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ANTIBACTERIAL ACTIVITY OF GUYABANO (Annona muricata) LEAVES

EXTRACT ON Staphylococcus epidermidis

A Science Investigatory Project Proposal

Submitted by:

Wendell Flores

II-Daffodil

BICOL REGIONAL SCIENCE HIGH SCHOOL

TUBURAN, LIGAO CITY

IN PARTIAL FULFILLMENT OF THE REQUIREMENT IN RESEARCH I

Submitted to:

Mrs. Ester Llobet-Maligaya

S.Y 2012-2013

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TABLE OF CONTENTS

I. Introduction

a) Statement of the Problem

b) General Problem

c) Specific Problem

d) Hypothesis

Operational

Null

e) Scope And Limitations

f) Definition of Terms

g) Conceptual Framework

h) Endnotes

II. Review of Related Literatures

a) Annonaceous acetogenins

b) Guyabano

c) Antibacterial

d) Staphylococcus Epidermidis

e) Biofilms

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f) End Notes

III. Methodology

a) Methods of research

b) Methods of collecting data

c) Statistical Treatment

d) Materials

e) Procedure: Extraction

Concentration 1

Concentration 2

Concentration 3

Concentration 4

Concentration 5

Concentration 6

Concentration 7

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Introduction

BACKGROUND OF THE STUDY

Guyabano (Annona muricata) are slender trees that are 5 to 10 meters in height and 15

centimeters in diameter. The trunk of the guyabano are mostly straight , the barks are smooth

and the leaves are alternately 7.6-15.2 cm long and 2.5-7.6 cm wide .Guyabano is commonly

used as herbal medicines especially its leaves. It contains different minerals and vitamins like

vitamin C, B1, B2, phosphorous, potassium, fiber and iron. 1 Because of these minerals, the

leaves were effective in curing acne, scars, and other common skin problems.2 Annonaceous

acetogenins are waxy substances consisting of C32 or C34 long chain fatty acids which have

been combined with a 2-propanol unit at C-2 to form a lactone are only found in several genera

of the plant family, Annonaceae like guyabano trees. Its diverse bioactivities are as antitumor,

immunosuppressive, pesticidal, antiprotozoal, anti-feedant, anthelmitic, antibacterial and

antimicrobial agents.3

Staphylococcus epidermidis is a gram-positive and coagulase-negative staphylococci and

commonly present in human skin.4 .It is approximately 0.5 to 1.5 micrometers in diameter and

one of the most common pathogen that causes nosocomial infections (infections that are acquired

through the hospital) . Those most susceptible to these infections are drug users , newborns and

elderly with catheters. S. epididermis also causes infections to wounds. Infections and

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inflammation of wounds caused by the bacteria needs astringents with higher content and

solutions.

Most astringents are expensive and yet not effective. The pathogen like S. epidermidis

are still unaffected by these products. Due to this, the researcher will conduct this research to

determine the antibacterial activity of guyabano (Annona muricata) leaves extract to reduce skin

inflammation or infection and to contribute to the society a raw material which can be made as

an astringent product.

Statement of the Problem

This study was conducted to determine the effectiveness of guyabano (Annona muricata)

Leaves extract against the pathogenic bacteria S. epidermidis that is commonly found in the skin.

Specifically the proponent will answer the following problems and questions:

General Problem:

Is the guyabano leaves extract effective against the pathogenic bacteria

Staphylococcus epidermidis?

Specific Problem:

1. What concentration of guyabano extracts to distilled water will be the most effective in

killing the bacteria staphylococcus epidermidis?

a. 20 ml distilled water

b. 30 g guyabano leaves

c. 10 grams of guyabano leaves and 20 ml of water

d. 20 grams of guyabano leaves and 30 ml of water

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e. 30 grams of guyabanoleaves and 30 ml of water

f. 40 grams of guyabanoleaves and 30 ml of water

g. 50 grams of guyabanoleaves and 30 ml of water

Hypothesis

Is the guyabno leaves extract effective against the pathogenic bacteria Staphylococcus

epidermidis?

Operational Hypothesis

The guyabano leaves extract is effective against pathogenic bacteria Staphylococcus

epidermidis

Null Hypothesis

The Annona Muricata leaves extract is not effective in killing the pathogenic bacteria

Staphylococcus epidermidis.

Significance of the study

This study will be beneficial to the following:

Botany. This study provides prominent information about guyabano leaves. It may also lead to

the discovery of other species of the plants that can be used for further studies related to the

present studies

Department of Health.

They can endorse the use of this natural and herbal product in minimizing the cases of infection

caused by S. epidermidis in our country.

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Medicine and Health and other related fields.

The antibacterial substance of plant establishes other properties which may lead to further

discoveries of other biological and medical products and techniques which may be developed to

cure other diseases.

Other Researchers.

This may serve as a related study as a reference and guide in conducting further studies.

Scope and Limitations

This study will be conducted to determine if the Guyabano leaves extract is effective in

killing the bacteria Staphylococcus epidermidis. It is only concerned on one specific bacteria

Staphylococcus epidermidis and not any another bacteria found in the skin. This study will also

be conducted to determine the proportion of guyabano extract to distilled water that will be the

most effective in killing the bacteria.

Definition of Terms

1. Annona Muricata

-Annona muricata is a member of the family of Custard apple trees called Annonaceae

and a species of the genusAnnonaknown mostly for its edible fruits Anona.

-This is the raw material that will be used in the study

2. Staphylococcus epidermidis

-a common skin and mucosal inhabitant in humans and occasionally in animals living in

association with humans.

-This is the bacteria that will be acted by guyabano

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3. Sensitivity Test

-a procedure which determines how responsive the subject is to a certain outside stimuli

- this procedure will be used to determine how effective is the proportion of leaves

extract to water in killing Staphylococcus epidermidis

4. Zone of inhibition

- The clear region around the paper disc saturated with anti microbial agent on the agar

surface.

-This is the one that is being measured to determine the effectiveness of the raw material.

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CONCEPTUAL FRAMEWORK

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END NOTES

1. Miss Pinky Soursop Medicinal Uses.2012. http://www.all-about-philippine-fruits-and-

herbs.com/index.html.

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SET-UPS

SETUP 1

SETUP 2

SETUP 3

SETUP 4

SETUP 5

SETUP 6

SETUP 7

Replicate 1

Replicate 2Replicate 3


Replicate 3


Replicate 3


Replicate 3


Replicate 3


Replicate 3


Replicate 3
http://www.all-about-philippine-fruits-and-herbs.com/index.htmlhttp://www.all-about-philippine-fruits-and-herbs.com/index.htmlhttp://www.all-about-philippine-fruits-and-herbs.com/index.htmlhttp://www.all-about-philippine-fruits-and-herbs.com/index.html

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2. Guyabano Herbal Medicine .Medicinal Health Guide.

2011.http://www.medicalhealthguide.com/herb/guyabano.htm

3. Muller,Viana. Graviola (Annona Muricata). 8 Jan 2010 . Whole World Botanicals.

http://wholeworldbotanicals.com/herbal_graviola

4. Staphylococcus epidermidis. 2010. Medpedia.inc.

http://wiki.medpedia.com/Staphylococcus_epidermidis.

Review of Related Literature

Annonaceous acetogenins

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Annonaceous acetogenins are powerful phytochemicals that are found in guyabano

plants.Many bioactive compounds and phytochemicals have been found in guyabano as scientists

have been studying its properties since the 1940's. Its many uses in natural medicine has been

validated by this scientific research. The earliest studies were between 1941 and 1962. Several

studies over the years have demonstrated that leaf, bark, root, stem and seed extracts of Graviola

are antibacterial in vitro against numerous pathogens and that the bark has antifungal properties.

Graviola seeds demonstrated active antiparasitic properties in a 1991 study, and a leaf extract

showed to be active against malaria in two other studies in 1990 and 1993.1. Annonaceous

acetogenins are only found in the Annonaceae family (to which Guyabano belongs). In general,

various Annonaceous acetogenins in the plant family have been documented with antitumorous,

antiparasitic, pesticidal, antiprotozoal, antifeedant, anthelmintic, and antimicrobial activities.

Guyabano

Guyabano is a small, upright evergreen tree, 5-6 m high, with large, glossy, dark green

leaves. It produces a large, heart-shaped, edible fruit that is 15-23 cm in diameter, is yellow-

green in color, and has white flesh inside. Guyabano is indigenous to most of the warmest

tropical areas in South and North America, including the Amazon and Asia. The fruit is sold in

local markets in the tropics, where it is called guanabana in Spanish-speaking countries and

graviola in Brazil. The fruit pulp is excellent for making drinks and sherbets and, though slightly

sour-acid, can be eaten.

All parts of the Guyabano tree are used in natural medicine in the tropics, including the

bark, leaves, roots, fruit, and fruit seeds. Different properties and uses are attributed to the

different parts of the tree. Generally, the fruit and fruit juice are taken for worms and parasites, to

cool fevers, as a lactagogue (to increase mother's milk after childbirth), and as an astringent for

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diarrhea and dysentery. The crushed seeds are used as a vermifuge and anthelmintic against

internal and external parasites, head lice, and worms. The bark, leaves, and roots are considered

sedative, antispasmodic, hypotensive, and nervine, and a tea is made

for various disorders toward those effects. Researchers verified Guyabano leaf's hypotensive

properties in rats again in 1991. Many bioactive compounds and phytochemicals have been

found in Guyabano, as scientists have been studying its properties since the 1940s. Its many uses

in natural medicine have been validated by scientific research. Several studies over the years

have demonstrated that leaf, bark, root, stem, and seed extracts of Guyabano are antibacterial in

vitro against numerous pathogens, and that the bark has antifungal properties. Guyabano seeds

demonstrated active antiparasitic properties in a 1991 study, and a leaf extract showed to be

active against malaria in two other studies (in 1990 and 1993). The leaves, root, and seeds of

Guyabano demonstrated insecticidal properties, with the seeds demonstrating strong insecticidal

activity in an early 1940 study.2.

Antibacterial

An antibacterial is an agent that inhibits bacterial growth or kills bacteria. The term is

often used synonymously with the term antibiotic(s); today, however, with increased knowledge

of the causative agents of various infectious diseases, antibiotic(s) has come to denote a broader

range ofantimicrobial compounds, including anti-fungal and other compounds.

The term antibiotic was first used in 1942 by Selman Waksman and his collaborators in journal

articles to describe any substance produced by a microorganism that is antagonistic to the growth

of other microorganisms in high dilution. This definition excluded substances that kill bacteria,

but are not produced by microorganisms (such as gastric juices and hydrogen peroxide). It also

excluded synthetic antibacterial compounds such as the sulfonamides. Many antibacterial

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compounds are relatively small molecules with a molecular weight of less than 2000 atomic

mass units.

With advances in medicinal chemistry, most of today's antibacterials chemically are

semisynthetic modifications of various natural compounds. These include, for example, thebeta-

lactam antibacterials, which include thepenicillins (produced by fungi in the genusPenicillium),

the cephalosporins, and the carbapenems. Compounds that are still isolated from living

organisms are the aminoglycosides, whereas other antibacterialsfor example, the

sulfonamides, the quinolones, and the oxazolidinonesare produced solely by chemical

synthesis. In accordance with this, many antibacterial compounds are classified on the basis of

chemical/biosynthetic origin into natural, semisynthetic, and synthetic. Another classification

system is based on biological activity; in this classification, antibacterials are divided into two

broad groups according to their biological effect on microorganisms: bactericidal agents kill

bacteria, andbacteriostatic agents slow down or stall bacterial growth3.

Staphylococcus Epidermidis

Staphylococcus epidermidis is a gram positive bacterium and one of 33 known species

belonging to the genus Staphylococcus. It is part of the human skin flora and consequently part

of the human microbiome. Although S. epidermidis is not usually pathogenic, patients with

compromised immune systems are often at risk for developing an infection. These infections can

be both nosocomial or community acquired, but they pose a greater threat to hospital patients. S.

epidermidis is also a major concern for people with catheters or other surgical implants because

it is known to causebiofilms that grow on these devices.4. When S. epidermidis causes illness in

humans, it most commonly results in many clinical conditions like blood stream infections,

Endocarditis, Cerebrospinal fluid (CSF) shunt infection, Peritoneal dialysis catheter infection,

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Urinary Tract Infection, Infection inprosthetic joints, Infection of vascular grafts (tubes inserted

into blood vessels to bypass areas of blockage or damage), Infection in newborn children and

Infection of breast implants5.. The natural environment ofS. epidermidis is the human body and

usually originates from disease. Since the bacteria usually lives on the skin and nares of all

human beings and is a nosocomial pathogen, it is imporant to be able to identify the specific

strains. S. epidermidis is the most common staphylococcus on the human skin. In addition, S.

epidermidis also covers 90%-100% staphylococci from the nares when S. aureus is not present.

When S. aureus is present the S. epidermidis amount decreases drastically. The formation of

biofilm allows S. epidermidis to attach and grow on biomedical devices and be released into the

blood to infect new areas. The increase use of intravascular catheters has caused a similar

increase in S. epidermidis infections. The increase causes a problem since S. epidermidis is

resistant to methicillin and all penicillins, penems, carbapanems, and cephalosporins which are

commonly used antibiotics. S. epidermidis has also been found to be more resistant to antibiotics

than other species

Although there are no one specific viruelence factors ofS. epidermidis, the ability to form

biofilm is one of the virulence factors. The biofilm allows the bacteria cells to adhere to an inert

or living areas .When a biofilm has formed it becomes harder to treat since the cells inside the

biofilm are guarded from antibiotics and the immune system . Biofilm also releases a host

immune response to antigens which prevents the removal of the biofilm and may also result in

tissue damage. The bacteria can be released into the blood from biofilms and start new infections

by attachment to the medical devices, thus devices will be required to be removed .

Some preventive strategies for infections are to provide prohylatic antibiotic therapy to cover

surgical insertions from temporary intravascular devices. There are also reports that warn to not

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use antibiotic prohylaxis, especially vancomycin for dialysis. Aseptic techniques should be used

for catheter infections to prevent contamination. New techniques concentrate on physical

electrical barriers for colonization and using biomaterials with antimicrobial agents already

inside. However these new methods have not been tried in the clinical settings6..

Biofilms

Biofilms are communities of microbial cells that grow on living or inert surfaces and

surround themselves with secreted polymers. Many bacterial species like staphylococcus

epidermidis form biofilms, and their study has revealed them to be complex and diverse. The

structural and physiological complexity of biofilms has led to the idea that they are coordinated

and cooperative groups, analogous to multicellular organisms.7.. Biofilms are usually found on

solid substrates submerged in or exposed to an aqueoussolution, although they can form as

floating mats on liquid surfaces and also on the surface of leaves, particularly in high humidity

climates. Given sufficient resources for growth, a biofilm will quickly grow to be macroscopic

(visible to the naked eye). Biofilms can contain many different types of microorganism,

e.g. bacteria,archaea,protozoa, fungi and algae; However, some organisms will form single-

species films under certain conditions.7. Biofilms have been found to be involved in a wide

variety of microbial infections in the body, by one estimate 80% of all infections. Infectious

processes in which biofilms have been implicated include common problems such asurinary

tract infections, catheterinfections, middle-ear infections, formation ofdental plaque,gingivitis,

coating contact lenses, and less common but more lethal processes such asendocarditis,

infections in cystic fibrosis, and infections of permanent indwelling devices such as

jointprostheses andheart valves. More recently it has been noted that bacterial biofilms may

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impair cutaneous wound healing and reduce topical antibacterial efficiency in healing or treating

infected skin wounds.

It has recently been shown that biofilms are present on the removed tissue of 80% of

patients undergoing surgery for chronicsinusitis. The patients with biofilms were shown to have

been denuded ofcilia and goblet cells, unlike the controls without biofilms who had normal cilia

and goblet cell morphology. Biofilms were also found on samples from two of 10 healthy

controls mentioned. The species of bacteria from interoperative cultures did not correspond to

the bacteria species in the biofilm on the respective patient's tissue. In other words, the cultures

were negative though the bacteria were present.

Biofilms can also be formed on the inert surfaces of implanted devices such as catheters,

prosthetic cardiac valves and intrauterine devices.

New staining techniques are being developed to differentiate bacterial cells growing in living

animals, e.g. from tissues with allergy-inflammations . 8

END NOTES

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1. Annonaceous Acetogenins. 2011.Pub Med, National Library of Medicine.

http://www.immupro.com/graviola.htm.

2. Guyabano(Annona Muricata). http://guyabano.com/Page_2.html.

3. Antibacterial.19 Nov 2012.Wikimedia Foundation, Inc.

http://en.wikipedia.org/wiki/Antibacterial

4. Staphylococcus Epidermidis 21 February 2013. Wikimedia Foundation, Inc.

http://en.wikipedia.org/wiki/Staphylococcus_epidermidis

5. Staphylococcus epidermidis. 2010. Medpedia.inc.

http://wiki.medpedia.com/Staphylococcus_epidermidis.

6. Staphylococcus Epidermidis. 22 April 2011 . Microbewiki Foundation Inc.

http://microbewiki.kenyon.edu/index.php/Staphylococcus_epidermidis.

7. Biofilm Bacteria 2 Jan 2012. Autoimmunity Research Foundation.

http://mpkb.org/home/pathogenesis/microbiota/biofilm

8. Biofilm. 23 Feb 2013 . Wikimedia Foundation Inc.

http://en.wikipedia.org/wiki/Biofilms

Methodology

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Methods of research

The methods of research used in the study are experimental and observation method.

Experimental method will be used to test the effectiveness of guyabano (Anonna muricata)

leaves against the bacteria Staphylococcus epidermidis and observation method to determine the

range of the zone of inhibition of the proportions. This will determine what proportion is the

most effective in killing the pathogenic bacteria Staphylococcus epidermidis.

The study is mainly PHBA(Potentially Hazardous Biological Agents) because it focuses

in killing pathogenic bacteria that can harm living organism. Thus this study focuses in killing

S.epidermidis using the extract from guyabano leaves

Methods of Collecting Data

The methods used for collecting data is experimental method and library method . The

experimental method used in the study is concerned with the extraction of the guyabano leaves

against S. epidermidis. It is more appropriate to use the experimental method in collecting

specific and reliable data. Library method is gathering information through books, literatures and

articles and is used to find related literatures to help answers of the specific problems.

Statistical Treatment

The statistical treatment will be used to show the results of experimentations through

mathematical graphs.Each proportion has 3 replicates in which we will get the mean and

compare which proportion more effective.

Materials

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To get the extract, the researcher will be utilizing a total of 180 grams guyabano leaves,

and 120 mL distilled water. Mortar and pestle, beaker, cheese cloth, weighing scale and plastics

bags will also be used.

Procedure: Extraction

Concentration 1 (negative control)

First, the 20ml distilled water will be measured using the beaker. Then it will be poured

into the small bottle using a funnel and will be labeled as CONCENTRATION 1.

Concentration 2 (30 grams of guyabano leaves) (Pure extract)

First, the 30g of guyabano leaves will be labeled and will be placed inside a plastic bag

and pounded using the mortar and pestle. Using the cheese cloth, the pounded leaves will be

squeezed until the pure extract is obtained. The extract will be then placed in test tube B and will

be transferred into a small bottle and will be labeled as CONCENTRATION 2.

Concentration 3 (10 grams of guyabano leaves and 20 ml of water)

The 10g of guyabano leaves will be labeled and placed inside a plastic bag. Then, a 50ml

beaker will be filled up with 20 ml of distilled water and will be poured in the plastic bag with

the leaves in it and will be pounded. To get the extract, same process in concentration 2 will be

used. The extract will be then placed in test tube C and will be transferred into a small bottle and

will be labeled as CONCENTRATION 3.

Concentration 4 (20 grams of guyabano leaves and 30 ml of water)

First, the 20g of guyabano leaves will be labeled and placed inside a plastic bag. Then, a

50ml beaker will be filled up with 30 ml of distilled water and will be poured in the plastic bag

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with the leaves in it and will be pounded. To get the extract, same process in concentration 2 will

be used. The extract will be then placed in test tube D and will be transferred into a small bottle

and will be labeled as CONCENTRATION 4.

Concentration 5 (30 grams of guyabanoleaves and 30 ml of water)

First, the 30g of guyabanoleaves will be labeled and placed inside a plastic bag. Then, a



be used. The extract will be then placed in test tube E and will be transferred into a small bottle



First, the 40g of guyabano leaves will be labeled and placed inside a plastic bag. Then, a



be used. The extract will be then placed in test tube F and will be transferred into a small bottle



First, the 50g of guyabanoleaves must bewill be labeled and placed inside a plastic bag.

Then, a 50ml beaker will be filled up with 20 ml of distilled water and will be poured in the

plastic bag with the leaves in it and will be pounded. To get the extract, same process in

concentration 2 will be used. The extract will be then placed in test tube G and will be

transferred into a small bottle and will be labeled as CONCENTRATION 7.

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