TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES

ABSTRACT

Chapter I INTRODUCTION 1.1 Research question

Which species of banana peel extract has greater antibacterial activity against Staphylococcus aureus?

1.2

Research hypothesis
1.2.1 Working hypothesis

The peel extracts obtained from the different species of banana have comparable antibacterial activity against Staphylococcus aureus.

1.2.2

Null hypothesis

The peel extracts obtained from the different species of banana do not have comparable antibacterial activity against Staphylococcus aureus.

1.3

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 3

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 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 sapientum species 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.

1.4

Rationale for 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 4

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.

1.5

Review of literature and conceptual framework

1.5.1 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. aureus among patients. A study 5

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 communityacquired 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)

1.5.2

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 by the World 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 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)

1.5.3

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.

1.5.3.1

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 and antioxidant activity. Among the specific compounds isolated from green banana peel, d-malic acid and 12hydroxystearic exhibited the most active antimicrobial response.(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 paradisiaca and Musa sapientum showed 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 7

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 Musa sapientum, L. subsp. Sylvestris fruits (MSSE). The methanolic extract of Musa sapientum peel 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 (Psidium guajava); ripe, unripe and leaves of starfruit (Averrhoa carambola); ripe and unripe banana (Musa sapientum variety 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 (Baccaurea motleyana) peel were evaluated for antimicrobial activity against Gram-positive bacteria, Gram-negative bacteria, yeast and fungi (Staphylococcus aureus, Bacillus subtilis, 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 8

yeast or fungi. Extracts from bananas, papayas, passionfruit peel, Lansium domesticum peels and rambutan peels showed activity against Candida lypolytica while extracts from guava showed strong activity against Saccharomyces cerevisiae. Unripe banana showed activity against all the bacteria except towards P. vulgaricus.(17) The study conducted by Mokbel and Hashinaga used both fresh green and yellow banana peel of Musa, cv. Cavendish fruits which were treated with 70% acetone; and afterwards, partitioned with chloroform (CH[Cl.sub.3]) and ethyl acetate (EtOAc). 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. Antimicrobial activities of the extracts and isolated components were evaluated using paper disk methods and minimum inhibition concentration (MIC). The EtOAc and water soluble fractions of green peel showed high antimicrobial and antioxidant activity. The antioxidant activity of water extracts was comparable to those of synthetic antioxidants such as butylated hydroxyanisole and butylated hydroxytoluene. Among all isolated components, B-sitosterol, malic acid, succinic acid, palmatic acid, 12-hydroxystrearic acid, d-malic and 12-hydroxystrearic acid were most active against the Gram-negative and Gram-positive bacteria species tested. The MIC of d-malic and succinic acid varied between 140-750 ppm, respectively. (11) 1.5.3.2 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. 9

1.5.3.3

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. 1.5.3.4 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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1.5.4

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 extract

Characteristics of the factor Type of solvent used Type of phytochemicals present which contributes to antibacterial activity Concentration of the extract

Inhibition of growth of Staphylococcus aureus

Characteristics of the factor Inherent defense mechanisms of the sample of interest

POSSIBLE CONFOUNDING FACTORS Exposure to environmental factors Preparation and storage techniques

CONTAMINATION

Figure 1. Conceptual framework

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Chapter II OBJECTIVES
Given the previous data, the study conducted was generally aimed at determining which species of banana peel extract has greater antibacterial activity against Staphylococcus aureus. In order to achieve the general objective, the following specific objectives were met: 1. To measure the zones of inhibition of peel extracts obtained from Musa acuminata, Musa balbisiana, and Musa paradisiaca using the disk diffusion method. 2. To establish the respective minimal inhibitory concentrations of peel extracts b from Musa acuminata, Musa balbisiana, and Musa paradisiaca using broth dilution test. 3. To compare the obtained zones of inhibition and minimal inhibitory concentrations of the peel extracts against Staphylococcus aureus.

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Chapter III METHODOLOGY

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Chapter IV RESULTS 4.1 Descriptive statistics

The antibacterial activity of the peel extracts obtained from Musa acuminata, Musa balbisiana, and Musa paradisiaca were initially determined using the disk diffusion method. Each species were prepared in three concentrations (30 g, 300 g, and 3,000 g) and were observed for their respective antibacterial activity as evidenced by the zones of inhibition produced. However, paper disk diffusion results revealed absence of inhibition in all species. Difficulty in replicating the methods from previous studies wherein zones of inhibition were observed brought about the need to conduct trials in determining the antibacterial activity of the peel extract. In each trial, the extract concentration used was set at 3,000 g. Several variables such as characteristics of the banana peel (e.g. color and storage duration), duration of blending the chopped peels, and the extract storage temperature were considered. Values for mean + standard deviation are then considered to be set at zero, since the values gathered per trial were null.(6) Table 1 shows the measurements of the zones of inhibition among the different banana species.

Table 1. Zone of inhibition (mm) of peel extracts of different banana species using paper disk diffusion in different trials

TRIAL 1 Musa sapientum Musa paradisiaca

TRIAL 2

TRIAL 3

TRIAL 4

TRIAL 5

TRIAL 6

14

Musa acuminata

4.2

Inferential statistics
Since the values obtained were set at zero, replacing these on the F-ratio (i.e. statistic used in utilizing ANOVA) would reveal that the p-value would progress towards a positive infinity, implying that the value of statistical significance is within the non-rejection area of the normal distribution curve.(7) A conclusion can then be made that there are no sufficient evidences to reject that the peel extracts obtained from the different species of banana do not have comparable antibacterial activity against Staphylococcus aureus, in favor of the alternative hypothesis.

(insert calculation; insert curve)

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