ii

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A Thesis Presented to the

Faculty of the College of Medical Laboratory Science
Liceo de Cagayan University
Cagayan de Oro City

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In Partial Fulfillment
of the Requirements for the Degree
Bachelor of Science in Medical Laboratory Science

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Maria BJ Theresa A. Aque

Alan Louise Y. Bualat

Umaynah E. Ahmed
Liceo de Cagayan University College of Medical Laboratory Science Page

JUNE 2023
iii

APPROVAL SHEET

This thesis entitled ANTIBACTERIAL ACTIVITY OF MANILA PALM (Adonidia

merrillii (Becc.) Becc.) FRUIT EXTRACT AGAINST Escherichia coli AND
Staphylococcus aureus, in partial fulfillment of the requirements for the Bachelor of Science
in Medical Laboratory Science prepared and submitted by ALAN LOUISE Y. BUALAT,
UMAYNAH E. AHMED and MARIA BJ THERESA A. AQUE has been examined,
accepted and recommended for Oral examination.

MARILOU O. HONCULADA, RMT, MA, MSMLS

Adviser

PANEL OF EXAMINERS

APPROVED in partial fulfillment of the requirements for the Bachelor of Science in

Medical Laboratory Science by the Panel of Examiners with a grade of ____.

DR. AMELDA C. LIBRES, RMT, MA, DODT

Chairman

MICHELLE T. MABASA, RMT, MA, MSMLS

Member

MARY ABIGAIL M. PAULAN, RMT, MSMLS

Member

Date of Final: :

DR. AMELDA C. LIBRES, RMT, MA, DODT

Liceo de Cagayan University College of Medical Laboratory Science Page

Chairman
iv

ABSTRACT

The use of plants as medicine for fighting infections is controversial. The purpose of
this study was to determine the effectiveness of Manila palm (Adonidia merrillii (Becc.)
Becc.) ethanolic fruit extract in the inhibition of growth of Escherichia coli and
Staphylococcus aureus. The ethanolic fruit extract was obtained through rotary evaporation
and was screened for phytochemical compounds using qualitative methods. Two
concentrations of the fruit extract were then impregnated to filter paper discs, namely 75%
and 100% concentration, with other discs containing ciprofloxacin as the positive control and
distilled water as the negative control.

Keywords: Escherichia coli, ethanolic extract, flavonoids, Manila palm, phytochemical

screening, Staphylococcus aureus, susceptible, zone of inhibition
Liceo de Cagayan University College of Medical Laboratory Science Page v

ACKNOWLEDGMENT

The researchers would like to share their deepest gratitude for the help and support to

following persons who in one way or another have contributed to the success in making the

study. 

Mrs. Marilou O. Honculada, RMT, MAEd, MSMLS their research adviser, for her

support, advice, guidance and suggestion that have benefited in the completion and success

of the study. The researchers are also immensely grateful for her overwhelming support,

patience and valuable comments that greatly improved the study. 

Dr. Amelda C. Libres, RMT, MA, DODT, Dean of the College of Medical

Laboratory Science, for approving the researchers’ request to conduct the experiment in one

of the MLS Laboratories. The researchers also appreciate her support, understanding and for

providing them her insights and expertise that also contributed to the success of the study. 

Mrs. Michelle T. Mabasa, RMT, MAEd, Academic chair of the College of Medical

Laboratory Science, for the approval of our research paper. The researchers also appreciate

her guidance and suggestions that contributed to the success of the study. 

Mr. Rogin A. Alamban, RMT, their research adviser assistant, for guiding the

researchers during the implementation of their experiment. The researchers appreciate him

for sharing his knowledge and criticism, effort, time and guidance in our research procedure. 

Sir Patric Manuel Villaces and Maam Hannah Claudine Gallardo, the

researchers’ laboratory custodians, for their support and guidance in conducting the

experiment. There researchers are also sincerely thankful for sparring their time despite their

busy schedule. 
Liceo de Cagayan University College of Medical Laboratory Science Page vi

Dr. Dominic T. Polancos, PhD, LPT, their research instructor and statistician, for

helping and guiding the researchers in their study. 

Dr. Mercedita S. Aquino, RP, MA, DODT, Dean of the College of Pharmacy, for

approving the researchers’ request to conduct Rotavap and Soxhlet in their laboratory for the

extraction of Manila Palm (Adonidia merrillii) fruit extract. 

Sir Nikko Sabellano, for sharing his knowledge, effort, time and guidance in

operating the rotary evaporator and soxhlet. 

Mr. Mel Albert Ederango, Mr. Allein Josef Lumacad, Mr. Carl Israel Acero,

Mr. Jon Lee Castro, Mr. Brielle Kyle Serrano, Ms. Harshey Agcaoili, and Ms. Bea

Juliana Gelacio, the researchers’ classmates, who willingly helped them with their ability.

The researchers’ family, friends and loved ones, for their unconditional love, trust,

encouragement and financial assistance that made the study possible. The researchers are

also thankful for supporting them financially. 

Liceo de Cagayan University College of Medical Laboratory Science Page vii

TABLE OF CONTENTS

                                                                                                       Page

COVER PAGE i

TITLE PAGE ii

APPROVAL SHEET iii

ABSTRACT iv

ACKNOWLEDGEMENT v

TABLE OF CONTENTS       vii   

LIST OF FIGURES ix

LIST OF TABLES x

Chapter

       1 THE PROBLEM AND ITS SCOPE

Introduction 1

Conceptual Framework 4

Objectives of the Study 6

Hypothesis 6

Significance of the Study          7

Scope and Limitation of the Study 8

Definition of Terms 8

       2 REVIEW OF RELATED LITERATURE                               10   

 
       3 METHODOLOGY

Research Setting 20
Liceo de Cagayan University College of Medical Laboratory Science Page viii

Research Design 20

Research Instrument        21

Data Gathering Procedure 21

4 PRESENTATION, ANALYSIS, AND 27

INTERPRETATION OF DATA

5 SUMMARY, CONCLUSION, AND 36

RECOMMENDATIONS

REFERENCES 40

APPENDICES

A. Letter of Approval 46

B. Completion Report 48

C. Certification of Plant 50

D. Letter of Consent for Laboratory Use 51

E. Letter of Consent for Laboratory Use 53

F. Documentation 55

G. Descriptive Statistics for the zones of inhibition of 61

Adonidia merrilli extract in millimeters (mm)

against Staphylococcus aureus

H. Kruskall Wallis H Test 62

I. Summary of the Hypothesis Test 63

J. Curriculum Vitae 64
Liceo de Cagayan University College of Medical Laboratory Science Page ix

LIST OF FIGURES

FIGURE           TITLE                         PAGE

     1 Schematic diagram showing the 5

interplay of variables
Liceo de Cagayan University College of Medical Laboratory Science Page x

LIST OF TABLES
Table Title Page

1 27

2 28

3 Comparison between the zones of inhibition of Adonidia merrilli 30

extract in millimeters (mm) by the positive control, negative control
and the experimental control group against Escherichia coli and
Staphylococcus aureus

4 Significance level of the two concentrations from Kruskal Wallis H 32

Test

5 Kruskal Wallis H Test with the corresponding significance level of the 34

experimental groups and control groups and their interpretations
Liceo de Cagayan University College of Medical Laboratory Science Page | 1

Chapter 1

THE PROBLEM AND ITS SCOPE

Introduction

Plants remain imperative in the continuous survival of human beings. It fulfills our

basic needs for living, especially in the present day where environmental and health problems

seem to be the new normal. With the diverse purposes that plants have, one of them is being

a medicinal and therapeutic alternative. Although synthetic drugs are now available in the

market, it can still be an issue especially for those who are in the low-income brackets to

maintain prescribed medications. Moreover, people coming from rural areas tend to choose

Filipino traditional medicines over synthetic drugs due to it being culturally accepted for

centuries (Lazarte, 2020). Despite plants' beneficial attributes, there is still plenty of room in

the research community that will support plants’ effectiveness towards fighting infection,

specifically the utilization of the fruit of Manila Palm.

Manila palm (Adonidia merrillii) a type of palm tree belonging to the

Arecaceae/Palmae family, is known as the manila palm. One of the most well-liked

ornamental palms in the entire world is this palm tree. It is indigenous to East Malaysia's

Sabah and the Philippine islands of Palawan, where it is widely dispersed. It flourishes in

tropical areas, which explains it being bountiful in the Philippines. The tree measures 10 m

tall and 15-20 cm diameter. The fruit is oval and measures about 3–4 cm long. At maturity,

the fruit's color changes from pale green to vivid red. The leaves are pinnate with sharply

defined arches. The fruit are being utilized by some cultural practices as an alternative of the
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betel chewing nut and as a bead source (Sandoval, 2019). Phytochemical screenings of the

Manila palm fruit extract have been studied upon and revealed the cytotoxic activity of the

fruit extract, which is potential for antifungal and antibacterial activity (Essien, E.E., Antia,

B.S., Solomon, A.U., Cloudhary, M.I., 2017). 

Over the years, clinical and technological advancements gave us an array of options

on how we can deal with bacterial infections such as antibiotic drugs. However,

antimicrobial resistance (AMR) incidences are having a continuous rise as microorganisms

(such as bacteria, fungi, viruses, and parasites) are responding to the antimicrobial drugs and

developing AMR, making drugs lose their effectiveness (Zhu, Huang, and Yang, 2022).

Backed up by the study of Rai, Randhawa, and Kaur (2013), the global clinical problem

might just lead to the return of the pre-antibiotic era, or in other words, herbal or plant-based

medicine. 

The Philippines is known for its abundance of natural resources and utilizing the

nutritional advantages it can give to every living organism. Generations have passed,

practices and beliefs with usage of herbal plants for therapeutic and medicinal purposes still

continue to prosper and is even one of the most studied topics in the research community in

the modern days. Medicinal plant extracts should be given high priority to characterize them

as potential antimicrobial agents (Vao, Stavropoulou, Voidarou, Tsigalou, and Bezirtzoglou,

2021). Collecting (Adonidia merrillii) fruit extract as an antibacterial agent may produce the

answer to the problem. This plant might not be known by most people, but this tree’s fruit is

rich in a variety of phytochemicals which have been tested to have a strong cytotoxicity,

giving it a strong potential in having an antibacterial activity. 

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 According to Wu, Ding, Yao, Gao, and Wang (2021), extended spectrum beta

lactamase (ESBL), a plasmid-mediated β-lactamase that is capable of hydrolyzing and

inactivating β-lactams like as cephalosporins and monobactams, is a mechanism through

which E. coli strains can develop resistance to beta lactam antibiotics. E. coli is greatly

impenetrable to ampicillin, amoxicillin, tetracycline and trimethoprim & sulfamethoxazole.

These gram-negative bacteria are usually found in our guts, specifically in the lower intestine

of warm-blooded organisms. Most of E. coli are harmless, and are just normal dwellers in our

intestinal tract however, E. coli O157:H7, a certain type of this bacteria can cause clinical

manifestations such as abdominal cramps, bloody diarrhea, and even kidney failure (SGS,

2017). 

 A gram-positive bacterium, Staphylococcus aureus, is responsible for a wide range

of clinical illnesses. These bacteria frequently cause infections, both community and

nosocomial acquired. On healthy skin, S. aureus often does not cause illness, but if it is able

to reach internal tissues or the bloodstream, these microbes may cause numerous potentially

dangerous infections (Taylor and Unakal, 2022). 

Given the facts aforementioned, this research will greatly serve as an additional

reference for the research community about the potential of this plants’ fruit extract and to

also be encouraged in using the other parts of Manila palm for other beneficial purposes. This

study will also greatly contribute to the pharmaceutical section and may develop drugs

incorporating this plants’ fruit extract. Very few studies are available on Manila palm which

is why delving more to its capabilities as a potential medicine against S. aureus and E. coli
Liceo de Cagayan University College of Medical Laboratory Science Page | 4

infection is the purpose of this study with a goal of battling the worrying resistance of

microorganisms with the available medicinal drugs available today.

Conceptual Framework 

This study was anchored with the study of Essein, et.al. (2017), where the Manila

palm (Adonidia merrillii) and Archontophoenix tukeri methanolic fruit extract was utilized

and screened for phytochemical compounds and it’s in vitro cytotoxic, antioxidant, and

antimicrobial activity. The extract showed a promising result where it exhibited antioxidant

activity and cytotoxicity. In the phytochemical screening, it was found out that flavonoids,

phenols, and deoxy sugars were highly present in the methanolic fruit extract. According to

Xie, Yang, Tang, Chen and Ren (2015), flavonoids are well known for their antibacterial

effect on a wide range of pathogenic microorganisms by inhibiting nucleic acid synthesis,

cytoplasmic membrane function, and more. 

In this study, the research paradigm that is used is IV-DV model and is further

detailed in the diagram below.

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Scope and limitation of the Study

The actual testing of this research takes place at MLS Laboratory 2 & 3 of the

College of Medical Laboratory Science located at the Fourth Floor of Liceo de Cagayan

University, Paseo del Rio Campus, Rodelsa Circle, Macasandig, Cagayan de Oro City,

Philippines. A timeline of first and second semester of the academic year 2022-2023 were set

to cover the processes involved in this research. 

Liceo de Cagayan University College of Medical Laboratory Science Page | 6

Chapter 2

REVIEW OF RELATED LITERATURE

This chapter presents the related literature and studies after the thorough and in-depth

research done by the researchers. This also presents studies relating to Manila Palm,

Escherichia coli and Staphylococcus aureus and concepts that are relevant to the present

study.

Manila Palm

          A monotypic genus of flowering plants among the Arecaceae family is Adonidia which

includes the species, Adonidia merrillii (the Manila Palm). In recent years, a number of

biological properties have been reported in this family (Vafaei, 2013). In the study of Vafaei

et al. (2018), the results demonstrated that the fruit extracts from Adonidia merrillii have

significant anti-oxidant and anti-cytotoxicity capabilities. Therefore, future research into the

fruit could lead to the discovery of antioxidant and cytotoxic medications derived from

natural sources. Most people may be familiar with this plant, but they may not be aware that

its fruit is rich in a variety of phytochemicals that have been shown to have significant

cytotoxicity, giving it a high potential for antifungal and antibacterial activity.

Phytochemicals are plant-derived chemicals that act as a defense against harmful and

dangerous substances that can cause cell damage. According to multiple studies, medicinal

plants have bioactive components with particular physiological qualities that can be used to
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treat ailments. These bioactive constituents include alkaloids, phenolic compounds, tannins,

flavonoids, and terpenoids (Bala, Alon, Petiluna, 2019). Top Cultures in 2016 pointed out

that as long as plants exist then phytochemicals also take place. 

As stated in the study of Al-Shwyeh (2019), Date Palm (Phoenix dactylifera L.) with

the same family (Arecaceae), is widely consumed and has long been used for traditional

medicinal purposes. The fruits are nutrient-dense, as they contain dietary fibers, sugar,

protein, vitamins, minerals, flavonoid, and phenolic compounds. Date palm fruits are

antioxidant-rich and possess potent antibacterial bioactivities due to the presence of phenolic

compounds. On the basis of the evidence reviewed on Al-Shwyeh study, it was determined

that date fruits are an excellent source of natural antioxidants that can be used to treat

oxidative stress–related and infectious diseases.

Chemical elements are primarily derived from natural resources. They have been

employed in numerous traditions of complementary medicine. Following chemical profiling,

the most potent components of diverse plant extracts and essential oils from various plants

were discovered. (Bentrad et al., 2017). According to the study of Bentrad et al. (2017), when

date palm organic extracts were subjected to phytochemical analysis, three types of

flavonoids—anthocyanidins, C-glycosylflavones, and flavonols and flavones—were found.

As well as phenolic compounds, some of which have been found in the tegument of date

seeds, organic extracts of date seeds and pollen are antibacterial against both gram-positive

and gram-negative bacteria. Analyses of the antifungal capabilities of anthocyanidin

polymers have highlighted the significance of these compounds for a range of applications

using natural additives in the pharmaceutical and food sectors.

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The most diverse groups of phytochemicals are phenolics and flavonoids which are

able to protect the human body against reactive oxygen species (Galindo et al., 2010). A.

merrillii, due to its exotic appearance, is primarily grown for ornamental purposes. Several

ornamental palms have been used in traditional medicine and have been scientifically studied

to determine their medicinal value. Using standard protocols, A. merrillii methanol extract

was screened for phytochemicals and tested for cytotoxicity against various human

carcinoma cell lines, antioxidants, and antimicrobial activity. The presence of phenolics,

flavonoids, deoxysugars, cardiac glycosides, and saponins was revealed during the

phytochemical screening. The total phenolics, flavonoids, and tannin content of A. merrillii

were 52.05, 20.68, 2.58 mg/g, and 40.58, 32.73, 3.09 mg/g, respectively (Essien et al.,

2017). 

A comparable investigation was carried out to ascertain the total phenolic and

flavonoid content of methanol, ethyl acetate, and water as solvents, as well as the antioxidant

capacity and cytotoxicity of extracts from the A. merrillii fruit. RP-HPLC was also used to

analyze the qualitative and quantitative composition of phenolic and flavonoid compounds in

each extract. The study revealed that methanol extract produced the highest yield compared

to the other solvents employed. The analysis revealed that a 5 g sample of powdered dried

fruit from A. merrillii produced extract yields of 28.25 2.12%, 21 1.31%, and 14.75 1.83% in

methanol, ethyl acetate, and water, respectively. Analyses of the phenolics and flavonoids in

A. merrillii fruit extracts also revealed statistically significant differences (P0.05). The total

phenolic content of methanolic, ethanolic, and water extracts was determined to be 17.8, 7.6,

and 2.22 mg GAE per gram of dry weight, respectively. In contrast, the total flavonoid
Liceo de Cagayan University College of Medical Laboratory Science Page | 9

content of the methanolic, ethanolic, and water extracts was 5.43 mg Rutin/g DW, 3.12 mg

Rutin/g DW, and 1.11 mg Rutin/g DW, respectively. Gallic acid, pyrogallol, caffeic acid,

vanillic acid, and syringic acid were found to be the most prevalent phenolic acids according

to the HPLC investigation, but naringin and rutin are flavonoid components found in extracts

of the A. merrillii fruit. It was found that methanolic extracts had higher antioxidant activity

than ethyl acetate and water extracts using DPPH radical scavenging, NO scavenging, and

ABTS scavenging tests. It was determined that the IC50 concentrations of methanolic extract

for DPPH scavenging, NO scavenging, and ABTS scavenging activities were >1000 g/ml,

616.5 g/ml, and 884.8 g/ml, respectively. The compounds present in the extracts were non-

toxic, making them suitable candidates for anticancer drug development (Vafaei, A., 2013).

In vitro, the methanolic extract of A. merrillii nuts has a potential anthelmintic effect on F.

gigantica by affecting the fluke's motility and mortality time. The greater the concentration of

the extract, the greater its anthelmintic efficacy. Due to its ability to sever tegument and harm

reproductive organs, the extract is also extremely destructive to flukes (Vanda et. al., 2021). 

Cocos nucifera (L.) (Arecaceae) is the most naturally widespread fruit plant on Earth

and is commonly known as the coconut tree. C. nucifera contains compounds with various

biological effects, including antihelminthic, anti-inflammatory, antinociceptive, antioxidant,

antifungal, antimicrobial, and antitumor properties. Cocos nucifera is a plant with significant

pharmacological effects and low toxicity that is widely dispersed. The pharmacological

effects of the plant vary depending on which plant part or fruit is used. Antioxidant activity

predominated in the endocarp and coconut water constituents. The fiber also exhibited

antibacterial, antiparasitic, and anti-inflammatory properties. Only the ethanolic extract of the
Liceo de Cagayan University College of Medical Laboratory Science Page | 10

root had central nervous system depressant and anticonvulsant effects. Coconut water

appears to have protective effects, such as on the kidney and heart, as well as antioxidant and

hypoglycemic properties (Lima, et al., 2015). 

According to Palmco (2020), it is unfortunate that the Manila palm is underutilized

given its numerous attractive and useful characteristics. Manila palm is a flowering plant

species belonging to the Arecaceae family. The fruits of the Arecaceae family, despite being

rich in bioactive compounds with potential health benefits, have been underexplored. There

has also been little research into their composition, bioactive compounds, and the effects of

their consumption on health. It is critical to understand these underutilized fruits in order to

promote production, commercialization, processing, and consumption. It can also encourage

their full utilization, thereby improving the economy and social conditions of the people who

live in areas where these fruits are found. Furthermore, it may help future research on the

composition, health effects, and development of new products. 

Studies routinely report lauric acid as a major in the plants of this family, which

makes it a potential compound to cure or assist in the treatment of various diseases (Silva et

al., 2021). They are high in phenolic compounds, carotenoids, anthocyanins, tocopherols,

minerals, vitamins, amino acids, and fatty acids, according to studies. Further to that, these

compounds have been linked to anti-inflammatory, antiproliferative, anti-obesity, and

cardioprotective effects. Despite their potential, some of them have rarely been studied, with

very few research on their composition, biological effects, and applications. As a result, in

vitro and/or in vivo studies on the composition and mechanism of action of Arecaceae family

fruits are necessary (Morais et al., 2022). 

Liceo de Cagayan University College of Medical Laboratory Science Page | 11

Plants are regarded as a vital and primary source of beneficial properties for living

organisms. Secondary metabolites and bioactive compounds of plants have been in high

demand as therapeutic agents since the past century, as synthetic drugs have been shown to

have a wide range of adverse side effects. It is undeniable that naturally occurring

phytochemicals generated from plants have a range of biological functions, such as

inhibitory, cancerous tumor and microbial-fighting characteristics. In recent studies, the

activity of flavonoid and phenolic compounds has been discovered. These are confirmed as

the major secondary metabolites in plant extracts with biological activities. It has been

demonstrated that plant phenols are multifunctional antioxidants that act as singlet oxygen

quenchers, hydrogen-donating antioxidants, and reducing agents. In addition, a number of

important biological effects have been confirmed for flavonoids, such as antioxidative,

antitumor, antiviral, antifungal, antibacterial, and platelet aggregation-inhibiting properties.

They inhibit a wide range of diseases of the heart, circulatory system, kidneys, muscles,

lungs, and brain, and are incredibly beneficial for delaying the aging process, etc. (Delphine

et al., 2019).

As specified by Othman et al. (2019), several plant secondary metabolites and

derivatives have been identified as potential antimicrobial agents. Alkaloids have shown

significant antimicrobial activity among the secondary metabolites studied and are one of the

most common plant-derived natural compounds with antiviral properties (Majnooni et al.,

2021). The ability of some plant secondary metabolites to act as resistance-modifying agents

is a promising field in preventing the spread of bacterial resistance. The mechanisms of

action of phenolic compounds on bacterial cells have been linked to membrane damage,
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inhibition of virulence factors such as enzymes and toxins, and inhibition of bacterial biofilm

formation. It has antimicrobial and antiviral activity, as well as promising biological

properties, such as enhancing cell proliferation, tissue regeneration, and wound healing.

Tannic acid is an excellent natural compound that can be used to eliminate pathogenic

factors.  Terpenes and their derivatives, on the other hand, are secondary metabolites found

in EOs that have antimicrobial properties against susceptible and resistant pathogens

(Mahizan, 2019). Monoterpenes and phenylpropanoids under study are potentially used as

antimicrobials against Escherichia coli and Staphylococcus aureus, with the mechanism of

action involving changes in membrane permeability without cell lysis (Nogueira et al., 2021).

Flavonoids, one of the most abundant phytochemicals in Veitchia arecina, were

identified by Ullah et al. (2020) as phytochemical compounds found in many plants, fruits,

vegetables, and leaves that have potential applications in medicinal chemistry. Flavonoids

have a variety of medicinal properties, including anticancer, antioxidant, anti-inflammatory,

and antiviral properties. Additionally, there is growing proof that flavonoids have

antibacterial properties that are effective against both Gram-positive (Staphylococcus aureus)

and Gram-negative (Escherichia coli) bacteria.(Miklasińska-Majdanik et al., 2018).

Furthermore, steroids are recognized for their antimicrobial properties, which is specifically

associated with membrane lipids and causes leakage from liposomes. Related studies of

antimicrobial activity indicate that crude extracts containing steroids have significant activity

against various strains of Staphylococcus aureus, Streptococcus faecalis, and Escherichia

coli (Mujeeb et al., 2014). 

Liceo de Cagayan University College of Medical Laboratory Science Page | 13

Escherichia coli

Studies have revealed that date palm leaf extracts in methanol and ethanol have E.

coli, Streptococcus pneumoniae, Klebsiella pneumoniae and other gram-positive and gram-

negative bacteria are all susceptible to the antibiotic's antibacterial activity (Abdulmajeed et

al., 2013). Similar to this, date palm (Phoenix dactylifera) leaf extracts in methanol and

acetone have shown antibacterial activity against E. coli, P. aeruginosa,Shigella flexneri,

Staphylococcus aureus, Streptococcus pyogenes, and Bacillus subtilis (Perveen, Bokhari, &

Soliman, 2012).

Staphylococcus aureus

Multiple studies have demonstrated that oil palm leaf extract possesses antimicrobial

properties. According to previous research, oil palm leaf extracts inhibit the growth of

Escherichia coli and Staphylococcus aureus (Febriani et al., 2020).

At 30 g/mL, A. merrillii extract demonstrated significant anti-proliferative activity

against cervical (HeLa), lung (H460), breast (MCF-7), and prostate (PC-3) cancer cells. The

extracts' minimum inhibitory concentrations (25–100 g/mL) against Proteus mirabilis,

Salmonella typhi, Shigella dysenteriae, and Candida albicans were determined (Essien et al.,

2017). 

In an effort to discover new antimicrobial agents, plant products were studied as

alternatives or supplements to antibiotics against which bacteria had already developed

resistance (Monte et al., 2014). According to Monte et al. (2017), 7-HC and I3C are the

phytochemicals that have the best chances of fending off E. S. coli and E. aureus. MICs
Liceo de Cagayan University College of Medical Laboratory Science Page | 14

against E. coli are 800 and S. aureus200 g/mL. One of the phytochemicals evaluated to be

most efficient against E. aureus was 7-HC. S. coli and E. aureus. In terms of preventing

biofilms, the exposure of S. The percentages of inactivation of aureus biofilms to 7-HC at

various concentrations were considerably varied (39 at the MIC and 47 at 5MIC). The MIC

between I3C and E. S. coli and E. 400 g/mL of aureus and 800 g/mL, respectively. In

contrast, dual combinations of all antibiotics and I3C had a synergistic effect against S.

aureus resistant strains. I3C and 7-HC, two phytochemicals, both influenced cell motility and

QS activity, indicating that they might be very important in preventing biofilms and

interfering with cell-cell interactions. Phytochemicals have also shown a great deal of

promise in reversing antibiotic resistance.

 
Liceo de Cagayan University College of Medical Laboratory Science Page | 15

Chapter 3

METHODOLOGY

This chapter discusses the research setting, design, instrumentation, protocol, and

specific methods employed by the researchers in the data gathering and analysis. 

Research Setting

The Manila palm fruit (Adonidia merrillii) was collected at Barangay Victoria,

Oroquieta City in the province of Misamis Occidental. The experiment took place at MLS

Laboratory 2 & 3 of the College of Medical Laboratory Science located at the Fourth Floor

of Liceo de Cagayan University, Paseo del Rio Campus, Rodelsa Circle, Macasandig,

Cagayan de Oro City, Philippines. The procedures were performed by the Medical

Laboratory Science researchers and guided by a registered medical technologist. The

Pharmaceutical Laboratory of Liceo de Cagayan University was utilized to perform rotary

evaporation and soxhlet with the company of registered Pharmacist. 

Research Instrument

The researchers utilized the following materials and equipment during the

experiment: Manila palm fruit (Adonidia merrillii), pure culture of Escherichia coli and
Liceo de Cagayan University College of Medical Laboratory Science Page | 16

Staphylococcus aureus, mortar and pestle, refrigerator for the bacterial culture and

preservation, laboratory oven and ethanol for the extraction of Manila palm fruit, rototary

evaporator (rotovap), soxhlet, petri plates, inoculating loops, alcohol lamps, electronic single

pan balance, electric stove, incubator, autoclave, autoclave tape, Whatman filter paper,

Mueller-Hinton agar, water bath, Bunsen burner, wire gauze, evaporating dish,

Erlenmayer/Florence flask, beakers, volumetric flask, stirring rods, pipettes, test tubes and

test tube racks, forceps, ethanol or isopropanol and sodium hypochlorite for the disinfection

of working areas, Personal Protective Equipment (PPE), gadgets: laptop for data input and

storage and camera for documentation. 

Data Gathering Procedure

Collection of Manila palm fruit (Adonidia merrillii)

After sample collection, the sample was sent to Central Mindanao University for Taxonomy

to certify the authenticity of the plant. The fruit has a small, ovoid, orange to red colored

appearance. The branch of the fruit was removed using a sharp object since the fruit is very

small and it is attached to it. The fruit was then transported in the laboratory to start the

actual experiment. 

Preparation of the Manila palm (Adonidia merrillii) fruit extract

The researchers washed the Manila palm with distilled water to remove dirt and

residues. After washing, the fruit was then air-dried. When drying was complete, it was

powderized using mortar and pestle. The powdered fruit was placed in a jar with ethanol as

an extracting solution for 72 hours. After 72 hours of soaking the powdered fruit, a
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Whatmann filter paper is then used to filter inside the beaker. A soxhlet was also performed

for further extraction. Through rotary evaporation, the ethanol was removed from the

mixture. The flask of Manila palm fruit soaked in ethanol was attached to the bump guard for

absorption. Once the rotary evaporation starts spinning the extract is made. Evaporation to

dryness technique is then performed to obtain the crude extract of the Manila palm fruit

(Adonidia merrillii). The extract was then tested at the Department of Pharmacy, Liceo de

Cagayan University for the Phytochemical screening for the identification of the active

chemicals present in the extract.  The extract was then stored in the refrigerator to preserve

and prevent degradation. 

Mueller-Hinton Agar Preparation

Mueller-Hinton agar was used in the study to perform the susceptibility test. The

MHA was heated, boiled and autoclaved prior to dispensing into the petri dish and cooled

down to solidify the media. It was then stored in the refrigerator to solidify for further

procedures. 

Preparation of standardized inoculum

The purely cultured bacteria were prepared in 1-3 loops mixed with Normal Saline

compared into a 0.5 MacFarland Standard turbidity to standardize the liquid suspension. The

suspension was prepared for inoculation when the turbidity is the same.

Inoculation of Agar Plates

The sterile swab was dipped in the standardized suspension with Escherichia coli

(ATCC 25922) and Staphylococcus aureus (ATCC 25923) separately. Gently press the swab

on the wall of the test tube to remove excess inoculum and spread to the Mueller-Hinton
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Agar using the streaking method. This procedure is done thrice to ensure proper distribution

of bacteria in the Agar plate. Allow the surface to dry for better absorption of excess

moisture. 

Disc Impregnation

The Whatman filter paper no. 3 was soaked in different concentrations of Manila

palm (Adonidia merrillii) fruit extract, placed on the surface of the Mueller-Hinton Agar

using a sterile forceps or a disc dispenser. The discs were placed 24 mm apart at a center-

center on the Mueller-Hinton agar. 4 discs were placed on a 150 mm plate. The positive

control and negative control were also impregnated in the Mueller-Hinton agar for the

comparison of the effectivity from the prepared inoculum. The procedure was performed in

triplication to ensure the efficacy of the antimicrobials. The researchers observed proper

spacing to prevent overlapping and detect the zone of inhibition. 

Incubation of Agar Plates

The plates were placed inside the incubator for 24 hours at 37 degrees Celsius in an

inverted position to allow growth of Escherichia coli (ATCC 25922) and Staphylococcus

aureus (ATCC 25923).

Measurement of Zones and Interpretation of Discs Susceptibility

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The result depends on the diameter of the zone of inhibition <7mm is Resistant, 7-

9mm is Intermediate and >10mm is Susceptible. (Iyasele, Uadia, Akhigbe, Jacob, Ogbeide,

2022). The results were interpreted as Resistant (R), Intermediate (I), or Susceptible (S).

  In accordance with the protocol for this research, the researchers sought the approval

from the adviser after careful assessment and review of the manuscript for the thesis. The

researchers started conducting the experiment starting from the collection of the Manila palm

fruit (Adonidia merrillii) at Barangay Victoria, Oroquieta City. After sample collection, the

sample was sent to Central Mindanao University for Taxonomy Certification. The

researchers proceed with the actual experiment. The Manila palm fruit was powdered and

soaked with ethanol for extraction. The extract was tested at the Department of Pharmacy

Liceo de Cagayan University for Phytochemical screening. Once the extraction was made, it

was diluted into different concentrations which are 75% and 100% Manila palm fruit extract.

The Mueller-Hinton Agar were used for the antimicrobial susceptibility testing. The

suspension is compared with 0.5 McFarland Standard when the turbidity matches, the

suspension is ready for inoculation. The inoculum was suspended in the Mueller-Hinton

Agar using the Streaking method to evenly distribute the bacteria in the plate. The discs with

different concentrations of Manila Palm fruit extract, positive control, negative control and

vehicle control were impregnated in the plate following the proper placement to prevent

overlapping. The researchers incubated the plates for 24 hours. After incubation the

researchers measured the zone of inhibition using a ruler. The results were interpreted into

Resistant (R), Intermediate (I) and Susceptible (S). Once results are released the researchers

proceed in discussion and provide a conclusion to the experiment.

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Furthermore, the researchers also made use of non-parametric tests such as Wilcoxon-

Mann-Whitney U testing along with the Kruskal-Wallis t-test. These tests are contributory in

cases where two of the variables are not normally distributed. A comparison between the

effectiveness of two of the independent groups in a clinical trial will be easily noticed and

distinguished. This assessed whether the values in one group are greater or less than the

values in the other group.

Chapter 4

PRESENTATION, ANALYSIS, AND INTERPRETATION OF DATA

The fruit of Adonidia merrillii, which was used as a sample in this study, initially

grows green, then turns orange to red when ripe, and has a small, ovoid appearance. In the

study of Essien et al. (2017), they described Adonidia merrillii  fruit as consisting of a thin

epicarp, a dry, yellowish, thin-fleshy mesocarp, and a thin, fragile endocarp. Adonidia

merrillii fruit is used as a masticatory when ripe but is an inferior substitute for betel nuts.

Adonidia merrillii was collected in the vicinity of Misamis Occidental, specifically in

Barangay Victoria, Oroquieta City, and has undergone plant authentication on May 24, 2023.

The plant sample was identified and confirmed by Dr. Maria Melanie M. Guiang, a Botanist
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and member of the Pool of Taxonomists at Central Mindanao University's Museum. The

Manila palm (Adonidia merrillii) plant is classified in Table 1.

Objective 3. Determine the antibacterial activity of Manila palm (Adonidia merrillii)

fruit extract using the experimental groups which are 75% and 100% concentration and

control groups which are positive control (Ciprofloxacin) and negative control (Distilled

water).

According to Iyasele et al., (2022), the result is determined by the diameter of the

zone of inhibition; a zone of inhibition measuring <7mm is resistant, 7-9mm is intermediate

and >10mm is susceptible. The results were interpreted as resistant (R), intermediate (I), or

susceptible (S).

Table 3 shows the comparison between the zones of inhibition of the experimental

group using different concentrations (100% and 75%), a positive control group

(Ciprofloxacin), and a negative control group (distilled water) of the Manila palm (Adonidia

merrillii) fruit’s ethanolic crude extract against Escherichia coli and Staphylococcus aureus.

On measuring the inhibitions of Escherichia coli, the three replicates utilizing the 75% and

100% concentrations and the negative control (distilled water) showed no antibacterial

inhibition, with only 6mm diameter, indicating that the replicates were resistant to the

ethanolic crude extract. Only the positive control (Ciprofloxacin) showed a powerful average

of inhibition of 29.7 mm, indicating that the replicates were susceptible to crude extract. On

the microorganism Staphylococcus aureus (ATCC 25923), the three replicates that used a

75% concentration showed an average inhibition of 15.67 millimeters, indicating that all
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replicates were susceptible to the extract. The three replicates using 100% concentration

showed an average inhibition of 15 millimeters, indicating that the replicates also exhibited

antimicrobial efficacy up to susceptible level. The three replicates utilizing the negative

control (distilled water) showed no antibacterial inhibition (6 millimeter), which makes it

resistant to the crude extract, while the positive control showed an average inhibition of 28

millimeters, indicating that all replicates were susceptible to the extract.

From the results of the table, it can be implied that of the two microorganisms, only

Staphylococcus aureus (ATCC 25923) exhibited antimicrobial activity as being susceptible

to the Manila palm (Adonidia merrillii) fruit’s ethanolic crude extract. According to studies,

the cell walls of gram-positive bacteria are more sensitive to many antimicrobial chemical

compounds and even many herbal drugs. Gram-negative bacteria's relative resistance can be

attributed to their lipopolysaccharide layer and periplasmic space. Between the two

concentrations the 75% showed a higher mean of 15.6mm whereas the the 100% showed a

mean of 15mm having a difference of 6mm. The findings of the study by Paz, Contreras,

Mungua, Aguilar, and Inungaray (2018) demonstrate that a higher percentage yield in extract

extraction is not a reliable sign of stronger antibacterial activity. The three extracts were

generally 75% effective at removing S. bacteria and S. 50% of typhi for S. E gets paratyphi

and 100%. coli. The percentage activity was 100% and the Index of Bacterial Susceptibility

(IBS) was 75% in the three extracts prepared using various techniques. Furthermore, in the

study of Swiner (2022), he explained that since 75% have more water compared to 100% that

do not have water at all, it has the advantage of allowing it to dissolve more slowly, penetrate

cells, and kill bacteria.

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Objective 4. Determine the significant difference in the antibacterial activity of Manila

palm fruit extract in terms of 75% concentration and 100% concentration. 

Table 4

Table 4 shows the significant difference of 100% and 75%, which are both .368. This

indicates that both concentrations have no significant difference. Which accepts Ho 1,

indicating that there is no significant difference in the anitibacterial activity of Manila palm

(Adonidia merrillii) fruit extract with the different concentrations. According to the study of

Ambon et al. (2019), using three different concentrations: 50%, 75%, and 100%, found that

there was no significant difference among the concentrations.

The three replicates employing a 75% concentration, however, showed an average

inhibition of 15.67 millimeters when the zone of inhibition of Staphylococcus aureus (ATCC

25923) was assessed, showing that all replicates were susceptible to the extract. In addition,

the three replicates using 100% concentration showed an average inhibition of 15

millimeters, indicating that the replicates also exhibited antimicrobial efficacy up to the

susceptible level.

Objective 5. Determine the significant difference in the antibacterial activity of

Manila palm (Adonidia merrillii) fruit extract against the experimental and the control

groups.
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Table 5 contains the Kruskal-Wallis H test result, and it showed that there was no

statistically significant difference in pain score between the two different concentrations,

namely 100% and 75% concentrations, having both a .368 pain score. Moreover,

Ciprofloxacin has a pain score of 1.000. In the study of Ambon, Guerra, Macapagal, Noble,

Param, and Sta. Rosa (2019), using three different concentrations: 50%, 75%, and 100%,

found that there was no significant difference among the concentrations.

According to Tenny and Abdelgawad (2022), The null hypothesis is not refuted and

the results are not statistically significant if the p value is greater than the significance level.

If the p value is lower than the significance level, the results are interpreted as refuting the

null hypothesis and reported as statistically significant.

Chapter 5

SUMMARY, CONCLUSION, AND RECOMMENDATIONS

This chapter presents the summary, conclusion, and recommendations of the study.

Summary

Furthermore, it aimed to determine whether there is a significant difference in zone of

inhibition in each experimental group using the following concentrations of the fruit's

ethanolic crude extract: 100% and 75%.

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The fruit of Manila palm was collected in an area within the vicinity of Misamis

Occidental, specifically Barangay Victoria, Oroquieta City. The test organism, Escherichia

coli (ATCC 25922) and Staphylococcus aureus (ATCC 25923) was obtained from Cotabato

Regional Medical Center.

To achieve the objectives of the study, the researchers assigned the microorganisms

of interest into two experimental groups. Both bacteria were subjected to the ethanolic crude

extract, and their susceptibility was measured separately. The amount of crude extract, the

number of trials and replicates, and the time and day when it was subjected to the

formulation were all the same for the two microorganisms. This is to determine which of

them is more sensitive to the ethanolic crude extract. The two bacteria were subcultured

separately on nutrient agar medium, and their exposure to different concentrations (100% and

75% ethanolic crude extract), the positive control (Ciprofloxacin), and the negative control

(distilled water) was monitored. The differences in their susceptibility, measured by the

diameter of inhibition in millimeters, were attributed to the antimicrobial property of the said

extract.

Based on the results, only Staphylococcus aureus (ATCC 25923) showed

antibacterial activity at any of the two concentrations of the crude extract. The zones of

inhibition based on the antimicrobial property of the ethanolic crude extract against

Staphylococcus aureus (ATCC 25923) using three replicates of 75% concentration revealed

an average inhibition of 15.67 millimeters, indicating that all replicates were susceptible to

the extract. The three replicates using 100% concentration showed an average inhibition of
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15 millimeters, indicating that the replicates also exhibited antimicrobial efficacy up to

susceptible level. The two concentrations from all the replicates of Escherichia coli (ATCC

25922), on the other hand, showed no antibacterial inhibition with 0 millimeter, making it

resistant to the crude extract. Only the positive control shows antibacterial activity in all of

the replicates. The mean of the susceptible was then computed. In 100% and 75%

concentrations, the statistical data utilizing the Kruskal-Wallis H test showed that there was

no statistically significant difference in pain score between the two different concentrations,

namely 100% and 75% concentrations having both a .368 significance level.