals, but now those plants are
inclined to bring forth its therapeutic level of importance. The
indigenous peoples in different parts of the world are practicing
since then the significance of medicinal plants in their day to day
living. Here in the Philippines, alternative medicines are highly
appreciated by the locals to combat against any health
complications.
As mentioned by Olowa et al. (2012) in their study entitled:
Medicinal plants used by the Higaonon Tribe of Rogongon, Iligan
City, Mindanao, Philippines, that nowadays the indigenous
knowledge on medicinal plants is fast diminishing because as
more plants are lost, so is the knowledge of their value to
humanity. They came up with a research that can provide
insights on the management and conservation of medicinal
plants in the area. One of the significant medicinal plants
mentioned was Ficus minahassae.
Ficus minahassae, local name Hagimit, is used to prevent
rheumatism and the sap is used as a beverage. Reddish color of

the bark extract suggests astringent properties and is used to

treat

kidney

ailment

(http://www.philstar.com/cebu-

news/719101/native-tree-month-part-2-hagimit).
The general objective of this study is for the evaluation of
Ficus minahassae through its antioxidant activity, phytochemical
screening and cytotoxicity potential using brine shrimp lethality
assay. Furthermore, this study will be specific only to the ethanol
leaf, fruit, bark and root extracts using DPPH method of
antioxidant

testing.

Qualitative

analysis

for

alkaloids,

anthraquinones, flavonoids, polyphenols, saponins, tannins and

terpenoids

will

be

investigated

following

the

standard

phytochemical screening procedure.

This

study

will

serve

as

baseline

information

for

researchers who wants to know the cytotoxicity potential of Ficus

minahassae. It will also contribute more to the evaluation of
phytochemical compounds present in the plant. For the benefit
of the society, this study will showcase the importance of health
through wellness enhancement and disease prevention.

CHAPTER 2
REVIEW OF RELATED LITERATURE
Ficus minahassae
Ficus

minahassae

local

name

Hagimit,

has

wide

canopy. With stem more or less buttressed at the base, it grows

to 15 meters or more in height. The branchlets are long and
bristly

hairy

(www.philstar.com/cebu-news/719101/native-tree-

month-part-2-hagimit).

Alkaloids
Alkaloids are basic nitrogenated compounds comprising the
largest class of secondary metabolites. This class of compounds
has attached much interest in the study of natural products and
a number of plants have been surveyed for its presence.
Alkaloids have been found to exhibit dramatic physiological

effects making them valuable in the field of medicine (Guevara

et al., 2005).

Terpenoids
Terpenoids constitute the largest class of natural products
and are found in abundance in higher plants. Many terpenoids
occur

as

glycosides

or

glycosyl

ester.

Terpenoids

are

commercially important as the basis of natural perfumes and of

spices and flavorings in the food industry (Guevara et al., 2005).
Anthraquinones
Anthraquinones

form

the

largest

group

of

naturally

occurring quinone pigments. They occur in plants usually as

hydroxylated,

methylated

or

carboxylated

derivatives

of

anthraquinones, anthrones, anthranal or dianthrone. They are

commonly

used

as

dyes

and

cathartics

or

purgatives.

Anthraquinone derivatives are often orange-red compounds that

are soluble in hot water and in dilute alcohol (Guevara et al.,
2005).

Flavonoids
Recent reports of the antiviral anti-fungal, anti-inflammatory and
cytotoxic activities of flavonoids have generated interest in

studies of flavonoid-containing plants. Flavonoids include the

anthocyanins and the leucoanthocyanins. Other flavonoids are
the cathechins, aurones and chalcones. Anthocyanins make up
the most important and widespread group of coloring matter in
plants. The anthocyanins are glycosides and on hydrolysis yield
sugars

and

colored

aglycones

known

as

anthocyanidins.

Extracting the plant material with 1% hydrochloric acid, then

boiling the extract can identify anthocyanins. An orange-red to
blue-red

color

upon

boiling

indicate

the

presence

of

anthocyanins. The leucoanthocyanins have as their aglycone the

leucoanthocyanidins. These are colorless, but give intense red or
violet color after boiling the acid-treated extract for a longer
time. Chalcones and aurones give an immediate red color when
the defatted ethanolic extract is treated with just the acid
(Guevara et al., 2005).

Saponins
Saponins

are

steroid/triterpenoid

glycosides

that

are

characterized by their ability to froth when the aqueous solution

is agitated. Plant materials containing saponins have long been
used for their detergent properties. Saponins usually exert a
powerful hemolytic action on the red blood cells and are highly

toxic when injected into the blood streams. They are used as fish
poison, causing paralysis of the gills. When taken orally, saponins
are, however, relatively harmless. A good example is the widely
used beverage known as sarsaparilla that is rich in saponins.
Plant saponins have interesting biological activities such as
spermicidal activity and molluscidal activity. The interesting
pharmacological properties associated with the Chinese drug
ginseng, which is considered a drug for longevity, are attributed
to the various saponins present in ginseng (Guevara et al.,
2005).

Tannins
Tannins have the ability to react with and precipitate proteins
forming stable water-insoluble copolymers. The term tannins
was first applied to plant constituents capable of transforming
raw animal skin into leather because of their ability to cross-link
with protein. Tannins are used in pharmaceutical preparations
because of their astringent action. Recent reports show that
tannins

may

have

potential

value

as

cytotoxic

and/or

antineoplastic agents. Chemically, there are two main groups of

tannins, the hydrolysable tannins and the condensed tannins.
The former are heterogeneous polymers containing phenolic

compounds united to glucose units by ester linkages. The latter,

the condensed tannins, are polymers of phenolic compounds
related to flavonoid pigments, frequent constituents of woody
plants. On treatment with acid, the condensed tannins are
decomposed

into

red

insoluble

compounds

known

as

phlobaphene or tannin reds. Tannins are detected in plant

extracts by the gelatin test. In such test, the tannins precipitate
the gelatin protein. The reaction is made sensitive by the
addition of sodium chloride to enhance the salting-out of the
protein-tannin complex. Positive results are confirmed by the
ferric chloride test. Hydrolyzable tanning give blue or blue-black
color while condensed tannins give a brownish-green color to the
test (Guevara et al., 2005).

Polyphenols
Polyphenols, on the other hand, also give characteristic
color reactions with the ferric chloride test. Polyphenols include a
wide range of plant substances, which possess in common, an
aromatic ring bearing two or more hydroxyl groups. These
substances tend to be water-soluble, since they most frequently
occur combined with sugar glycosides. Polyphenols, however,
give no precipitate with the gelatin test and because of their

similarity to tannins in terms of structure, are used as additives

in tanning agents in the leather industry (Guevara et al., 2005).

Antioxidants in plants
A compound that contains double bonds, allylic or benzylic
hydrogens, or tertiary hydrogens is susceptible to air oxidation, a
process called auto-oxidation. Antioxidants inhibit auto-oxidation.
Auto-oxidation of a fat yields a mixture of products that includes
low-molecular

weight

and

foul

smelling

carboxylic

acids.

Polyunsaturated fatty acids are converted by reactions with

oxygen to compounds containing oxygen and conjugated double
bonds,

such

as

hydroperoxides.

The

formation

of

such

compounds is responsible for the development of rancidity in

soils in soils and therefore for the spoilage of any food that
contains oil. The Not only do rancid oils taste bad, they are also
toxic. The reaction of unsaturated acid with oxygen involves freeradical intermediates, molecular oxygen being a diradical having
two unpaired electrons. The role of free-radical chemistry in the
body and especially its relation to aging are subjects of vigorous
debate. Polyunsaturated fatty acids mostly as their esters are
components of the body. Phenols, compounds containing an OH
group attached to an aromatic ring carbon, are effective anti-

oxidants. They break up the free-radical chain reactions by

reacting themselves with any radicals that form. Substituted
phenols are used as anti-oxidants in processed foods.

The Brine Shrimp Assay

The brine shrimp assay is a rapid, reliable and inexpensive
general bioassay tool for active plant extracts. The procedure
allows determination of the LC50 values in g/mL of active
constituents in the brine medium. This bioassay has been used in
the analysis of natural products, pesticides residues and other
chemical pollutants in marine environments. The eggs of brine
shrimp, Artemia salina Leach, are readily available in pet shops,
in the dry state, as food for tropical fish. When placed in a brine
solution, the eggs hatch within 48 hours, providing large
numbers of larvae (nauplii).

CHAPTER 3
METHODOLOGY
Preparation of plant samples. Fresh samples of Ficus
minahassae leaves, fruits, barks and roots will be collected from
Hagimit Falls, Island Garden City of Samal, Davao del Norte,
Philippines. Garden tools such as pruning scissors, scalpel trowel,
bolo and dissecting needle will be used for the plant collection;
and the ethical standards regarding plant collection will be
followed using the guidelines stated by Guevara et al. (2005). All
the

collected

parts

of

the

plant

will

be

identified

and

authenticated in the Biology Department Laboratory, Ateneo de

Davao University, E. Jacinto St., Davao City, Philippines.
The plant samples will be washed with distilled water,
air-dried, pulverized using an electric blender and will be stored
in clean and sealed plastic bags until analysis (Lagunay & Uy,
2015).

Preparation of plant extracts. Ethanol extract of each parts

will be prepared by soaking 50 g dried powdered plant parts with
adequate amount of 80% ethanol for 48 hours. The extracts will
be filtered using a filter paper in a canonical flask (Lagunay & Uy,
2015).

Antioxidant screening of the plant extracts

2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging
assay. The radical scavenging activity of the extracts will be
performed using DPPH spectrophotometric method.

Inhibition

(%) of free radical DPPH will be calculated using the equation:

% of inhibition = absorbance of control-absorbance of sample
100
absorbance of control
(MSU-Gensan Research
Workshop, 2016)

and Product

Development

Seminar

Ferric Reducing Antioxidant Power (FRAP) assay. This

assay gives an indication of the reducing ability of the plant
extract. This assay will developed using the experimental
protocol described by Benzie and Strain (1996), but will be

modified in terms of time lapse; the reaction will be evaluated at

4, 30 and 60 min. The FRAP reagent will be freshly prepared and
contained 1020 L of 300 mM sodium acetate pH 3.6, 100 L of
10 mM TPTZ (Sigma Chemical), and 100 L of 20 mM ferric
chloride. The FRAP reagents will be mixed with 10 L aliquots of
each extract. Then the mixture will be incubated at 37 C for 4,
30 and 60 min; after this time, the absorbance will be read at
593 nm using a UV-Vis spectrophotometer (Unicam Heio ). The
FRAP value will be determined by plotting in a standard curve
produced by the addition of ferrous sulphate (Merck, Darmstadt,
Germany) to the FRAP reagent. Results will be expressed as mol
Fe+2 per gram of fresh weight (mol Fe+2 g-1 FW) (Henriquez et
al., 2010).

Phytochemical screening. Phytochemical screening will be

carried out on the ethanol extracts of plant parts. Qualitative
analysis for alkaloids, anthraquinones, flavonoids, polyphenols,
saponins, tannins and terpenoids will be investigated following
the phytochemical screening procedure described by Guevarra et
al (2005); Chichioco-Hernandez, Paguigan and Ramiro (2010).

Test for Alkaloids. The presence of alkaloids will be tested by

dissolving 5 mL of the extract in 2 mL distilled water added with
3 drops of Wagners reagent (2 g iodine and 6 g potassium iodide
in 100 mL water). Formation of a blue black precipitate confirmed
the presence of alkaloids (Chichioco-Hernandez, Paguigan and
Ramiro, 2010).

Test for Anthraquinones. The presence of anthraquinones will be

tested using the test tube screening method through the
Borntrgers test. 35.7 mL of 20% ammonia will be diluted with
enough distilled water to make a total volume of 100 mL. An
equivalent of 1 g plant material will be taken from the stock plant
extract and will be evaporated to incipient dryness over a steam
bath. The residue will be taken up with 10 mL distilled water and
will be filtered; residue will then be discarded. The aqueous
filtrate will be extracted twice with 5 mL portions of benzene,
combining the benzene extracts. The combined benzene extracts
will be divided into two portions. One portion will be reserved as
the control. The other portion will be treated with 5 mL ammonia
solution will be shaken. It will then be compared with the control
tube. A red coloration in thje lower ammoniacal layer indicates
the presence of anthraquinones (Guevara et al., 2005).

Test for Flavonoids. The presence of flavonoids will be tested

by dissolving 2 mL of the extract in diluted NaOH, followed by
the addition of HCl. A yellow to orange solution with NaOH that
turned colorless upon addition of HCl denotes the presence of
flavonoids (Chichioco-Hernandez, Paguigan and Ramiro, 2010).

Test for Polyphenols. Screening method for polyphenols by dry

distillation will be tested by placing about 2 g of the finely cut
plant samples at the bottom of a test tube fixed for a support.
The mouth of the tube will be covered with a disk of air-dried
filter paper impregnated with 2,6-dichloroquinone-4-chloroimine
in benzene. The bottom of the test tube will be heated for 30
minutes. The paper filter disk will be removed from the mouth of
the test tube, air cooled and the paper disk will be held over a
bottle of strong ammonia. The result will be compared with a
standard, a pinch of ellagic acid will be placed in a test tube and
will be treated similarly.
Test paper disk for polyphenols. A piece of filter paper
1x1 will be cut. The paper disk will be completely wet with a
solution of 50% (w/v) 2,6-dichloroquiinone-4-chloroimine in
benzene. For positive result, a brown-violet spot will appear on

the disk which gradually fades on standing. Color is restored by a

renewed exposure to ammonia vapors (Guevara et al., 2005).

Test for Saponins. The presence of saponin will be tested by

boiling 5 mL of the extract in 5 mL distilled water. Cool the
mixture then shake vigorously. Frothing would indicate the
presence

of

saponins

(Chichioco-Hernandez,

Paguigan

and

Ramiro, 2010).

Test for Tannins. To test for tannins, about 2 mL of the extract

will be dissolved in 5 mL distilled water followed by dropwise
addition of 15% FeCl3 solution. Formation of a blue-black
precipitate indicates the presence of hydrolysable tannins while
brownish-green precipitate indicates the presence of condensed
tannins (Chichioco-Hernandez, Paguigan and Ramiro, 2010).

Test for Terpenoids. The presence of terpenoids will be tested

by treating 2 mL of the extract with 2 mL CHCl3 followed by
layering with H2SO4. The formation of a reddish brown coloration
in the interface indicates the presence of terpenoids (ChichiocoHernandez, Paguigan and Ramiro, 2010).

Brine Shrimp Lethality Test (BSLT). Brine shrimp lethality

assay will be performed by preparing artificial sea water through
dissolving a 3.8 g of rock salt per 100 mL distilled water
(Guevara et al., 2005). Brine shrimps (Artemia salina Leach) will
be hatched using brine shrimp eggs in a glass tank with two
unequal-sized

compartments

filled

with

brine

water.

One

compartment of the tank will be covered with aluminum foil and

will be fully aerated. The airstone will carefully be place at the
bottom of the tank to ensure sufficient aeration of the eggs. The
brine shrimp eggs will be incubated for approximately 24 hours
at room temperature and will be illuminated. Subsequently,
hatched active nauplii will be attracted to the other side of the
tank with a light source and will be collected using a Pasteur
pipette then consequently it will be used for the assay.
Percentage

mortality

at

each

test

solution

was

determined using the following formula: % mortality = (no. of

dead nauplii / initial no. of live nauplii) X 100 (Del Socorro, Bendoy &
Dacayana, 2014).