CHAPTER I

INTRODUCTION

BACKGROUND OF THE STUDY

The entire human population is overwhelmed with an incalculable number of
annual hospital cases. Most of them are caused by sickness causing bacteria that are
widespread, hovering around just waiting for its next victim.
An example of pathogenic bacteria is Escherichia coli. There are many types of
E. coli but most of them are harmless. But some can cause bloody diarrhea called
enterohemorrhagic E. coli (EHEC). Some species of E. coli may also cause severe
anemia and kidney failure that may result to death.
Staphylococcus aureus is another bacterium that is frequently found in the human
respiratory tract and on the skin. It is a common cause of skin infections (boils),
respiratory diseases (sinusitis), and life-threatening diseases such as pneumonia and
meningitis. It is estimated that 20% of the population are lifelong carriers of S. aureus.
Another sickness causing bacteria is Staphylococcus haemolyticus which accounts
for urinary tract infection,
In this connection, this study is being conducted by the researcher not only as a
part of the required curriculum and to support the research study utilizing the plants God
has given us, but also to satisfy her interest in determining the prophylactic property of
Calabur against the mentioned pathogenic bacteria. In the course of the study, the
researcher aims to gain a broader knowledge about the plant not only for her own benefit,
but also to be a source of information to other people.

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STATEMENT OF THE PROBLEM

This study aimed to determine the prophylactic property of Calabur extract on
selected pathogenic bacteria Staphylococcus haemolyticus, Staphylococcus aureus and
Escherichia coli. Specifically, it sought to answer the following questions:
a) Does Calabur (Muntingia calabura) exhibit a prophylactic property based on the
zone of inhibition of the selected pathogenic bacteria (Staphylococcus
haemolyticus, Staphylococcus aureus and Escherichia coli)?
b) Is there a significant difference between the effect of Calabur (Muntingia
calabura) leaves extract on the selected pathogenic bacteria (Staphylococcus
haemolyticus, Staphylococcus aureus and Escherichia coli) compared to the
effect of the standard?
c) Among the selected pathogenic bacteria, on which of the following is Calabur
extract most effective based on their zones of inhibition?

STATEMENT OF HYPOTHESIS
The hypothesis is tested at 0.1 level of significance.
Ho Calabur (Muntingia calabura) does not exhibit a prophylactic property based on the
zone of inhibition of the selected pathogenic bacteria (Staphylococcus
haemolyticus, Staphylococcus aureus and Escherichia coli).
Ho There is no significant difference between the effect of Calabur (Muntingia
calabura) leaves extract on the selected pathogenic bacteria (Staphylococcus
haemolyticus, Staphylococcus aureus and Escherichia coli) compared with the
effect of the standard.

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SIGNIFICANCE OF THE STUDY

This study aimed to determine whether Calabur extract exhibits a prophylactic
property on selected pathogenic bacteria (Staphylococcus haemolyticus, Staphylococcus
aureus and Escherichia coli). If proven, Calabur extract can be used to prevent the spread
and reproduction of the pathogenic bacteria and could benefit a wide-range of people
especially microbiologists, makers of medicine and consumers. It can also provide a
backbone for further studies about Muntingia calabura, its properties and other uses.

SCOPE AND DELIMITATIONS

This study started on January 2013 and was completed on July 2013 at the
Dagupan City National High School, at the researchers residence and at the University
of Pangasinan, Dagupan City Medical Laboratory Science Department.
The pathogenic bacteria Staphylococcus aureus, Escherichia coli, and
Staphylococcus haemolyticus used in the study were obtained and cultured at the
University of Pangasinan Medical Laboratory Science. This study aimed to determine
the prophylactic property of Calabur (Muntingia calabura Linn) extract on selected
pathogenic bacteria. The study covers the investigation of the prophylactic property of
Calabur on common pathogenic bacteria Staphylococcus haemolyticus, Staphylococcus
aureus and Escherichia coli. Determining the chemical component of the plant which
exhibits the prophylactic property is not covered by the study because of the availability
of facilities and cost of experimentation. Effect of Calabur on other pathogenic bacteria
are not covered in the study but is included in the recommendations page for future
studies.

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DEFINITION OF TERMS

Calabur a small tree native to the Philippines which is about 7-12 meters tall with
tiered and slightly drooping branches. It has serrated leaves 2.515 cm long and 1
6.5 cm wide.

Escherichia coli is a Gram-negative, facultative anaerobic, rod-shaped bacterium

that is commonly found in the lower intestines of warm-blooded organisms.

Pathogenic bacteria bacteria that cause diseases to humans, animals or even plants.
Some pathogenic bacteria can cause one particular host to be ill; others cause trouble
in a number of hosts; it depends on the host specificity of the bacteria.

Prophylactic acting to defend against or prevent something especially diseases or

the spread of bacteria.

Prophylaxis something that defends against or prevent diseases or the spread of

bacteria.

Staphylococcus

haemolyticus

is

non-motile, non-sporulating, facultatively

anaerobic, coagulase-negative and Gram-stain positive. Cells are typically coccusshaped and range from 0.8-1.3 m in diameter.

Staphylococcus aureus is a Gram-positive spherical bacterium that occur in

microscopic clusters which is the cause of diseases such as Pneumonia, Meningitis
and Toxic shock syndrome (TSS).

Zone of Inhibition It is the area on an agar plate where growth of a control organism
is prevented by an antibiotic usually placed on the agar surface. If the test organism is
susceptible to the antibiotic, it will not grow where the antibiotic is.

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CHAPTER II
REVIEW OF LITERATURE AND STUDIES

This chapter presents the literature related to the study of Calabur (Muntingia
calabura Linn). It includes a number of reference materials which are critical to the
study.

RELATED LITERATURE

General Description of the Plant

Common name:

Calabur

Scientific name:

Muntingia calabura

Family:
Other names:

Eleocarpaceae
Aratiles (Tagalog)
Cherry tree, Panama berry (English)

Botanical Description
Calabur is a fast growing tree, 5 to 10 meters high, with spreading branches.
Leaves are hairy, sticky, alternate, distichous, oblong-ovate to broadly oblong-lanceolate,
8 to 13 centimeters long, with toothed margins, pointed apex and inequilateral base, one
side rounded and the other acute. Flowers are about 2 centimeters in diameter, white,
extra-axillary, solitary or in pairs. Sepals are 5, green, reflexed, lanceolate, about 1
centimeter long. Petals are white, obovate, 1 centimeter long, deciduous and spreading.
Fruit is a berry, rounded, about 1.5 centimeter in diameter, red on ripening, smooth,
fleshy, sweet and many seeded.

(www.stuartxchange.com, Philippine Medicinal Plants)

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Pathogenic Bacteria
Staphylococcus haemolyticus is a coagulase-negative member of the genus
Staphylococcus. The bacteria can be found on normal human skin flora and can be
isolated from axillae, perineum, and ingunial areas of humans. S. haemolyticus is also the
second most common coagulase-negative staphylococci presenting in human blood.
Lacking coagulase, an enzyme-like protein that was traditionally associated with virulent
potential of staphylococci, coagulase-negative staphylococci are usually considered lowvirulent

pathogens

comparing

to

the

well-known

pathogenic

coagulase-

positive Staphylococcus aureus. However, recent studies indicate that coagulase-negative

staphylococci have emerged as a major cause of opportunistic infection. Staphylococcus
haemolyticus itself is also a remarkable opportunistic baterial pathogen that is wellknown for its highly antibiotic-resistant phenotype. The bacteria can cause meningitis,
skin or soft tissue infections, prosthetic join infections, or bacteremia. Common
antibiotics that are subject to resistance in S. haemolyticus include methicillin,
gentamycin, erythormycin, and uniquely among staphylococci, glycopeptide antibiotics.
Escherichia coli is the head of the large bacterial family, Enterobacteriaceae,
the enteric bacteria, which are facultatively anaerobic Gram-negative rods that live in the
intestinal tracts of animals in health and disease. The Enterobacteriaceae are among the
most important bacteria medically. A number of genera within the family are human
intestinal pathogens (e.g. Salmonella, Shigella, Yersinia). Several others are normal
colonists of the human gastrointestinal tract (e.g.Escherichia, Enterobacter, Klebsiella),
but these bacteria, as well, may occasionally be associated with diseases of humans.
Physiologically, E. coli is versatile and well-adapted to its characteristic habitats. E.

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coli is a consistent inhabitant of the human intestinal tract, and it is thepredominant

facultative organism in the human GI tract; however, it makes up a very small proportion
of the total bacterial content.
Staphylococcus aureus is a bacterium that is a member of the Firmicutes, and is
frequently found in the human respiratory tract and on the skin. Although S. aureus is not
always pathogenic, it is a common cause of skin infections (boils), respiratory disease
(sinusitis), and food poisoning. Disease-associated strains often promote infections by
producing potent protein toxins, and expressing cell-surface proteins that bind and
inactivate antibodies. The emergence of antibiotic-resistant forms of pathogenic S.
aureus (e.g. MRSA) is a worldwide problem in clinical medicine. S. aureus is responsible
for many infections but it may also occur as a commensal. The presence of S. aureus does
not always indicate infection. S. aureus can survive from hours to weeks, or even months,
on dry environmental surfaces, depending on strain.

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RELATED STUDIES

Ricky Raguindins study entitled, Synergistic effect of Jackfruit fruit and

Chrysanthemum flower extract on selected pathogens provided an experimental
framework on how the extraction of plant material and testing of the product on the
pathogens is to be done. The abstract of his study is included below:
The study entitled, Synergistic effect of Jackfruit (Artocarpusheterophyllus)fruit
and Chrysanthemum (Chrysanthemum morifolium) flower extract on selected pathogens
was conducted to determine if there is a synergistic effect of Jackfruit fruit and
Chrysanthemum flowers on Klebsiella pneumoniae, Staphylococcus aureus and
Escherichia coli. Specifically, the study aimed to determine the effect of Jackfruit
(Artocarpus heterophyllus) fruit and Chrysanthemum (Chrysanthemum morifolium)
flower extract on the means of zone inhibition of the selected pathogens.
The collected freeze-dried unripe Jackfruit fruit and Chrysanthemum flowers
were gathered and were subjected to maceration in 100ml of 70% ethyl alcohol for 24
hours with ratio 1:1. Then, the separated extracts were subjected for water bath until 25%
(1/4) of the obtained extracts evaporated. The treatment was subjected to Antimicrobial
Sensitivity Test.
Results show that each of the selected pathogens (Klebsiella pneumoniae,
Staphylococcus aureus and Escherichia coli) are moderately sensitive to the treatment
since there is 7 mm, 7 mm and 8.5 mm zone of inhibition displayed by the bacteria,
respectively. Moreover, the antimicrobial index of Jackfruit fruit and Chrysanthemum
flowers is near the standard antibiotic (0.7) for the Staphylococcus haemolyticus. Also,

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results show that Jackfruit fruit and Chrysanthemum flowers have moderate synergistic
effect on each of the selected pathogens (Klebsiella pneumoniae, Staphylococcus aureus
and Escherichia coli).
Based from the results, the researcher have concluded that Jackfruit fruit and
Chrysanthemum flowers extract possess moderate synergistic effect on each of the
selected pathogens (Klebsiella pneumoniae, Staphylococcus aureus and Escherichia
coli).

Synergistic effect of Jackfruit fruit and Chrysanthemum flower extract on selected

pathogens; Ricky Raguindin, Dagupan City National High School, 2012
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RELATED READINGS
This chapter includes laws, resolutions and acts of the Philippine Constitution
involving that of the research objective.

Republic Act No. 9502

An Act Providing for Cheaper and Quality Medicines, Amending for the Purpose of
R.A. 8293 or the Intellectual Property Code, R.A. 6675 or the Generics Act of 1988, and
R.A. 5921 or the Pharmacy Law, and for other purposes

Section 1. Short Title. - This Act shall be known as the "Universally Accessible Cheaper
and Quality Medicines Act of 2008".

Section 2. Declaration of Policy. - It is the policy of the State to protect public health and,
when the public interest or circumstances of extreme urgency so require, it shall adopt
appropriate measures to promote and ensure access to affordable quality drugs and
medicines for all.

Section 3. Construction in Favor of Protection of Public Health. - All doubts in the

implementation and interpretation of the provisions of this Act, including its
implementing rules and regulations, shall be resolved in favor of protecting public health.

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Section 4. Definition of Terms. - For purposes of this Act, the following terms are to
mean as follows:
(a) "Compulsory License" is a license issued by the Director General of the
Intellectual Property Office to exploit a patented invention without the permission
of the patent holder, either by manufacture or through parallel importation;
(b) "Drug outlet" refers to drugstores, pharmacies, and any other business
establishments which sell drugs and medicines;
(c) "Drugs and medicines" refers to any chemical compound or biological
substance, other than food, intended for use in the treatment, prevention or
diagnosis of disease in humans or animals.

Approved on June 6, 2008

(Source: www.lawphil.net)

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RELEVANCE OF THE STUDY

This study entitled, The Prophylactic Property of Calabur (Muntingia calabura
Linn) Extract on Selected Pathogenic Bacteria aimed to determine if Calabur extract
exhibits a prophylactic property on selected pathogenic bacteria (Staphylococcus
haemolyticus, Staphylococcus aureus and Escherichia coli) on which the test is
administered on. It also aimed to determine the effect of Calabur extract on some
pathogenic bacteria.
The concepts used in Raguindins previous study influenced the
methodology of this study especially in the extraction and antimicrobial sensitivity
testing. But unlike the present study, Raguindins study focused on the combined
antibacterial ability of Jackfruit and Chrysanthemum on the selected pathogens.
If this study would be approved, it would be a great help to our community,
especially to the world of pharmacy and microbiology. The approval and wideacceptance of this project would also provide a base for future studies about Calabur, its
properties and other uses.

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CHAPTER III
METHODOLOGY

This chapter presents the research methods and procedures used. The study made
used experimental research approach and the actual results to be interpreted. The actual
experiment followed strict laboratory safety procedures throughout the conduct of the
study.
I. Materials and Equipment
Plant Collection and Extraction

Calabur leaves

vial

beaker

graduated cylinder

funnel

weighing scale

ethanol

Preparation of Culture Media and Sensitivity Test

Staphylococcus aureus

standard (commercial antibiotic)

Escherichia coli

petri dish

Staphylococcus haemolyticus

alcohol lamp

Mueller Hinton Agar plates and Nutrient Broth (NB)

forceps

cotton swabs

inoculating loop
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Gathering of Laboratory Equipment and Collection of Plant

Material

Extraction of Plant Material

Collection and Preparation of Culture Media

Sterilization of Materials

Subculture of Pathogenic Bacteria and Incorporation in Nutrient

Broth

Antimicrobial Sensitivity Test

Computation of Antimicrobial Indices of the Extract on the

Pathogenic Bacteria

Interpretation of Results

Summary of Findings and Conclusion

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II. Extraction of Plant Material

The plant extract was obtained by maceration method. The Calabur leaves was
cut into smaller portions, measured to 100g using the weighing scale and mixed with
the ethanol in the glass bowl. The botanical material was soaked in ethanol. The plant
extract was obtained by separating the botanical material using a white cloth. Then,
the mixture was boiled until the ethanol has evaporated. The obtained extracts was
put into a vial and is ready for testing.

III. Collection of Test Organisms

The test organisms: Staphylococcus haemolyticus, Staphylococcus aureus and
Escherichia coli were obtained and cultured at the medical laboratory of University of
Pangasinan PHINMA.
First, the inoculating loop and the opening of the screw cap tube containing
the nutrient broth was flame-sterilized using alcohol lamp to avoid contamination and
spread of fungus. The bacteria in nutrient broth was inoculated across the surface of
the agar with the use of sterile inoculating loop. After this, the bacteria in the plate
were incubated for about at 38 degree Celsius. The process was be done on each of
the selected pathogenic bacteria.

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IV. Subculture of Pathogenic Bacteria

Alcohol lamp, inoculating loop, and Nutrient Broth were used in sub-culturing
the test organisms. The inoculating loop and the opening of the screw scrap tube
containing the nutrient broth were flame sterilized using the alcohol lamp. The
bacteria on the plate after incubation was placed on the nutrient broth using the
inoculating loop. After this process, the sub-culture of bacteria was incubated prior to
the sensitivity test.

V. Antimicrobial Sensitivity Test

Paper disc method was followed in conducting the sensitivity test. The
subculture of bacteria was applied using sterile cotton swabs on the agar plates. The
plates were incubated overnight and were observed thereafter.
After incubation, the absence or presence of circular inhibition zones were
used as bases in determining the activity of the sample being tested. The length of
zones of inhibition will be recorded and interpreted using The Interpretative
Guidelines for Nonfastidious Aerobic bacteria Zone Diameter Interpretative
Standards published in the book Introduction to Diagnostic Microbiology by Maria
Damnessa Delost. The results were given descriptive equivalents of Susceptible,
Moderately Susceptible, Intermediate and Resistant.

VI. ANTIMICROBIAL INDEX OF THE EXTRACT

For the antimicrobial index of the extract, the following formula is used:
Antimicrobial Index =

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CHAPTER IV
RESULTS AND DISCUSSION

This chapter presents the discussion, analysis and interpretation of actual data
gathered.
Staphylococcus haemolyticus
Zone of Inhibition

Descriptive Equivalent

Calabur Extract

11.7

Moderately Susceptible

Vancomycin (positive
control)

12.6

Susceptible

Oxacillin (negative control)

Resistant

Staphylococcus aureus
Zone of Inhibition

Descriptive Equivalent

Calabur Extract

10.9

Intermediate

Vancomycin (positive
control)

10.5

Intermediate

Oxacillin (negative control)

Resistant

Escherichia coli
Zone of Inhibition

Descriptive Equivalent

Calabur Extract

Resistant

Chloramphenicol (positive
control)

10.0

Intermediate

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The length of the zones of inhibition were recorded and interpreted using The
Interpretative Guidelines

for

Non-fastidious

Aerobic bacteria Zone Diameter

Interpretative Standards published in the book Introduction to Diagnostic Microbiology

by Maria Damnessa Delost. The results were given descriptive equivalents of
Susceptible, Moderately Susceptible, Intermediate and Resistant.
Based on the table, Staphylococcus haemolyticus is moderately susceptible to
Calabur extract; susceptible to Vancomycin; and resistant to Oxacillin based on the
11.7mm, 12.6mm, and 0mm zones of inhibition respectively. Staphylococcus aureus is
intermediate to Calabur extract; intermediate to Vancomycin; and resistant to Oxacillin
based on the 10.9mm, 10.5mm and 0mm zones of inhibition respectively. Escherichia
coli is resistant to Calabur extract; and intermediate to Chloramphenicol based on the
0mm and 10.0mm zones of inhibition respectively.

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CHAPTER V
SUMMARY, CONCLUSION, AND RECOMMENDATIONS

Summary of Findings
The main goal of this study is to determine the prophylactic property of Calabur
(Muntingia calabura) extract on selected pathogenic bacteria Staphylococcus
haemolyticus, Staphylococcus aureus and Escherichia coli. After a series of research,
experiment and investigation, and based on the results found on Chapter 4, the researcher
was able to answer the specific questions stated in Chapter 1.

a) Does Calabur (Muntingia calabura) exhibit a prophylactic property based on the

zone of inhibition of the selected pathogenic bacteria (Staphylococcus
haemolyticus, Staphylococcus aureus and Escherichia coli)?
Calabur exhibits a prophylactic property on Staphylococcus haemolyticus
and Staphylococcus aureus since there are 11.7 mm

and 10.9 mm zone of

inhibition respectively. Calaburs effect on Staphylococcus haemolyticus was

given a descriptive equivalent of moderately susceptible while its effect on
Staphylococcus aureus was given a descriptive equivalent of intermediate. The
descriptive equivalents were identified using the table on The Interpretative
Guidelines for the Nonfastidious Aerobic Bacteria Zone Diameter Interpretative
Standards in the book Introduction to Diagnostic Microbiology by Maria
Damnessa Delost.

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b) Is there a significant difference between the effect of Calabur (Muntingia

calabura) leaves extract on the selected pathogenic bacteria (Staphylococcus
haemolyticus, Staphylococcus aureus and Escherichia coli) compared to the
effect of the standard?
There is no significant difference between the effect of Calabur leaves
extract on Staphylococcus aureus and Staphylococcus haemolyticus since their
antimicrobial indices are 1.04 and 0.93, respectively. For S. aureus, Calabur
extract and the control (Vancomycin) acquired a descriptive equivalent of
intermediate. For S. haemolyticus, Calabur extract acquired a descriptive
equivalent of moderately susceptible while the control (Vancomycin) acquired a
descriptive equivalent of susceptible.

c) Among the selected pathogenic bacteria, on which of the following is Calabur

extract most effective based on their zones of inhibition?
Calabur extract is most effective on Staphylococcus haemolyticus since it
is susceptible to the extract based on its zone of inhibition of 11.7 mm compared
with the 10.9mm zone of inhibition in Staphylococcus aureus and 0mm zone of
inhibition in Escherichia coli. The effect of Calabur extract is also comparable to
the effect of the standard since the extracts antimicrobial index is 0.93 which is
close to 1.00.

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Conclusion
The researcher had concluded that Calabur leaves extract exhibits a prophylactic
property on Staphylococcus haemolyticus and Staphylococcus aureus. It was also found
out that there is no significant difference between the effect of Calabur leaves compared
with the effect of standard antibiotics used.

Recommendations
The researcher would like to propose the following recommendations for further
study by people who are willing to investigate on the same subject matter in determining
the prophylactic property of Calabur (Muntingia calabura) on selected pathogenic
bacteria:
1. The use of other agents other than ethanol in the extraction of plant material.
2. The use of other plant extraction methods.
3. The use of other culture of bacteria to determine if Calabur exhibits a prophylactic
property on them.

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