2edited research output 7-edited

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RESEARCH OUTPUT 7: Research Protocol

Adviser: Dr. I.A. Ilano

Submitted by: GROUP 9-A

Leader:

Holgado, Anna Victoria

Members:

Alcantara, Jan Christopher

Balandan, Patricia

Buenafe, Jonas Joaquin

Constantino, Erwin

Delos Santos, Kathrine Aira

Flores, Marie Felle

Hernandez, Kristeen Khae

Lopez, Edison

Date:

November 8, 2011

Topic: "A comparative study on the antibacterial activity peel extracts obtained from Musa

acuminata, Musa balbisiana, and Musa paradisiacaagainst Staphylococcus aureus


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Table of Contents

I. Introduction 2

A. Research Question ........... 2

B. Research Hypotheses ... 2

C. Background of the Research Question .. 2

D. Significance of the Study .. 3

II. Research Objectives .. 3

III. Literature Review ........... 4

A. Epidemiology of Disease of Interest 4

B. Epidemiology of Exposure of Interest .. 4

C. Summary of Related Studies 5

D. Conceptual Framework . 6

IV. Methodology 9

A. Research Design 9

B. Experiment Procedure . 10

C. Sampling ............ 14

D. Data Collection ............. 17

V. Bibliography ............... 19

VI. Appendices ............... 20


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I. INTRODUCTION

A. Research question

Which species of banana peel extract has greater antibacterial activity against

Staphylococcus aureus?

B. Research hypotheses

i. Working hypothesis

The peel extracts obtained from the different species of banana have comparable

antibacterial activity against Staphylococcus aureus.

ii. Null hypothesis

The peel extracts obtained from the different species of banana do not have

comparable antibacterial activity against Staphylococcus aureus.

C. Background of the research question

The contribution of various plants or their parts (roots, stems, leaves, fruit) in the

treatment and management of certain health conditions has been growing in recognition.

At present, the use of herbal medicine are becoming more common both in developing

and developed countries. The World Health Organization (WHO) has estimated that about

70-95% of the citizens in majority of the developing countries utilize traditional medicine

(including the use of herbal medicines) in managing their health and incorporate the

practice in their primary health care to address emerging health-related needs.(1)

Industrialized countries, such as Canada, France, Germany and Italy, share similar

percentages in terms of the proportion of individuals who make use of traditional

medication.(1) In the Philippines, the use of plant extracts as medication has been passed

on from one generation to another, and has established its importance in health delivery,

considering the expensive Western treatment that most Filipinos cannot afford or cannot

easily access.(2)

The banana fruit (Musa sapientum), has been commonly known for its nutritional

value; however, its medicinal properties have only been recently investigated, mostly in

tropical and subtropical countries wherein the banana fruit is considered as one of their

major agriculture products, such as India and other Southeast Asian countries. In these

regions, other parts of the banana plant such as their young shoots and peels have been


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utilized as an alternative source of treatment for ulcers and wounds, especially in areas

where access to conventional treatment is difficult.(3) In addition, majority of the studies

conducted in these countries have been focusing on the most common variants of the

Musa sapientumspecies available in their locality.

The study aims to investigate the antibacterial activity of the most common local

variants of banana grown in the province of Cavite, namely Musa acuminata(lakatan),

Musa balbisiana (saba), and Musa paradisiaca (latundan)(4), and determine if the

species variant plays a significant role in their respective antibacterial activity, specifically

against Staphylococcus aureus, a common Gram-positive agent found in most health

care-related infections.

D. Significance of the study

This study may provide additional information regarding the antibacterial activity of

the common variants of bananas in the region, and thus provide possible plant leads that

can be used as alternative and less expensive sources of treatment for the benefit of the

local residents. The additional information obtained may also contribute in evoking interest

for further research on the phytochemical profile and antibacterial activity of the local

banana variants which may be used as active ingredients against drug-resistant

microorganisms such as Staphylococcus aureus.

II. RESEARCH OBJECTIVES

A. General objective

To determine which species of banana peel extract has greater antibacterial activity

against Staphylococcus aureus.

B. Specific objectives

1. To measure the zones of inhibition of peel extracts obtained from Musa acuminata,

Musa balbisiana, and Musa paradisiacaagainst Staphylococcus aureususing the

disk diffusion method.

2. To establish the respective minimum inhibitory concentrations of peel extracts

obtained from Musa acuminata, Musa balbisiana, and Musa paradisiaca against

Staphylococcus aureususing broth dilution test.

3. To compare the obtained zones of inhibition and minimum inhibitory concentrations

of the peel extracts from different banana species against Staphylococcus aureus.


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III. LITERATURE REVIEW

A. Epidemiology of disease of interest

Staphylococcus aureus is considered as one of the medically important pathogens,

commonly causing abscess formation, various pyogenic infections (e.g. endocariditis),

food poisoning, and toxic shock syndrome. It has also been among the prevalent

causative agent for majority of hospital-acquired infections (e.g. pneumonia), septicemia,

and surgical-wound infections.(5)(6) Infections caused by the bacteria have been more

prevalent in the health care setting where these are treated with more frequent and

intensive antimicrobial therapy as compared in the community setting. (6)

Throughout the evolution of antimicrobial therapy against S. aureus, various strains

of resistance have been developed by the agent, thus amplifying the disease burden. (6) In

some countries in the Western Pacific Region, such as the Philippines, a growing trend in

methicillin-resistant S. aureus (MRSA) had been recorded within a span of 7 years. (7)

According to the 2005 Philippine Antimicrobial Resistance Surveillance report, wherein

twelve out of the agencys 17 sentinel health care institutions contributed to the data

output, the overall MRSA rate among admitted patients significantly increased, from 17%

in 2004 to 31% in 2005.(8) An increase of 34% was also observed in urban areas such as

Metro Manila.(8)

Health care institutions in the Philippines constantly deal with the disease burden

brought about by various strains of methicillin-resistant S. aureusamong patients. A study

conducted in a tertiary medical institution revealed the presence of hospital-acquired

MRSA (HA-MRSA) among patients suffering from chronic kidney disease and were

undergoing renal replacement therapy, such as hemodialysis. Strains of community-

acquired MRSA (CA-MRSA) were also isolated and were identified among patients with

no other underlying co-morbidities. CA-MRSA strains were commonly seen in skin and

soft tissue infections.(9)

B. Epidemiology of exposure/factor of interest

Traditional medicine has been highly adopted throughout the world due to their

availability, affordability and cultural familiarity. It has been estimated bytheWorld Health

Organization that in some Asian and African countries, 80% of the population depends on

traditional medicine.(10) They also identified the use of herbal treatments as the most

popular form of traditional medicine. Herbal medicines include herbs, herbal materials,

herbal preparations, and finished herbal products that contain parts of plants or other plant


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materials as active ingredients. Among the components included in the current day

pharmaceuticals, it has been estimated that about seven thousand active ingredients are

of herbal origin. (10)

The antimicrobial properties of the banana plant (Musa sapientum) are not as widely

commercialized as compared to other local preparations. Nonetheless, its young leaves

have been used for a long time in local folk medicine as a cold dressing for inflamed and

blistered surfaces.(3) Scientific studies have presented evidences regarding the

antimicrobial activities of the banana plant. Commonly utilized parts among the studies

were its leaves, stem, peel, and fruit. Studies conducted by Mokbel and Hashinaga,

Fagbemi et. al, and Scott et. al showed high antimicrobial acitivity in peel and pulp extracts

of unripe bananas against certain bacteria, including S.aureus.(11)(12)(13) Further discussions

regarding the designs and methods, as well as results of these studies are covered in the

next portion of the study, Summary of related/similar studies.

C. Summary of related/similar studies

Majority of the experiments conducted made use of an analytical experimental

design wherein the exposure variable under observation was assigned particularly to a

treatment group and was compared to a control group. The method of extract preparation

and the different solvents used were some of the factors identified that may have

influenced the extracts potency and enhanced its antimicrobial activity.

Method of preparation and solvents used for banana extracts

Banana (Musa sapientum), belonging to Musa species is considered as one of the

most useful plant species that carries a number of beneficial pharmacological effect such

as ulcer protective activity, antioxidant activity and mutagenic effect, antibacterial activity

and wound healing activity.(14)

Several studies have been conducted to show that banana extracts do have

antibacterial properties. In a study by Mokbel and Hashinaga, the antimicrobial and

antioxidant activity of fresh green and yellow banana peel extracts obtained from the

Cavendish variant were compared. Chloroform, ethyl acetate and water were used as

solvents for the peel extracts. Results showed that ethyl acetate and water soluble

fractions of green banana peel displayed high antimicrobial activity. Among the specific

compounds isolated from green banana peel, d-malic acid and 12-hydroxystearic


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exhibited the most active antimicrobial response, with an MIC varying between 140-750

ppm, respectively. (11)

Findings were supported in a phytochemical and pharmacologic review conducted

by Akter et al. wherein it was demonstrated that the banana peel extract obtained from

Musa paradisiacaand Musa sapientumshowed better antibacterial activity against the test

bacteria (Staphylococcus and Pseudomonas species) than the banana leaf extract. The

peel extract was also shown to be more active against Staphylococcus (Gram-positive)

than Pseudomonas species (Gram-negative). Furthermore, the review of pharmacological

activities suggests that the traditional uses of the banana plant in diarrhea, dysentery,

ulcer, diabetes, hypertension and cardiac diseases are scientifically valid.(15)

Other studies investigated if the difference in the subspecies of the Musa sapientum

would yield varying degrees of antibacterial activity against a range of microorganisms.

Akter et. al aimed to evaluate the antimicrobial and cytotoxic activities of different extracts

of Musasapientum, L. subsp. Sylvestris fruits (MSSE). The methanolic extract of Musa

sapientumpeel was investigated for antimicrobial activity by disk diffusion method and for

cytotoxic activity by Brine shrimp lethality bioassay. The findings of the study

demonstrated that the methanolic extract of Musa sapientum possessed good

antimicrobial activity against Gram-positive and Gram-negative bacteria as well as against

pathogenic fungi and affirmed the traditional use of the fruit to treat dysentery and

diarrhea.(16)

In addition, studies conducted by Hamid et al. as well as Mokbel et al. showed how a

certain development of the banana fruit may contribute to its antibacterial activity by using

both ripe and unripe banana peel extract. The type of solvent used was not

specified. Extracts of ripe, unripe and leaves of guava (Psidiumguajava); ripe, unripe and

leaves of starfruit (Averrhoacarambola); ripe and unripe banana (Musasapientumvariety

Montel); ripe and unripe papaya (Carica papaya); passionfruit (Passiflora edulis F.

Flavicarpa) peel; two varieties of Lansium domesticum peels; rambutan (Nephelium

lappaceum) peel and rambai (Baccaureamotleyana) peel were evaluated for antimicrobial

activity against Gram-positive bacteria, Gram-negative bacteria, yeast and fungi(Staphylococcusaureus, Bacillussubtilis, Bacillus cereus, Lactobacillus bulgaricus; E. coli,

Proteus vulgaricus, Pseudomonas aeruginosa, Salmonelli typhi; Saccharomyces

cerevisiae, Candida lypolytica; Rhizopus spp., Aspergillus niger, and Chlamydomucor

spp). The antimicrobial activities were tested using both the disk diffusion and tube dilution

assays. Most of the fruits showed some activity towards bacteria but poor activity against


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yeast or fungi. Extracts from bananas, papayas, passionfruit peel, Lansiumdomesticum

peels and rambutan peels showed activity against Candida lypolyticawhile extracts from

guava showed strong activity against Saccharomycescerevisiae. Unripe banana showed

activity against all the bacteria except towards P. vulgaricus.(17) In the study conducted by

Mokbel and Hashinaga, fresh green and yellow banana peel of Musa, cv. Cavendishfruits

were treated with 70% acetone; and afterwards, partitioned with chloroform and ethyl

acetate. The antioxidant activities of the extracts were evaluated by using the thiocyanate

method, beta-carotene bleaching method and 1,1-diphenyl-2-picrylhydrazyl (DPPH) free

radical elimination whereas the antimicrobial activities of the extracts and isolated

components were evaluated using paper disc methods and minimum inhibition

concentration (MIC). Results of the study showed that the ethyl acetate and water soluble

fractions of green peel showed higher antimicrobial activity than yellow banana peel. The

antioxidant activity of the water extracts from green banana peel was comparable to those

of synthetic antioxidants such as butylated hydroxyanisole and butylated hydroxytoluene.

(11)Biases

Although there were no significant experimental biases recognized in the related

literatures that were reviewed, a possible researcher bias, wherein the prior knowledge of

the researchers might affect the analysis of the results, may be encountered in the study.

Observational bias may also be encountered among the researchers along the course of

the experimental proper, wherein there may be discrepancies in measuring the outcome

observed.

Limitations

It can be noted that since almost all of the studies were conducted in vitro, the

occurrence of possible side effects or interactions of the extracts on actual clinical

infections cannot be identified.

Recommendations

Results from related literatures showed that the banana pulp and peel exhibits high

antimicrobial activity whereas in terms of solvent to be used, extracts using ethyl acetate,

ethanol and methanol exhibits high antimicrobial activity. These results should be taken

into consideration when choosing which part of the banana and what kind of solvent

should be used for extraction.


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D. Conceptual framework

Figure 1 depicts the possible relationship of the exposure variable (application of plant

extract) with that of the outcome variable (inhibition of microbial growth). The

characteristics of the exposure variable must be taken into consideration as to how it

would influence the outcome variable. Similarly, the characteristics of the outcome

variable should also be taken into consideration as to how it may counteract the exposure

variable and affect the result. Possible confounding factors such as exposure to

environmental factors as well as experimental protocol (preparation and storage methods)

were derived from literatures documenting various experimental processes in determining

the antimicrobial activities of the plant extracts.

Antibacterial activity

of banana peel

POSSIBLE CONFOUNDING

FACTORS

Exposure to environmentalfactors

Preparation and storagetechniques

CONTAMINATION

Characteristics of the

factor

Type of solvent used

Type of

phytochemicals

present which

contributes to

antibacterial activity

Concentration of the

extract

Characteristics of the

factor

Inherent defense

mechanisms of the

sample of interest

Inhibition of growth

ofStaphylococcus

aureus


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IV. METHODOLOGY

A. Researchdesign

The study will utilize an analytic experimental design, wherein the independent

variable under observation will be assigned particularly to a treatment group and will be

compared to a positive and negative control group. Figure 2 illustrates the design of the

study.

Operational definition of variables

1. Independent variable

The antibacterial activity of the different species of banana peel extracts on the

growth of Staphylococcus aureus serves as the independent variable for the study.

Antibacterial activity refers to the capacity of an agent to kill or suppress the growth of

microorganisms, specifically bacteria.(18)

This property may be further classified into twomechanisms, bacteriostatic and bacteriocidal. Bacteriostatic activity results into the

inhibition of microbial growth within a certain period of time. Microbial growth may be

observed once environmental elements become suitable, or the microorganism has

gained resistance to counteract the stimulus presented by the agent. (18) On the other

hand, bacteriocidal activity results into the complete eradication of the species. In the

study, significant bacteriostatic activity of the different species of bananas will be observed

through disk diffusion method and broth dilution test.

Musa acuminata (locally known as lakatan), Musa balbisiana (saba), and Musa

paradisiaca(latundan) are considered as the most common group of species grown and

commonly sold in the province of Cavite. Because of their wide availability and easy

accessibility, these species will be chosen as samples for the study.

2. Dependent variable

The expected result from the study will be the inhibition of the growth of

Staphylococcus aureuson nutrient agar medium. Inhibition denotes a temporary cessation

in microbial growth processes. This implies that there are still possible chances for growth,

given that the environment becomes favorable once again for microorganism

propagation.(18) Therefore, strict compliance with the incubation of disk diffusion plates

within the allotted time of 24 hours for the Staphylococcispecies should be observed so

as to achieve reliable results and prevent possible growth of the organism. (19)


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Inhibition of growth will be determined qualitatively using the disk diffusion method,

as represented by zones of inhibition. Broth dilution test will also be employed to quantify

the degree of the antibacterial activity by determining the peel extracts minimum inhibitory

concentration. Minimum inhibitory concentration (MIC) of an antibiotic is the smallest

amount per unit volume that will inhibit the growth of a certain organism. In broth dilution

test, the end point tube will represent the peel extracts MIC. It is the tube wherein there is

complete inhibition of growth of bacteria with the least concentration of the peel extract,

which can be visually observed as absence of turbidity. (21)

3. Confounding variables

Both the independent and dependent variables may face possible contamination

brought about by 1) their exposure to environmental factors, such as temperature and

foreign body contamination; 2) as well as their subjection to certain preparation and

storage techniques executed in the duration of the study. Contamination of the variables of

interest may significantly influence the accuracy and analysis of the results. (18)(19)(21)

B. Experiment procedure

For the study, microbial growth inhibition will be determined using the disk diffusion

method, also known as the Kirby-Bauer test. The principle behind the disk diffusion

method depends on the formation of a concentration gradient as the antimicrobial agentdiffuses instantaneously into the agar. The drug concentration decreases at increasing

distances from the disk. At a critical point, the amount of drug at a specific location in the

medium is unable to inhibit the growth of the test organism, thus forming well-demarcated

borders, resulting in a distinct area known as the zone of inhibition.(18) This method only

gives a qualitative value of an agents antimicrobial activity against a particular bacterial

species, as determined by the resulting zone of inhibition. The zone of inhibition refers to

the clear area surrounding an antimicrobial disk following overnight incubation that result

from the diffusion of the antimicrobial molecules into the agar and inhibition of growth ofthe test bacterium. This will be the parameter used in the study to reflect the presence of

microbial growth inhibition.(18)

Furthermore, control groups will be set up to validate that the microbial growth

inhibition is indeed attributed to the antibacterial activity of the banana peel extract. A

positive control will confirm that the treatment applied is competent to produce the


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intended effect, thus minimizing the probability of false negatives. (20) For the study, the

drug of choice for Staphylococcus aureus, Vancomycin, will be utilized as the standard for

evaluating the antibacterial activity of the plant extracts. A negative control will also be set

up in order to confirm that the effect produced was not influenced by extraneous factors,

thereby minimizing the probability of false positives.(20) For the study, distilled water will be

used as a reference reflecting microbial growth, indicating that the treatment applied has

not been effective.

In addition, a solvent group will also be set up to evaluate the influence of the solvent

on the observed microbial growth inhibition. For the study, methanol will be used as

reference.

Quantitative measures, such as the minimal inhibitory concentration (MIC), will also

be applied to validate the degree of the antibacterial activity of the banana peel extracts.

Minimal inhibitory concentration refers to the smallest amount per unit volume that will

inhibit the growth of a certain organism.(18)(21) Broth dilution tests, wherein tubes containing

decreasing concentration of the plant extract tested are prepared by serial dilution, will be

conducted to determine the plant extracts respective MIC.(21)

Steps to be undertaken

a) Physical set-up

The study will use nutrient agar plates in observing the activity of peel extracts fromdifferent banana species (Musa paradisiaca, Musa balbisianana and Musa acuminata)

compared with the positive control, negative control and solvent control. Three replicates

will be used. Figure 3 illustrates the distribution of the treatments and control among the

bacterial culture.

Figure 3:Allocation of treatment and control

SPECIES

(A)

SPECIES

(B)

SPECIES

(C)

(+)

CONTROL

(-)

CONTROL

SOLVENT

ONLY


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b) Procurement of plant materials

Peels from different species of banana (Musa paradisiaca, Musa balbisianana

and Musa acuminata) will be collected from Dasmarias, Cavite and will be identified

taxonomically.

c) Preparation of extract

The banana peels (300 g for each variety) will be washed with water then coarsely

chopped. The peels will then be placed in a solvent, methanol, at a ratio of 1 gram banana

peel per 4.5 mL of methanol. The mixture will then be homogenized using a blender then

transferred into their respective reaction tanks which will be left for 3-48 hours at room

temperature. The mixture will be agitated or mixed periodically to prevent separation of

masticated peel from the solvent. Change of color from yellow to amber to an opaque

black liquid will indicate that the reaction is already complete. Upon completion of the

reaction step, the mixture will then be filtered through a screen mesh. The collected filtrate

will be further filtered through flat filter paper. The volume of the solution obtained after

filtration will be recorded. The extracts will also be stored in a desiccator at 5C. (22)

d) Preparation of peel extract stock solution

The crude extract with an initial concentration of 22 g/mL will be diluted with

distilled water to 0.001 g/mL (1000 ug/mL). To obtain the 1000 ug/mL final concentration

the concentration-volume ratio will be used.

(Initial Concentration) (Initial Volume) : (Final Concentraton) (Final Volume)

( 220 000 ug/mL) (Initial Volume) : (1000 ug/mL) (X)

e) Preparation of bacterial suspension

Staphylococcus aureus maintained on nutrient agar slants at 4C will be

aseptically transferred using a sterilized needle into fresh broth culture. The tubes will be

incubated at 37C for 24 hours. For standardization of bacterial density, the turbidity of the

fresh 24hour old cultures will be adjusted to 0.5 MacFarland standard (1 108 cells/ml)

by diluting with the sterile broth. (18)(21)

f) Paper disk diffusion assay

The plates with nutrient agar will be divided into 4 equal parts with a marking

pencil on the bottom of the plates. A sterile cotton swab will be aseptically dipped into the


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S. aureusbroth culture. The cotton swab will be rotated against the inside wall of the tube

to remove excess broth, then the swab will be streaked evenly over the surface of the

plate and in three directions, leaving no gaps in between strokes. The plate will be allowed

to dry for 3-5 minutes with the lid in place. Filter paper disks (6mm) will be impregnated

with the respective treatments and controls by dropping 1mL to each disk using a

micropipette then will be allowed to dry for 10 minutes. The individual disks will then be

placed and pressed lightly on the surface of the nutrient agar in the different sections

marked on the bottom of the plates. The plates will be incubated at 37C for 24 hours. The

zone of inhibition will be measured (in millimeters) after overnight incubation. (19)(23)

g) Determination of MIC

Two-fold broth dilution test method will be used in determining the minimum

inhibitory concentration of the plant extracts. In ten sterile tubes labeled 1 through 10,

0.5ml of sterile broth will be aseptically added. Then, 0.5 ml of the peel extract will be

added to tubes 1 and 2. Serial dilution of the peel extracts will be carried out from tube 2

until tube 9. All the tubes will then be inoculated with 0.5 mL of S. aureussuspension and

incubated at 37C for 24 hours. Tube 10, containing only the sterile broth and S. aureus

suspension will serve as the control tube. Bacterial growth in each tube will be examined

by observing turbidity. The highest dilution without growth is the minimal inhibitory

concentration.

Possible biases and limitations

Although there were no significant experimental biases recognized in the related

literatures that were reviewed, a possible researcher bias, wherein the prior knowledge of

the researchers might affect the analysis of the results (24), may be encountered in the

study. Observational bias may also be encountered among the researchers along the

course of the experimental proper, wherein there may be discrepancies in the manner of

implementing the experiment procedure as well as in measuring the outcome observed.

The in vitrosetting of the study may be accounted to the limitation of the external

validity of the findings.(25) Extract activity observed in a controlled setting may be observed

differently when applied in the clinical setting.

The possible sources of errors in the study may come from faulty observation of

preparation and storage techniques of both the inoculum and plant extracts. Application of

the inoculum may contribute to the inaccuracy of results. Thickly applied inoculum onto the


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agar may result to a small zone of inhibition; whereas, a thinly applied inoculum may result

to a large zone of inhibition.(18)(21) Inadequate or haphazard application of plant extracts as

well as those of the controls on to the paper disks also contribute to the inaccuracy of

results.(18)

Control plan for biases and possible errors

The study will be conducted in an isolated controlled unit set at the temperature of

37C to minimize the possible effect of environmental factors. A facilitator with

microbiology expertise will be supervising the study in order to monitor errors in

preparation and storage techniques throughout the duration of the study. The principle of

blinding will be applied in the execution of the study and data collection. Three members

will be assigned solely to the execution of the experiment, observation, and data

collection. Another subgroup of three members will determine what type of treatment will

be assigned to a particular sample. The remaining three members will be responsible for

the result analysis, so as to minimize the occurrence of possible researcher bias.

Assigned members will also be oriented with the flow of the experiment proper (as

illustrated in the procedure schematic diagram) and proper data collection in order to

minimize the occurrence of observational bias.

C. Sampling

Selection of treatment and control

Selection for the type of banana species that will be utilized for the study will be

based on the species availability and accessibility to the public. Different banana species

that were common in the province of Cavite were determined. Based on the provinces

consolidated production report for the month of August 2011, Musa acuminata (lakatan)

garnered the top sales, followed by Musa balbisiana (saba) and Musa paradisiaca

(latundan).(4) Choices for banana species were further specified as to their distribution

among the districts of the province. Although majority of the local banana cultivars are

grown in the upland districts (i.e. Districts V VII), the purchase of the banana samples

will be done at the most convenient district for the study, the 4 th district of Dasmarinas City

(26). Figure 4 depicts the selection process for the independent variable of the study.


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Figure 4: Selection process for banana species

One subgroup consisting of three members will be in-charge of treatment allotment.

Nutrient agar plate will be used for observing the activity of extracts from different banana

species (Musa paradisiaca, Musa balbisianana and Musa acuminata) in different

concentrations compared with a positive and negative control. Control groups as well as a

solvent group will be set up so as to validate that the intended effect is indeed attributed to

the independent variable of the study. Vancomycin, the drug of choice for Staphylococcus

aureuswill be used as a positive control, evaluating the antibacterial activity of the banana

peel extracts (27). On the other hand, distilled water will be used as the negative control for

the study to serve as a reference reflecting microbial growth (11). The disk diffusion assay

will be replicated three times.

Sample size

The type of significance test to be done depends on the hypothesis. In this study,

since the null hypothesis states that the peel extracts obtained from the different species

of banana do not have comparable antibacterial activity against Staphylococcus aureus,

therefore a two-tailed test will be employed because the null hypothesis does not tell the

specific direction of difference.(28)

PROVINCE OF CAVITE

BANANA-GROWING

DISTRICTS

DISTRICT V DISTRICT VI DISTRICT VII

DISTRIBUTION/EXPORT OF

BANANAS TO OTHER DISTRICTS

DISTRICT IV


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In comparing the antibacterial activity of different species of banana, three controls

and three treatments will be used. Antibacterial activity of the peel extracts obtained from

Musa acuminata, Musa balbisiana, and Musa paradisiaca will be observed. For the

control, a positive control containing Vancomycin and a negative control containing

distilled water will be used. Methanol will be used for the solvent group. The zone of

inhibition observed for each concentration per treatment and control group will be

measured using a 6-inch ruler.

Determining the number of replicates to be used in an experiment is a matter of

judgement and the available resources. The following formula incorporating the

probabilities for type I and type II errors as well as the variance and true difference

anticipated will be utilized in computing for the number of replicates: (29)

# of replicates = 2 (Z/2

+ Z)(/)2

Wherein Z/2 isassociated with Type I error; Z is associated with Type II error; is

associated with standard deviation; and is associated with the anticipated true difference

that might be detected among replicates.

Values obtained for the formula were based from previous experiments on the

antibacterial activity of a particular species.(11) The study will be set a confidence level of

95%, with a power of 80%. Based on these percentages, the following Z values for Type I

and Type II error are the following:

Z/2: = 0.05 (at 95% confidence level) Z: = 0.80/2= 0.025 Z value = 0.84

= 1 - 0.025= 0.975

Z value = 1.96

Value for standard deviation is set at = 0.1, based on previous experiments. Avalue of 0.14 is set for the anticipated true difference.

Calculation:

# of replicates = 2 (Z/2+ Z )(/)2

= 2 (1.96+0.84)(0.1/0.14)2

= 2 (2.8)(0.51)

= 2.86

~ 3 replicates will be used


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Reference citations

In several studies investigating the antibacterial activity of unripe banana peels

conducted by Alisi, et. al and Sulaiman, et. al, n=3 was used for the number of replicates

with a significance of p = 0.05. Alisi, et. al made use of a two-way analysis of variance

(ANOVA) to determine the inhibition of bacterial dehydrogenase activity reflected in

dehydrogenase assays. (30) On the other hand, Sulaiman, et. al utilized one-way ANOVA

testing to establish the correlations between total phenolic and mineral contents with the

antioxidant activities of the pulp and peel obtained from 8 different cultivars. (31)

D. Data collection

Sources of data (variables to be measured)

The independent variable in the study is the antibacterial activity of the banana peel

extracts from different species whereas the dependent variable is the inhibition of growth

of Staphylococcus aureus. Paper disk diffusion method and broth dilution tests will be

performed to collect the data needed in determining the antibacterial activity of different

banana species against Staphyloccous aureus. The zones of inhibition and minimal

inhibitory concentration can be obtained, respectively.

Method to be used

Results from the aforementioned tests will be determined through observation of the

presence or absence of microbial growth in the agar plate for the disk diffusion method, as

well as in the series of test tubes for MIC. The observation method is the preferred method

in the context of a laboratory experimental study. Data collection is done by making use of

standardized measurement methods and utilizing replications to achieve precision. (32) In

the study, a schematic diagram of the step-by-step procedure will be developed to ensure

that uniformity in the observation process and data collection will be implemented among

the assigned observers. Three replicates per banana species for the disk diffusion method

will be prepared to assure the precision of the observed antibacterial activity.

Prior to the experiment proper, the group will be divided into three subgroups for the

following tasks: 1) preparation of plant extract; 2) execution of antibacterial susceptibility

tests and data collection; and 3) data analysis. Members assigned in a particular subgroup

will be oriented to the schematic process to ensure a smooth execution of the study and

uniformity in the observation process, thus reducing a possible researcher and


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observational bias. The principle of blinding, as well as plans for controlling bias and other

possible errors have been discussed in the studys Research Design section under the

main point, Methodology.

In addition, available calipers or rulers will be calibrated to warrant accurate

measurement of zones of inhibition and extract concentration for the disk diffusion method

and broth dilution tests, respectively.

Data collection tool

Collection tools provide the necessary data for analysis; in this respect, tools should

be strong enough in order for a research to yield a claim. Data collection tools are mainly

categorized into three: secondary participation, in-person observations, case studies and

analysis content.(33)In experimental designs however, these tools are rarely used. Instead,

the researchers use an array of experimental procedures to obtain the data.

The size of the zone of inhibition obtained from the disk diffusion method is directly

proportional to the sensitivity of the organism to the antibiotic,(18)(19) which in this particular

study is the sensitivity of Staphylococcus aureus to peel extracts of different banana

species. Infections due to organisms designated as sensitive to a given antibiotic are more

likely to respond clinically to that antibiotic.(34) The measured zones of inhibition from the

three banana test species will be recorded in millimeters. (21)

Antibiotic sensitivity expressed in the context of the minimal inhibitory concentration

gives quantitative data not obtainable with the disk diffusion method. MIC is identified as

the smallest concentration of antibiotic that inhibits the growth of the test bacterium; thus,

these quantitative results are useful in predicting the amount of antibiotic that must be

attained to assure inhibition.(19)Results of MIC will be recorded in g/mL.

The data obtained will then be organized in tables, as shown in the following section

(Tables I III). This functions as the data sheet but also aids in the analysis of the data.

The tables provide the analyst of the experiment the specific data required for the

evaluation of the hypotheses. Data collected from disk diffusion method will be recorded in

Tables I and II, for the treatment and control respectively. The measured zone of inhibition(mm) from the three replicates of different concentrations and species will be recorded and

their respective means standard deviation will be computed. In this way, the analyst can

assess the variation among the zones of inhibition observed due to possible errors and

discrepancy of the measurements.


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Data collected from MIC, on the other hand, will be recorded in Table III. The

resulting concentration of peel extracts after serial dilution using a series of ten tubes as

well as observation for microbial growth in each tube will be recorded. Presence of

turbidity in the tube indicates growth. (36)

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(8) Carlos, C.C. (2006) The 2005 antimicrobial resistance surveillance data. PIDSP Journal, 10, (9 pages).

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(18) Mahon, C.R., Lehman, D.C., and Manuselis, G. (2007). Textbook of Diagnostic Microbiology. Missouri:Saunders.

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(21) Department of Microbiology and Parasitology, DLS-HIS College of Medicine (2011). Laboratory Manual onMicrobiology and Parasitology.

(22) Edwards, B. G. (2003). Banana peel extract composition and method for extraction. Delft PharmaInternational, Ogden Utah. Retrieved from:http://www.freepatentsonline.com/5989559.pdf

(23) World Health Organization. (2006). Blood Safety and Clinical Technology: Guidelines on Standard OperatingProcedures for Microbiology. Retrieved from: http://www.searo.who.int/en/section10/section17/section53/section482_1786.htm

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Systems(Chapter 6: Experimentation). Retrieved from:http://www.fao.org/docrep/w3241e/w3241e07.htm#chapter 6: experimentation

(26) Republic Act No. 9272 An Act Reapportioning the Province of Cavite into Seven (7) Legislative Districts.Retrieved fromhttp://www.senate.gov.phlast September 6, 2011.

(27) World health Organization.http://www.who.int

(28) UCLA Academic Technology Services. What are the differences between one-tailed and two-tailed tests?.http://www.ats.ucla.edu/stat/mult_pkg/faq/general/tail_tests.htm. Retrieved last 11 September 2011.

(29) North Dakota State University. Siza of an experiment the number of replicates to use. Retrieved from:http://www.ndsu.edu/ndsu/horsley/ExptSize.pdf, 26 September 2011.

(30)Alisi, C.S., et. al. (2008) Inhibition of dehydrogenase activity in pathogenic bacteria isolates by aqueous

extracts of Musa paradisiaca(var. Sapientum). African Journal of Biotechnology. Vol. 7 (12), pp. 1821-1825.

(31) Sulaiman, S.F. et. al. (2011) Correlation between total phenolic and mineral contents with antioxidantactivity of eight Malaysian bananas (Musasp.). Journal of Food Composition and Analysis. Vol. 24 (1), pp1-10. Retrieved from:http://www.sciencedirect.com/science/article/pii/S0889157510001985, 12 September2011.

(32) McKubre, M.C.H. (2008) The Importance of Replication. ICCF-14 International Conference on CondensedMatter Nuclear Science.

(33) The Three Main Types of Data Collection Tools. (n.d.). Retrieved October 1, 2011, from Scienceray:http://scienceray.com/technology/information/the-three-main-types-of-data-collection-tools/

(34) Vasanthakumari (2007). Practical Microbiology. BI Publications Pvt Ltd. Copyright. http://books.google.com/

(35) Parija,S.C.(2009).Textbook of Microbiology & Immunology. Elsevier India

Copyright.http://books.google.com/

(36) Neelima Garg, K. L. Garg and K. G. Mukerji. (2010). Laboratory Manual of Food Microbiology. I. K.

International Pvt Ltd. Copyright. pp. 75-78. http://books.google.com/
http://encyclopedia.thefreedictionary.com/http://encyclopedia.thefreedictionary.com/http://encyclopedia.thefreedictionary.com/http://www.freepatentsonline.com/5989559.pdfhttp://www.freepatentsonline.com/5989559.pdfhttp://www.freepatentsonline.com/5989559.pdfhttp://www.pairbondpublications.com/for_students/online_glossaryhttp://www.pairbondpublications.com/for_students/online_glossaryhttp://www.pairbondpublications.com/for_students/online_glossaryhttp://www.senate.gov.ph/http://www.senate.gov.ph/http://www.senate.gov.ph/http://www.who.int/http://www.who.int/http://www.who.int/http://www.ats.ucla.edu/stat/mult_pkg/faq/general/tail_tests.htmhttp://www.ats.ucla.edu/stat/mult_pkg/faq/general/tail_tests.htmhttp://www.ndsu.edu/ndsu/horsley/ExptSize.pdfhttp://www.ndsu.edu/ndsu/horsley/ExptSize.pdfhttp://www.sciencedirect.com/science/article/pii/S0889157510001985http://www.sciencedirect.com/science/article/pii/S0889157510001985http://www.sciencedirect.com/science/article/pii/S0889157510001985http://scienceray.com/technology/information/the-three-main-types-of-data-collection-tools/http://scienceray.com/technology/information/the-three-main-types-of-data-collection-tools/http://books.google.com/url?id=F3tSac2PGicC&pg=PT57&q=http://www.bipgroup.com&clientid=ca-print-pub-7392917442895701&channel=BTB-ca-print-pub-7392917442895701+BTB-ISBN:8172253184&linkid=1&usg=AFQjCNFQuURFHID5U3RHCpaDOhRPBcbD3A&source=gbs_pub_info_s&cad=3http://books.google.com/books?idhttp://books.google.com/url?id=HcgGLfxDJSQC&pg=PA72&q=http://www.elsevierhealthindia.com&clientid=ca-print-reed_elsevier_india_pvt_ltd&channel=BTB-ca-print-reed_elsevier_india_pvt_ltd+BTB-ISBN:8131221636&linkid=1&usg=AFQjCNHSklgu2Bxdck2MwPtYxZ84C5sYPA&source=gbs_pub_info_s&cad=2http://books.google.com/books?id=HcgGLfxDJSQC&printsec=copyright&source=gbs_pub_info_s&cad=2http://books.google.com/http://books.google.com/http://books.google.com/http://books.google.com/url?id=8h4Ze5s6sFUC&pg=PA77&q=http://www.ikbooks.com&clientid=ca-print-pub-2792127120201681&channel=BTB-ca-print-pub-2792127120201681+BTB-ISBN:9380578016&linkid=1&usg=AFQjCNHqWlBEDx9hxlaHGnHzP0KasPTerA&source=gbs_pub_info_s&cad=3http://books.google.com/url?id=8h4Ze5s6sFUC&pg=PA77&q=http://www.ikbooks.com&clientid=ca-print-pub-2792127120201681&channel=BTB-ca-print-pub-2792127120201681+BTB-ISBN:9380578016&linkid=1&usg=AFQjCNHqWlBEDx9hxlaHGnHzP0KasPTerA&source=gbs_pub_info_s&cad=3http://books.google.com/books?id=8h4Ze5s6sFUC&printsec=copyright&source=gbs_pub_info_s&cad=3http://books.google.com/books?id=8h4Ze5s6sFUC&printsec=copyright&source=gbs_pub_info_s&cad=3http://books.google.com/url?id=8h4Ze5s6sFUC&pg=PA77&q=http://www.ikbooks.com&clientid=ca-print-pub-2792127120201681&channel=BTB-ca-print-pub-2792127120201681+BTB-ISBN:9380578016&linkid=1&usg=AFQjCNHqWlBEDx9hxlaHGnHzP0KasPTerA&source=gbs_pub_info_s&cad=3http://books.google.com/url?id=8h4Ze5s6sFUC&pg=PA77&q=http://www.ikbooks.com&clientid=ca-print-pub-2792127120201681&channel=BTB-ca-print-pub-2792127120201681+BTB-ISBN:9380578016&linkid=1&usg=AFQjCNHqWlBEDx9hxlaHGnHzP0KasPTerA&source=gbs_pub_info_s&cad=3http://books.google.com/http://books.google.com/books?id=HcgGLfxDJSQC&printsec=copyright&source=gbs_pub_info_s&cad=2http://books.google.com/url?id=HcgGLfxDJSQC&pg=PA72&q=http://www.elsevierhealthindia.com&clientid=ca-print-reed_elsevier_india_pvt_ltd&channel=BTB-ca-print-reed_elsevier_india_pvt_ltd+BTB-ISBN:8131221636&linkid=1&usg=AFQjCNHSklgu2Bxdck2MwPtYxZ84C5sYPA&source=gbs_pub_info_s&cad=2http://books.google.com/books?idhttp://books.google.com/url?id=F3tSac2PGicC&pg=PT57&q=http://www.bipgroup.com&clientid=ca-print-pub-7392917442895701&channel=BTB-ca-print-pub-7392917442895701+BTB-ISBN:8172253184&linkid=1&usg=AFQjCNFQuURFHID5U3RHCpaDOhRPBcbD3A&source=gbs_pub_info_s&cad=3http://scienceray.com/technology/information/the-three-main-types-of-data-collection-tools/http://www.sciencedirect.com/science/article/pii/S0889157510001985http://www.ndsu.edu/ndsu/horsley/ExptSize.pdfhttp://www.ats.ucla.edu/stat/mult_pkg/faq/general/tail_tests.htmhttp://www.who.int/http://www.senate.gov.ph/http://www.pairbondpublications.com/for_students/online_glossaryhttp://www.freepatentsonline.com/5989559.pdfhttp://encyclopedia.thefreedictionary.com/


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Documents