Chapter I

The Problem and Its Settings

This chapter introduces the problem of the study. And explains the nature of the study.
Background of the Study
Herbal medicines are the use of herbal plants to improve ones body or wellbeing.
According to a study of the ResearchGate.net, in the year 2014, almost 80% of the countries
across the world are using herbal plants as alternative for medical drugs. The Philippines are
one of those countries. Because of the rising prices of medical drugs in the country, more than
100 million Filipinos are pushed into poverty every year just because of pricey medical drugs.
High-priced drugs are sometimes the reason why bacterial diseases such as meningitis, ulcer,
gastritis and pneumonia are not treated and leads to death. Under the Republic Act (RA) 8423
or Traditional and Alternative Medicine Act of 2007 by the former Sec. Juan M. Clavier states
that the use of plants as alternative medicine is encouraged by the Philippine Government. The
Philippines Institute of Traditional and Alternative Health Care (PITAHC) was created to
promote and advocate the use of traditional, alternative, preventive and curative health care
modalities that have been proven safe, effective, cost-effective and consistent with government
standards on medical practice.
There are over 40 ethnic groups present in the Philippines. And 18 of those tribes are
in Mindanao. They can be classified as Tribal groups and Indigenous groups. The Blaan
are the major indigenous groups in Mindanao. They believe in their God or what they call
Dawata. Blaan have rituals for everything- including their medicines. They believe that all of
the plants on earth are planted by Dawata and these plants will help them to cure such illnesses
that will make them healthier. But they are not aware of what specific plant are they going to
use and what are the good or bad effects to them.
Plant-based natural constituents can be derived from any part of the plant like bark,
leaves, flowers, roots, fruits, seeds, etc i.e. any part of the plant may contain active components.
Tagisang bayauak (Ficus variegata blume) is a native plant which commonly grows on
primary forests and places with low and medium altitudes. It is a tall, spreading plant with pale
bark, broad leaves and fruits that grows or attached on its bark where the fruit are on its twigs.
The latex of its bark is commonly used in treating external wounds together with its leaves.
Native people in this area call by many names according to their tribe. Badyang (Alocasia
macorrhiza) are commonly found in Tboli, South Cotabato, although this plant can be seen
everywhere and is valued an ornamental. This is sometimes referred to as Elephant ears and
Giant taro because of its upright and large leaves standing straight up pointing skyward.
According to folktales, the plants stems is edible and are eaten as vegetable during scarce
times. Galinsoga (Galinsoga parviflora) is a forb and an abundant seed-producing plant with
hairy leaves and stem. The stem of this plant is erect, branched, grows up to 60 cm and is
slightly hairy. The leaves of this plant is slightly hairy, grows up to 6cm long and 4m wide
simple and ovate.
Statement of the Problem
This study aims to identify the Antibacterial activity of Tangisan bayuak (Ficus
variegata blume), Badyang (Alocasia macrorrhiza) and Galinsoga (Galinsoga parviflora)
extracts on the growth of Staphylococcus aureus. More specifically the study aims to answer
to following problem:

1. What is the level of antibacterial activity using the mean zone of inhibition of
Tagisang bayauak (Ficus variegata blume), Badyang (Alocasia macorrhiza) and
Galinsoga (Galinsoga farviflora) plants extract against the reference strain S. aureus?
2. What is the level of efficacy of the antibacterial activity using the mean zone of
inhibition of Tagisang bayauak (Ficus variegata blume), Badyang (Alocasia
macrorrhiza) ointment and Galinsoga (Galinsoga farviflora) against the reference
strain S. aureus?

Hypotheses
This study will be conducted to prove the following hypotheses:
Null
1. The zone of inhibition of Tagisang bayauak (Ficus variegata blume), Badyang (Alocasia
macrorrhiza) and Galinsoga (Galinsoga farviflora) plants extract are the same with the
positive control, Mupirocin.
2. The mean zone of inhibition of Tagisang bayauak (Ficus variegata blume), Badyang
(Alocasia macrorrhiza) and Galinsoga (Galinsoga parviflora) is the same with the positive
control, Mupirocin.

Alternative
1. The zone of inhibition of Tagisang bayauak (Ficus variegata blume), Badyang
(Alocasia macrorrhiza) and Galinsoga (Galinsoga farviflora) plants extract are greater
than the positive control, Mupirocin.
2. The mean zone inhibition of the positive control is lesser than the mean zone of inhibition
of the Tagisang bayauak (Ficus variegata blume), Badyang (Alocasia macrorrhiza) and
Galinsoga (Galinsoga parviflora).
.
Significance of the Study
According to a study of Centers for Disease Control and Prevention, allmost 91% of
the deaths in the year 2013 are caused by bacterial diseases which were untreated because of
poverty. The use of medicinal plant extract for the treatment of human diseases is ancient
practice. Folktales and beliefs that herbal plants found in the community which can be used as
medicines are widespread in rural places and in tribes like Blaan.
The results of this study is useful the Blaan people in understanding why certain plants
like Tangisang bayawak, Badyang and Galinsoga, which are present in their community, can
heal their illnesses. It gives them scientific explanations to support their beliefs. It also creates
a spot for the Blaan people to discover plants in their community which can aid them in their
illnesses.
This study created an option to those people who cant afford medicines prescribed by
their doctors for they can substitute these safe herbal plants as medicines as they can use this
study as a basis in creating alternative medicines.
Lastly, this study is an addition to the list of the usage of natural resources, aiding the
government in lowering the high percentage of incurable bacterial diseases due to poverty. The
government can now provide medicines which the populace can afford.
Scope and Limitation
This study will focus on the effectiveness of Tagisang bayawak (Ficus variegata
blume), Badyang (Alocasia macorrhiza), Galinsoga (Galinsoga farviflora) in inhibiting the
growth of bacteria, on the testing of the bioactive compunds present in the plant and the
antioxidants present in the plants. This study will only test the effectivity of Tagisang bayawak
(Ficus variegata blume, Badyang (Alocasia macorrhiza), Galinsoga (Galinsoga farviflora) to
lessen the growth of Staphylococcus aureus-.This study also focuses on the capacity of the
Blaan plants extract in the ointment formulation.
Operational Definition of Terms
The following terms are define to provide clarity and better understanding of the words
frequently used in the following pages.
Antibacterial- refers to the ability of the selected Blaan plants extract to suppress or inhibit
the growth of harmful bacteria through the measurement of the mean zone of inhibitions.
Antioxidant- a substance that inhibits oxidation or reactions promoted by oxygen, peroxides,
or free radicals.
Ascorbic Acid- the standard used in the reducing property test to determine the reducing
property of the selected Blaan plants.
Badyang (Alocasia macrorrhiza)- one of the selected Blaan plants in South Cotabato that are
manipulated in the study.
Bioassay- refers to the determination of the strength of a drug or biological product by testing
its effect on living organisms using aseptic techniques.
Galinsoga (Galinsoga farviflora)- one of the selected Blaan plants in South Cotabato that are
manipulated in the study.
Staphylococcus aureus- common gram-positive bacteria which will be used to test the
antibacterial property of the extract.
Tagisang bayuak (Ficus variegata blume)- one of the selected Blaan plants extract in South
Cotabato that are manipulated in the study.
Zone of inhibition- it is the area to be measured in order to know the potential of the plant,
Tagisan bayauak to inhibit the growth of a bacteria.
 Chapter II
Review of Related Literature
This chapter presents the literature taken from various works and researchers which are
significant in conducting this study.
Tagisang Bayauak
Ficus variegate blume is a tall spreading tree with pale bark. It commonly grows in primary
forests and can also grow in countries in secondary forests. It is also widely distributed in moist
areas of low to medium elevations like forest, forest thickets, farmlets, along gullies and
rivebanks. The leaves of this plant are broad or elliptic-ovate, that measure 10 to 17 centimeters
long and are yellowish-brownish. Its receptacles are about 1 cm in diameter and are clustered
on long branches. The wood are known to be used in temporary construction, mouldings,
interior works and etc. It is known or believed to treat external wounds using its boiled leaves
extract. People sometimes use this plant as decoration. Their young shoot-tips and young fruits
are sometimes eaten raw. (http://www.stuartxchange.org/Agi.html).
Badyang
Badyang (Alocasia macrorrhiza) or sometimes called Giant Taro is a large evergreen
plant with extremely large erect or spreading leaves; cultivated widely in tropics for its edible
rhizome and shoot and are used in wet warm regions as a stately ornamental. Any plant of the
genus Alocasia has large showy basal leaves and boat-shaped spathe and reddish leaves. This
plant is commonly used in rituals not only by the Blaan people but also of the Folk-Healers of
Siquijor. This plant is involved in the minasa ritual where it is believed to further bestow
potency to the brew. (Fuentes, Evelyn & Mascunaa, Rolando V., 2004).
Galinsoga (Galinsoga parviflora)
Galinsoga parviflora is an herbaceous plant in the Asteraceae (daisy) family. It is a herb
and a leafy vegetable used in some cuisines in Colombia and on East African countries.
Galinsoga has medicinal benefits yet it still considered as noxious weed (Cultural History of
Plants, 2005). The leaf juices of the plant is used as astringent for stings, cuts, wounds (Invasive
Alien Plants: An Ecological Appraisal for the Indian Subcontinent, 2012).
Galinsoga parviflora is a forb and an abundant seed-producing summer annual with
hairy leaves and stems. At seedling stage, it produces club-shaped cotyledons with slightly
indented tip. The stem below the cotyledon (hypocotyl) is very short, green, becoming maroon
with age. Young leaves are opposite, triangular with slightly toothed margins, and covered with
hairs. It has a shallow fibrous root system. The stem is erect, branched, grows up to 60cm high
and is slightly hairy. Leaves are opposite, up to 6cm long and 4cm wide, simple, ovate and
slightly hairy. Three distinct veins mark the leaf, converging at the base and the margin is
shallowly toothed. The inflorescence is flower heads 5-8mm in diameter on stalks 12-25mm
long. The leafy inflorescence is regularly branched at the stem apex and from upper leaf axils.
Flower heads consist of many yellow tubular florets, and 4-5 white 3-lobed ray florets
surrounded by membranous bracts. Fruit is an achene and propagation is by seed. (http://agron-
www.agron.iastate.edu/~weeds/weedbiollibrary/517%20student%20pages/2000/Galinsogad..
html).
Anti-bacterial
Antibacterials are topical agents that eliminate the bacteria that causes epidermal
infections. The antibiotic neomycin is frequently used in ointments for this purpose. (Allan,
David M., Buchman, Michelle A. & Lockyer, Karen D., 2003).
The basics of bacterial structure describing the basis for your understanding
mechanisms of antibacterial action. Antibacterial may refer to anything that destroys bacteria
or suppresses their growth or ability to reproduce. It covers the characteristic features of
bacterial pathogenicity, the genetic basis of resistance to anti-bacterial drugs, the biochemical
mechanisms of action of antibacterial drugs, how antibacterial drugs reach their targets in
gram-positive 3and gram-negative bacteria, and the wide range of human immune responses
against bacterial infection. Recent advances in research and development of new classes of
antibacterial drugs are examined. (Khakdan, Fatemeh & Khosro, Piri, 2013).
Ointment
For the past years, man has applied preparations to soothe and medicate the skin.
Creams, plasters and ointments were one of those. Ointments are thick liquid substance that
creates a soothing effect when applied to the skin. It is used especially when a person has burns,
insect bites and etc. Ointments are prepared in small preparations and are made to fill the
doctors prescribed formula.
The efficacy of different ointments have been proved through breakthroughs of
different studies. These ointments has different basis with different penetrations. According to
Ernest A. Strakoschs study, Penetration of Various Ointments Bases, ointments were revealed
to have different penetration with different bases. The relative intensity of the penetration into
the normal human skin of the different test substances listed in the order from the best to the
worst penetration, as revealed by this study is as follows: the base consisting of: liquid
petrolatum-peanut oil-steric acid-triethanolamine-acetyl alcohol and water; "Hydrosorb"
(Abbott), "Aquaphor" (Dule), lard, cold liver oil, stearyl alcohol-mineral oil-water-petrolatum,
lanolin, a base consisting of mannide monooleate-ceresin wax-petrolatum-mineral oil-lanolin,
lecithin ointment, petrolatum and lanolin to equal parts, petrolatum plus five per cent cetyl
alcohol, olive oil, rose water ointment, vaseline (Chesebrough), and finally petrolatum as
such. Because of multi-drug resistance among many bacterial species has been reported to be
on the increase due to inappropriate or widespread use of antimicrobials. Resistance to
antimicrobials has been observed in various parts of the world. Because of this, Alternative
ointments were also introduced in the modern years. Ointments formulated from herbal plants
with bioactive compounds were proven safe and effective. Herbal ointments were also
introduced to the market. (http://jpet.aspetjournals.org/content/78/1/65.abstract).
Extracts from different plant extracts like Aloe vera, Ficus infectoria, Ficus religiosa,
and Piper betel were studied for antibacterial activity on resistant and sensitive strains, isolated
from skin and soft tissue infections. The combined extract was formulated in different ointment
bases such as polyethylene glycol, gelatin, sodium alginate, carbopol, cream base and honey.
These were then evaluated to find a suitable base for preparation of an ointment. In vitro study
of the release of antimicrobials and kill-time studies of the herbal ointments was carried out
against multi-drug resistant isolate of Pseudomonas. The ointment showed bactericidal activity
within 2 h against the resistant strain of Pseudomonas spp.
(http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3003181/).
Staphylococcus Aureus
S. Aureus was first identified in 1880 by the surgeon Sir Alexander Ogston in pus from
a surgical abscess in a knee joint. It is estimated that 20% of the human population are long-
term carriers of S. aureus which can be found as part of the normal skin flora and in anterior
nares of the nasal passages. It is still one of the five most common causes of nosocomial
infections and is often the cause of postsurgical wound infections. Many patients in the hospital
are contracted to staphylococcal infection.
(http://en.wikipedia.org/wiki/Staphylococcus_aureus)
Staphylococcus aureus, the gold pigmented form, is a durable organism found on the
mucous membranes and skin of a considerable segment of the population. It produces infection
of the skin, but it is also noted for causing a variety of serious infections, particularly in the
debilitated. There is extensive information about the products, metabolites, enzymes, extracts,
structure and antigen of most cultivatable microorganisms. The knowledge about these
substances is used to identify the microorganisms, to interpret their virulence, to explain their
metabolic activities, and to control the microorganisms. However, due to the presence of
extrachromosomal genetic units called plasmids, antibiotic resistance in S. aureus is created.
Persistent use of an antibiotic favors the continued presence of the specific antibiotic resistance
plasmid in the bacterial population. In staphylococcal treatment it has been recommended that
the use of tropical antibiotics be eliminated. The presence of antibiotics at the carriage sites of
man only increases the probability of selection of resistant strains. Another involvement of
staphylococci is in the most prevalent type of food poisoning. Food preparers and handlers who
have staphylococcal lessions of the skin, especially on the hands are most likely to contaminate
the food. (Boyd, Robert F. & Hoerl, Ryan G., 2011).
Theoretical and Conceptual Framework
The concept of the research is a comparative study where the process is based upon
comparison of two independent variables with its effect to the dependent variable. The
independent variables, which are the Tagisan bayauak (Ficus variegate blume), Galinsoga
(Galinsoga parviflora) and Badyang (Alocasia macrorrhiza) leaves extract, are the ones to be
tested whether or not it will affect the growth of Staphylococcus aureus.

INPUT PROCESS OUTPUT

INDEPENDENT
DEPENDENT VARIABLE
VARIABLES
Zones of Inhibition
Tagisang bayauak
EXTRACTION in culture of
Galinsoga Staphylococcus
aureus
Badyang

CONTROL
Mupirocin.

Fig. 1 Diagram of Theoretical and Conceptual Framework

 Chapter III
Methodology
Research Site and Duration
This study will follow a simple research design. It assessed the selected Blaan plants
to show its properties through bioassay. The experiment will take place in the pharmacy lab of
General Santos Doctors' Medical School Foundation Inc., wherein the research instruments and
materials used for the experiment are already complete and provided by the school itself
Research Design
This study will employ a descriptive-quantitative design for assessing the antibacterial
properties of Tagisan bayauak (Ficus variegate blume), Galinsoga (Galinsoga parviflora) and
Badyang (Alocasia macrorrhiza) leaves extract and its efficacy against the growth of S.
Aureus.

Blaan plants extract

Bioassay Ointment

T1
S. Aureus

Zone of Inhibition

T2 R2
Scavenging Activity
R3

R1
T3 R2 Legend:
R3 T Treatment

R1 R Replicate

T4 R2 T1 Positive Control (Mupirocin)

R3 T2 Negative Control (Water)

R1 T3 50% concentration of Selected Blaan

T5 plants extract
R2
T4 75% concentration of Selected Blaan
R3 plants extract
T5 100% concentration of Selected Blaan
plants extract
A. Procedure for Bioassay

Blaan Plants Extract

Antibacterial Screening

Reference strains
S. Aureus

Preparation of Inoculum

Preparation of Plate

Preparation of Extract disks

Reading of Zone inhibition

Materials and Instruments
Table 1
List of Raw Materials and Laboratory Equipment for Pure Extract Extraction and Bioassay.
Pure Leaves Extract Extraction Bioassay

Raw Materials Agar medium

Tagisan bayauak plant Nutrient agar
Galinsoga plant S. aureus
Mani-manian plant Laboratory equipment
Laboratory Equipment Autoclave
Weighing scale Petri dishes
Scissors Beaker
Petri dishes Erlenmeyer flasks
Graduated cylinder (500 mL) Forceps
Volumetric flasks Incubator
Beaker Whattman discs
Stirring rod Vernier caliper
Funnel Screw capped test tube
Spatula/Ladle Test tube stand
Erlenmeyer flasks Sterile cotton swabs
Extractor setup Vortex mixer
Rotary evaporator Micropipettor
Filter paper
Solvents
Distilled water
95% ethanol

Experimentation and General Procedure

A. Preparation of Plants Leaves Extract
Tagisang bayuak (Ficus variegata blume), Badyang (Alocasia macorrhiza) and
Galinsoga (Galinsoga Farviflora) plant will be collected at South Cotabato. Five hundred fifty
grams (550 g) of freshly washed Tagisan bayauaks pericarp and Badyang and Galinsoga
leaves will be completely soaked in 1.5 L of 95% ethyl alcohol for 48 hours and will be filtered.
The crude ethanolic extract will then be concentrated by using a rotary evaporator at
temperature 60, resulting to a thick, viscous and syrupy extract which was later used for the
tests involved in the study.
B. Procedure for Bioassay: Antibacterial Activity of Tagisan bayauak (Ficus
variegate blume) Badyang (Alocasia macorrhiza) and Galinsoga (Galinsoga
Farviflora) Extract Against the Growth of Staphylococcus aureus

Preparation of Inoculum
Colonies of Staphylococcus aureus will be inoculated onto 2 different 5 mL Mueller-
Hinton broth (MHB) and incubated at 35C for 2 hours to allow bacteria to grow. It will be
adjusted using 0.5 McFarland standard solution with sterile Normal Saline Solution (NSS).
These will serve as the standardized inoculum. (Guevara et al, 2005)
Preparation of Seeded Agar Plate
Twenty (20) mL of Mueller-Hinton Agar (MHA) will be dispensed into sterile petri
dishes to a depth of 4 mm and allowed to cool and solidify. A sterile cotton swab was dipped
onto the standardized organism inoculums suspension, pressed and rotated firmly against the
inside wall of the tube just above the fluid to remove the excess liquid. The entire surface of
the solidified agar will be swabbed evenly to ensure even and complete distribution of the
inoculum. The plates will be allowed to dry for 15 minutes. This preparation will be done in
the biosafety cabinet. (Guevara et al., 2005)
Position of Paper Disks for Preliminary Screening of Antibacterial Activity
Twenty microliters (20 L) of each leaves extract was dispensed into sterile paper disks
with the use of a pipette. The impregnated paper disks was air-dried under for two hours the
biosafety hood. Using sterile forceps, the impregnated paper disks will be placed in a clockwise
order onto the seeded plate at equidistant positions. Tetracycline disk and distilled water was
used as positive and negative controls, respectively. The drugs was allowed to diffuse through
the agar for 60 minutes. The plates was inverted and incubated for 24 hours at 350C. The
diameter of the zone of inhibition was measured in millimeter (mm) using a ruler. The assay
will be done in triplicate and the means will be computed.
Figure 8. Position of Paper Disks for Preliminary Screening as Antibacterial Agent

Legend:
1, 2, 3 - replicates
2

1 3