The Prophylactic Property of Calabur on Selected Pathogenic Bacteria (Body)

7/23/2019 The Prophylactic Property of Calabur on Selected Pathogenic Bacteria (Body)

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1DAGUPAN CITY NATIONAL HIGH SCHOOL

Science, Technology and Engineering (STE) Program

School Year 2013-2014

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.

HoCalabur (Muntingia calabura)does not exhibit a prophylactic property based on the


haemolyticus, Staphylococcus aureus and Escherichia coli).

Ho There is no significant difference between the effect of Calabur (Muntingia


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 aboutMuntingia 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 CityMedical 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 bacteriabacteria 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 coccus-

shaped 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 InhibitionIt 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.
http://en.wikipedia.org/wiki/Leafhttp://en.wikipedia.org/wiki/Leaf


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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: Eleocarpaceae

Other names: 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 haemolyticusis 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. haemolyticusis 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 low-

virulent 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

haemolyticusitself is also a remarkable opportunistic baterial pathogen that is well-

known 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. haemolyticusinclude 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. TheEnterobacteriaceae 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. coliis versatile and well-adapted to its characteristic habitats. E.


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coliis 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 aureusis a bacterium that is a member of the Firmicutes, and is

frequently found in the human respiratory tract and on the skin. Although S. aureusis not

alwayspathogenic, it is a common cause of skin infections (boils), respiratory disease

(sinusitis), andfood poisoning. Disease-associated strains often promote infections by

producing potent proteintoxins, and expressing cell-surface proteins thatbind and

inactivate antibodies. The emergence ofantibiotic-resistant forms of pathogenic S.

aureus(e.g.MRSA)is a worldwide problem in clinical medicine. S. aureusis responsible

for many infections but it may also occur as acommensal.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.
http://en.wikipedia.org/wiki/Pathogenhttp://en.wikipedia.org/wiki/Boilshttp://en.wikipedia.org/wiki/Sinusitishttp://en.wikipedia.org/wiki/Food_poisoninghttp://en.wikipedia.org/wiki/Exotoxinhttp://en.wikipedia.org/wiki/Protein_Ahttp://en.wikipedia.org/wiki/Protein_Ahttp://en.wikipedia.org/wiki/Antibiotic-resistanthttp://en.wikipedia.org/wiki/Methicillin-resistant_Staphylococcus_aureushttp://en.wikipedia.org/wiki/Commensalhttp://en.wikipedia.org/wiki/Commensalhttp://en.wikipedia.org/wiki/Methicillin-resistant_Staphylococcus_aureushttp://en.wikipedia.org/wiki/Antibiotic-resistanthttp://en.wikipedia.org/wiki/Protein_Ahttp://en.wikipedia.org/wiki/Protein_Ahttp://en.wikipedia.org/wiki/Exotoxinhttp://en.wikipedia.org/wiki/Food_poisoninghttp://en.wikipedia.org/wiki/Sinusitishttp://en.wikipedia.org/wiki/Boilshttp://en.wikipedia.org/wiki/Pathogen


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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

andEscherichia 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 wide-

acceptance 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

Escherichia coli

Staphylococcus haemolyticus

Mueller Hinton Agar plates and Nutrient Broth (NB)

forceps

cotton swabs

inoculating loop

standard (commercial antibiotic)

petri dish

alcohol lamp


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A

Gathering of Laboratory Equipment and Collection of Plant

Material

BExtraction of Plant Material

CCollection and Preparation of Culture Media

DSterilization of Materials

E

Subculture of Pathogenic Bacteria and Incorporation in Nutrient

Broth

FAntimicrobial Sensitivity Test

G

Computation of Antimicrobial Indices of the Extract on the

Pathogenic Bacteria

H Interpretation of Results

ISummary 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 coliwere obtained and cultured at the medical laboratory of University of

PangasinanPHINMA.

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 inthe 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 datagathered.

Staphylococcus haemolyticus

Zone of Inhibition Descriptive Equivalent

Calabur Extract 11.7 Moderately Susceptible

Vancomycin (positivecontrol)

12.6 Susceptible

Oxacillin (negative control) 0 Resistant

Staphylococcus aureus


Calabur Extract 10.9 Intermediate

Vancomycin (positivecontrol)

10.5 Intermediate

Oxacillin (negative control) 0 Resistant

Escheri chia coli


Calabur Extract 0 Resistant

Chloramphenicol (positivecontrol)

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


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


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 aureusand 0mm zone of

inhibition inEscherichia 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 haemolyticusand 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.

Documents

The Prophylactic Property of Calabur on Selected Pathogenic Bacteria (Body)