Antibacterial Activity of Manila Palm (Adonidia Merrillii

ANTIBACTERIAL ACTIVITY OF MANILA PALM (Adonidia merrillii
(Becc.) Becc.) FRUIT EXTRACT AGAINST Escherichia coli AND

Staphylococcus aureus
Maria BJ Theresa A. Aque
Alan Louise Y. Bualat
Umaynah E. Ahmed
JUNE 2023
Liceo de Cagayan University College of Medical Laboratory Science Page ii
ANTIBACTERIAL ACTIVITY OF MANILA PALM (Adonidia merrillii

(Becc.) Becc.) FRUIT EXTRACT AGAINST Escherichia coli AND
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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
JUNE 2023
Liceo de Cagayan University College of Medical Laboratory Science Page iii
Certificate of Originality
I hereby declare that this submission is my own work and that, to the best of my
knowledge and belief, it contains no material previously published or written by another
person nor material to which to a substantial extent has been accepted for award of any other
degree or diploma of a university or other institute of higher learning, except where due
acknowledgement is made in the text.
I also declare that the intellectual content of the thesis is the product of my work,
even though I may have received assistance from others on style, presentation and language
expression.
MARIA BJ THERESA A. AQUE

Signature of advisee over printed name Date
ALAN LOUISE Y. BUALAT

UMAYNAH E. AHMED
MARILOU O. HONCULADA, RMT, MA, MSMLS

Signature of adviser over printed name
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English Editing Certification Form
English Editing Certification Form This is to certify that I have edited the thesis
manuscripted “ANTIBACTERIAL ACTIVITY OF MANILA PALM (Adonidia merrillii
(Becc.) Becc.) FRUIT EXTRACT AGAINST Escherichia coli AND Staphylococcus
aureus”, prepared by Maria BJ Theresa A. Aque, Alan Louise Y. Bualat, Umaynah E.
Ahmed and have found it thorough and acceptable with respect to grammar and composition.
DR. DOMINIC T. POLANCOS, PHD, LPT

Signature over printed name
Date
Liceo de Cagayan University College of Medical Laboratory Science Page v
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 degree Bachelor of Science in
Medical Laboratory Science by the Panel of Examiners with a grade of ____.
AMELDA C. LIBRES, PHDOD, RMT

Chairman
MICHELLE T. MABASA, RMT, MAEd MARY ABIGAIL M. PAULAN, RMT, MSMLS

Member Member
Date of Final Defense:
AMELDA C. LIBRES, PHDOD, RMT

Dean, College of Medical Laboratory Science
Liceo de Cagayan University College of Medical Laboratory Science Page vi
DEDICATION
This research paper is dedicated to the supportive parents of the researchers, who
wholeheartedly exerted their love and assistance which helped boost the morale of the
researchers to also exert their best.
In addition, the researchers also dedicate this paper to their research adviser, Ms.
Marilou O. Honculada, for her unfailing guidance throughout this journey. Her experience
and knowledge were greatly helpful in the completion of this research paper.
Moreover, we dedicate this research paper to Mr. Rogin Alamban, for his knowledge
in the laboratory procedures were greatly beneficial to ensure a quality and reliable data
gathering for this research study.
Last, but the most important of all, the researchers dedicate this study to God
almighty, for without Him and His unconditional support, the success of this research would
not have been possible
Liceo de Cagayan University College of Medical Laboratory Science Page vii
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.
Ms. 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, PHDOD, RMT 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 viii
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 ix
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. The bacterial colonies of E. coli and S. aureus were streaked in Mueller
Hinton Agar plates for susceptibility testing in hand with the experimental and control groups
and to compare the zone of inhibition of the fruit extract against E. coli and S. aureus.
Testing was done in triplication to ensure the efficacy of the fruit extract. The extract
exhibited a presence of flavonoids, tannins, and saponins. The fruit extract exhibited a zone
of inhibition against S. aureus with a mean of 15mm and 15.67mm for 100% and 75%
concentration respectively which is above 10mm; indicating that the bacteria was susceptible
against the ethanolic fruit extract of Manila palm.
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 x
TABLE OF CONTENT
Page
COVER PAGE i
TITLE PAGE ii
APPROVAL SHEET iii
DEDICATION iv
ACKNOWLEDGEMENT v
ABSTRACT vii
TABLE OF CONTENT viii
LIST OF FIGURES xi
LIST OF TABLES xii
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

Liceo de Cagayan University College of Medical Laboratory Science Page xi
3 METHODOLOGY
Research Setting 19
Research Design 19
Research Instrument 20
Data Gathering Procedure 20
4 PRESENTATION, ANALYSIS, AND 26
INTERPRETATION OF DATA
5 SUMMARY, CONCLUSION, AND 35

RECOMMENDATIONS
REFERENCES 39
APPENDICES
A. Letter of Approval 45
B. Completion Report 47
C. Certification of Plant 49
D. Letter of Consent for Laboratory Use 50
E. Letter of Consent for Laboratory Use 52
F. Documentation 54
G. Descriptive Statistics for the zones of inhibition of 60
Adonidia merrilli extract in millimeters (mm)
against Staphylococcus aureus
H. Kruskall Wallis H Test 61

Liceo de Cagayan University College of Medical Laboratory Science Page xii
I. Summary of the Hypothesis Test 62
J. Curriculum Vitae 63
Liceo de Cagayan University College of Medical Laboratory Science Page xiii
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 xiv
LIST OF TABLES
Table Title Page
1 Classification of Manila palm (Adonidia merrillii) Plant 26
2 Phytochemical Properties of Manila palm (Adonidia merrillii) Fruit 27
3 Comparison between the zones of inhibition of Adonidia merrilli 29

extract in millimeters (mm) by the positive control, negative control
and the experimental control group against Escherichia coli and
4 Significance level of the two concentrations from Kruskal Wallis H 31
Test
5 Kruskal Wallis H Test with the corresponding significance level of the 33

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, making it bountiful in the Philippines. The tree measures 10 m tall and 15-20
cm in 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 is being utilized by some cultural practices as an alternative to the betel
chewing nut and as a bead source (Sandoval, 2019); Phytochemical screenings of the Manila
palm fruit extract have been studied and revealed the cytotoxic activity of the fruit extract,
which the potential for antifungal and antibacterial activity (Essien, E.E., Antia, B.S.,
Solomon, A.U., Choudhary, M.I., 2017).
Over the years, clinical and technological advancements have given 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 pills lose their effectiveness (Zhu, Huang, and Yang, 2022).
Backed up by the study of Rai, Randhawa, and Kaur (2013), the global clinical problem
might 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 for utilizing the
nutritional advantages it can give to every living organism. Generations have passed, and
practices and beliefs about usage of herbal plants for therapeutic and medicinal purposes
continue to prosper and are 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 answer the
problem. This plant might not be known by most people, but its fruit is rich in a variety of
phytochemicals that have been tested to have potent cytotoxicity, giving it a strong potential
for antibacterial activity.

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 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, trimethoprim, and sulfamethoxazole.
These gram-negative bacteria are usually found in our guts, specifically in the lower intestine
of warm-blooded organisms. Most E. coli are harmless and are just average dwellers in our
intestinal tract; however, E. coli O157:H7, a specific 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 disease, but if it can
reach internal tissues or the bloodstream, these microbes may cause numerous potentially
dangerous infections (Taylor and Unakal, 2022).
Given the aforementioned facts, this research will significantly serve as an additional
reference for the research community about the potential of this plant’s fruit extract and also
encourage them to use the other parts of the Manila palm for other beneficial purposes. This
study will also significantly contribute to the pharmaceutical section and may develop drugs
incorporating this plant’s fruit extract. Very few studies are available on Manila palm, which
is why delving more into its capabilities as a potential medicine against S. aureus and E. coli
infections is the purpose of this study, to battle 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 its 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 deoxysugars 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 many 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.

Schematic Presentation
INDEPENDENT VARIABLES DEPENDENT VARIABLES
Antimicrobial activity of
Manila Palm
Figure
The schematic presentation presents the independent and dependent variables. The
Independent variables are composed of an experimental group which consists of 75%
concentration and 100% concentration and a control group which consists of positive control
and negative control. The Dependent variable is the antimicrobial activity of Manila palm.
Objectives of the study

The study aims to determine the antibacterial activity of Manila palm (Adonidia
merrillii) fruit extract against E. coli and S. aureus.
Specifically, it aims to:
1. Collect, identify, classify, and describe the Manila palm (Adonidia merrillii) plant.
2. Determine the phytochemical property of Manila palm (Adonidia merrillii) fruit.
3. Determine the antibacterial activity of Manila palm (Adonidia merrillii) fruit extract
using the following:
1. 75% concentration
3. Positive control (Ciprofloxacin)
4. Negative Control (Distilled water)
4. Determine the significant difference in the antibacterial activity of Manila palm fruit
extract in terms of:
1. 75% concentration; and
2. 100% concentration.
5. Determine the significant difference in the antibacterial activity of Manila palm
(Adonidia merrillii) fruit extract against the experimental and the control groups.
Hypothesis
Ho1: There is no significant difference in the antibacterial activity of Manila palm
(Adonidia merrillii) fruit extract with the different concentration:
Ho2: There is no significant difference in the antibacterial activity of Manila palm
(Adonidia merrillii) fruit extract against the experimental and the control groups.
Significance of the Study
The findings of this study might be expected to be essential and benefit the following
sectors:
Pharmaceutical. The study is expected to give pharmaceuticals the idea that there is
a natural herbal extract which provides an alternative antibacterial control which can fight
diseases caused by Escherichia coli and Staphylococcus aureus.
Community. The study is expected to provide awareness to people about the
antibacterial activity of Manila palm (Adonidia merrillii) fruit extract against Escherichia
coli (bacteria found in the intestines of healthy people and animals that can manifest
clinically as abdominal cramps, bloody diarrhea, and even kidney failure) and
Staphylococcus aureus (bacteria found in the nose and on the skin of humans, which is the
most common cause of skin and soft tissue infections including abscesses, furuncles, and
cellulitis), allowing the community to consider organic antibacterial controls.
Patients. The findings of study will help previously or currently infected patients
with Escherichia coli and Staphylococcus aureus to use plant-based antibacterial controls
instead of chemically synthesized ones.
Medical Laboratory Science Students. The study is expected to broaden the skills
of Medical Laboratory Scientists as well as researchers in terms of finding, analyzing,
considering different methods, and understanding the susceptibility of Escherichia coli and
Staphylococcus aureus to the antimicrobial activity of Manila palm (Adonidia merrillii) fruit
extract.
Future Researchers. Indeed, the progress and improvements of this study will be
possible with the help of the future researchers especially in developing another study that
will utilize the other parts of the Manila palm (Adonidia merrillii) for potential medicinal
use.
Scope and limitation of the Study
This study was confined to the determination of the antibacterial activity of Manila
Palm (Adonidia merrillii) fruit extract against Escherichia coli and Staphylococcus aureus
using two concentrations: 75% and 100%. The primary part that was utilized in this study is
the fruit of the Manila palm and was collected in Barangay Victoria, Oroquieta City, Misamis
Occidental, Philippines. 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.
Definition of Terms
The following terms are operationally defined for a better understanding of this study.
Antimicrobial activity – This refers to the effectivity of Manila Palm (Adonidia
merrillii) fruit extract to inhibit the growth of E. coli and S. aureus
Ciprofloxacin – This will be used as a positive control of this study.

Concentration – In this study, this will be differentiated into 75% and 100%.
Distilled Water – This will serve as the negative control of this study.
Escherichia coli – For this study, these bacteria will be experimented for the
antibacterial activity of Manila Palm (Adonidia merrillii) fruit extract.
Ethanol – This will be used as the vehicle control of this study.
Negative Control – It is a solution that cannot inhibit the growth of E. coli and S.
aureus.
Positive Control – It is a known antibiotic that can inhibit the growth of E. coli and
S. aureus.
Staphylococcus aureus – In this study, this will be the bacteria to be used for
experimenting the antibacterial activity of the fruit extract of Manila Palm (Adonidia
merrillii).
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, several 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 antioxidant and anti-cytotoxicity capabilities. Therefore, future research into the
fruit could lead to discovering antioxidant and cytotoxic medications derived from natural
sources. Most people may be familiar with this plant, still, they may not be aware that its fruit
is rich in various 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 defend 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 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,
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, containing dietary fibers, sugar, protein,
vitamins, minerals, flavonoids, and phenolic compounds. Date palm fruits are antioxidant-
rich and possess potent antibacterial bioactivities due to the presence of phenolic compounds.
Based on the evidence reviewed in Al-Shwyeh’s 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. The most potent components
of various plant extracts and essential oils from various plants were discovered through
chemical profiling. According to the study of Bertrand et al. (2017), when date palm organic
extracts were subjected to phytochemical analysis, three types of flavonoids—
anthocyanidins, C-glycosyl flavones, 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 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.

Phenolics and flavonoids are the most diverse groups of phytochemicals, and they can
protect the human body from reactive oxygen species (Galindo et al., 2010). Adonidia
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, Adonidia 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 Adonidia
merrillii were 52.05, 20.68, 2.58 mg/g, and 40.58, 32.73, and 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 Adonidia merrillii fruit. RP-HPLC was also
used to analyse 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. The analysis revealed that a 5 g sample of powdered
dried fruit from Adonidia merrillii had 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 Adonidia merrillii fruit extracts also revealed statistically significant
differences (P0.05). The total phenolic content of methanolic, ethanolic, and water extracts
was 17.8, 7.6, and 2.22 mg GAE per gram of dry weight, respectively. In contrast, the total
flavonoid 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. Still, naringin and rutin are flavonoid components
found in extracts of the Adonidia 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 Fasciola 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 highly 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
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 valuable characteristics. Manila palm is a flowering plant
species belonging to the Arecaceae family. Despite being abundant in bioactive compounds
with possible health benefits, the fruits of the Arecaceae family 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 to
promote production, commercialization, processing, and consumption. It can also encourage
their full utilization, thereby improving the economy and social conditions of the people
living 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 primary constituent 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. Furthermore, these compounds have been linked to anti-inflammatory, anti-
proliferative, anti-obesity, and cardioprotective effects. Despite their potential, some of them
have rarely been studied, with very little research on their composition, biological products,
and applications. As a result, in vitro and, or in vivo studies on the design and mechanism of
action of Arecaceae family fruits are necessary (Morais et al., 2022).
Plants are 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 for 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, some critical 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,
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 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 therapeutic properties, including anticancer, antioxidant, anti-inflammatory,
and antiviral properties. Additionally, there is growing proof that flavonoids have
antibacterial properties that are effective against Gram-positive (Staphylococcus aureus) and
Gram-negative (Escherichia coli) bacteria.(Miklasińska-Majdanik et al., 2018). Furthermore,
steroids are recognized for their antimicrobial properties, associated explicitly with
membrane lipids and cause 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).
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 that 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). The crude methanol, acetone, and water extracts of the leaves and pits of
three different types of Phoenix dactylifera were evaluated for their antibacterial activity
against Gram-positive and Gram-negative pathogens. With the exception of Enterococcus
faecalis, the acetone and methanol extracts exhibited potent antibacterial activity against
Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Shigella flexeneri,
Staphylococcus aureus, and Streptococcus pyogenes, whereas the water extract had a
negligible effect on all tested bacterial It was discovered that pit extracts from all three
varieties of P. dactylifera were more effective than leaf extracts. S.pyogenes was the most
sensitive pathogen to the crude extracts, as evidenced by the largest zone of inhibition. The
MIC for S. pyogenes was determined to be 1,3, 1,1, and 1,6 mg/ml for methanol leaves and
pits extracts and acetone leaves and pits extracts, respectively.
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). According to Bentrad et
al. (2017), date seeds contain an abundance of oleic (36.69%) and lauric (20.49%) acids, as
determined by GC/MS analysis. Nevertheless, the pollen is rich in palmitic (22.27%), linoleic
(33.4%), and linolenic (17.055%) acids. In addition, the organic extract of Deglet Nour with
strong antibacterial activity against Escherichia coli and pollen extract with strong inhibition
against Escherichia coli, Staphylococcus aureus, S. aureus MRSA, and Enterococcus faecalis
produced the largest inhibition zone. Aqueous extracts of date palm seeds and pollen appear
to have fongitoxique activity at concentrations of 6 mg. mL-1 and 12 mg. mL-1 of cyanidine;
and organic extracts of pollen at a concentration of 90 g.mL"1 inhibit the growth of five
unique strains of Fusarium oxysporum.
At 30 g/mL, Adonidia 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).
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. coli and S. aureus. MICs against E. coli are 800 and S.
aureus 200 g/mL. One of the phytochemicals evaluated to be most efficient against S. aureus
was 7-HC E. coli and S. aureus. The percentages of inactivation of aureus biofilms to 7-HC
at various concentrations varied considerably (39 at the MIC and 47 at 5MIC). The MIC
between I3C and E. coli and S. aureus. 400 g/mL of aureus and 800 g/mL, respectively. In
contrast, dual combinations of all antibiotics and I3C had a synergized effect against S.
aureus-resistant strains. I3C and 7-HC, two phytochemicals, influenced cell motility and QS
activity, indicating that they might be essential in preventing biofilms and interfering with
cell-cell interactions. Phytochemicals have also shown a great deal of promise in reversing
antibiotic resistance.
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
experiment used a pure culture of identified Escherichia coli ATCC 25922 and
Staphylococcus aureus ATCC 25923. The microorganisms were obtained from the Cotabato
Regional Medical Center. The Pharmaceutical Laboratory of Liceo de Cagayan University
was utilized to perform rotary evaporation and soxhlet with the company of registered
Pharmacist.
Research Design
The study employed an experimental research design for it to utilize controls to
compare the efficacy of the antibacterial activity of Manila palm (Adonidia merrillii) fruit
extract regarding different concentrations against Escherichia coli and Staphylococcus
aureus.
Research Instrument
The researchers utilized the following materials and equipment during the
experiment: Manila palm fruit (Adonidia merrillii), pure culture of Escherichia coli and
Staphylococcus aureus, mortar and pestle, refrigerator for the bacterial culture and
preservation, laboratory oven and ethanol for the extraction of Manila palm fruit, rotary
evaporator (rotavap), 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)
The researchers collected the Manila palm fruit (Adonidia merrillii) in an area within the
vicinity of Misamis Occidental, specifically Barangay Victoria, Oroquieta City. 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, oval, orange-to-red colored appearance. The
branch of the fruit was removed using a sharp object since the fruit is tiny and attached to it.
The fruit was then transported to 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
powdered using mortar and a 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
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. The 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 to identify 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 before dispensing into the petri dish and cooled
down to harden the media. It was then stored in the refrigerator to solidify for further
procedures.
Dilution of the Manila palm (Adonidia merrillii) fruit extract
In 6 ml of 100% extract of Manila palm (Adonidia merrillii) fruit extract, 3ml was
transferred to another beaker to be diluted in a 0.75ml distilled water to make a 3ml of 75%
concentration of Manila palm (Adonidia merrillii) fruit extract. The remaining 3ml will be
the 100% concentration of Manila palm (Adonidia merrillii) fruit extract.
Preparation of standardized inoculum
The purely cultured bacteria Escherichia coli (ATCC 25922) and Staphylococcus
aureus (ATCC 25923) 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
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. three was soaked in different concentrations of Manila
palm (Adonidia merrillii) fruit extract, placed on the surface of the Mueller-Hinton Agar
using sterile forceps or a disc dispenser. The discs were placed 24 mm apart at a center center
on the Mueller-Hinton agar. Four discs were placed on a 150 mm plate. The positive control
and negative control were also impregnated in the Mueller-Hinton agar to compare 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
After 24-hours of incubation, the plates were taken out and the researchers used a
ruler to measure the zone of inhibition. The researchers evaluated and observed the different
zones of inhibition. The fuzzy area of the zone was disregarded when measuring the zone's
diameter. The inner limit of the zone is measured. 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).
Following the protocol for this research, the researchers sought 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
researchers obtained the identified Escherichia coli (ATCC 25922) and Staphylococcus
aureus (ATCC 25923) at Cotabato Regional Medical Center. The researchers prepared the
inoculum with Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 25923).
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.
Statistical Tools
Different statistical tool was employed to answer each objective of the study:
The first objective utilized Descriptive statistics for the collection, identification,
classification and description of Manila palm (Adonidia merrillii) plant.
The second objective utilized Descriptive statistics for the determination of
phytochemical properties of Manila palm (Adonidia merrillii) fruit.
The third objective utilized Mean and Standard Deviation for the determination of
antibacterial effects of Manila palm (Adonidia merrillii) fruit extract in terms of different
concentrations.
The fourth objective utilized Wilcoxon-Mann-Whitney U Testing to compare the
efficacy of the antibacterial activity of Manila palm (Adonidia merrillii) fruit extract in terms
of different concentrations.
The fifth objective utilized Kruskal Wallis H Test to determine the significant
difference of the antibacterial activity of Manila palm (Adonidia merrillii) fruit extract
against the experimental and control groups.
Furthermore, the researchers also used non-parametric tests such as the Wilcoxon-
Mann-Whitney U testing along with the Kruskal-Wallis t-test. These tests are contributory in
cases where two variables are not normally distributed. A comparison between the
effectiveness of two independent groups in a clinical trial will be easily noticed and
distinguished. This assessed whether the values in one group were greater or less than those
in the other group.
Chapter 4
PRESENTATION, ANALYSIS, AND INTERPRETATION OF DATA
This chapter presents the results of the antibacterial activity of Manila palm
(Adonidia merrillii) fruit extract in different concentrations, the negative control (distilled
water), and the positive control (Ciprofloxacin) against Escherichia coli and Staphylococcus
aureus. The presentation of data follows the logical order of the objectives of the study.
Objective 1. Collect, identify, classify and describe the Manila palm (Adonidia
merrillii) plant.
Table 1.
Classification of Manila palm (Adonidia merrillii) Plant
Kingdom Plantae
Phylum Magnoliophyta
Class Liliopsida
Subclass Arecidae
Order Arecales
Family Arecaceae
Genus Adonidia
Species Merrillii
Other name Manila palm
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
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 2. Determine the phytochemical properties of Manila palm (Adonidia
merrillii) fruit.
Table 2
Phytochemical properties of Manila palm (Adonidia merrillii) Fruit
Phytochemicals Flavonoids Tannins Saponins
Ethanolic crude extract of Manila palm fruit + + +
Note. (+) = present; (-) = absent
Table 2. shows the phytochemical properties of Adonidia merrillii fruit. An array of
phytochemical screening tests and procedures were performed on the fruit's ethanolic crude
extract to detect the presence of common compounds known to have antibacterial properties.
The result of the phytochemical screening tests and procedures revealed the presence of
flavonoids, saponins, and tannins. Flavonoids, one of the most abundant phytochemicals in
Adonidia merrillii, 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. Also, there is mounting evidence that flavonoids, saponins, and tannins have
antibacterial activity against both gram-positive (Staphylococcus aureus) and gram-negative
(Escherichia coli) bacteria (Miklasińska-Majdanik et al., 2018).

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
ESCHERICHIA COLI
EXPERIMENTAL REP REP REP MEAN INTERPRETATION

GROUP 1 2 3
100% Concentration 6mm 6mm 6mm 6mm Resistant
75% Concentration 6mm 6mm 6mm 6mm Resistant
CONTROL
GROUP
Positive control 31mm 29mm 29mm 29.7mm Susceptible
(Ciprofloxacin)
Negative control 6mm 6mm 6mm 6mm Resistant
(Distilled Water)
Comparison between the zones of inhibition of Adonidia merrilli extract in millimeters (mm)
by the positive control, negative control and the experimental control group against
Escherichia coli and Staphylococcus aureus
STAPHYLOCOCCUS AUREUS
EXPERIMENTAL REP REP REP MEAN INTERPRETATION

GROUP 1 2 3
100% Concentration 14mm 16mm 15mm 15mm Susceptible
75% Concentration 13mm 17mm 17mm 15.67mm Susceptible

CONTROL
GROUP
Positive control 28mm 28mm 28mm 28mm Susceptible
(Ciprofloxacin)
Negative control 6mm 6mm 6mm 6mm Resistant

(Distilled Water)
Note. <7mm=Resistant; 7-9mm=Intermediate; >10mm=Susceptible
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 (ATCC 25922) and
Staphylococcus aureus (ATCC 25923). 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 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. In fact, in the study of
Koohsari, Ghaemi, Sheshpoli, Jahedi, and Zahiri (2015), it shows that gram-positive bacteria
are more sensitive to herbal extracts than gram-negative bacteria. This could be due to gram-
negatives' inherent tolerance as well as the nature and composition of herbs. 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. According to the results obtained in
the study of Paz, Contreras, Munguía, Aguilar, Inungaray (2018), prove that a higher
percentage yield in obtaining the extract is not an indicator of increased antibacterial activity.
In general, the three extracts had a 75% efficiency for S. aureus and S. typhi, 50% for S.
paratyphi and 100% for E. coli. In the three extracts obtained by different methods the
percentage activity was 100% and the Index of Bacterial Susceptibility (IBS) was 75%.
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.
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
Concentration Significance level
100% .368
75% .368
Significance level of the two concentrations from Kruskal Wallis H Test.
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. Table 4 shows the efficacy of
the antibacterial activity of Manila palm fruit extract in terms of 75% concentration
and 100% concentration against Escherichia coli (ATCC 25922) and Staphylococcus aureus
(ATCC 25923). In the zone of inhibition of Escherichia coli, the three replicates utilizing the
75% and 100% concentrations showed no antibacterial inhibition (0 millimeter), indicating
that the replicates were resistant to the ethanolic crude extract. In contrast, when the zone of
inhibition of Staphylococcus aureus (ATCC 25923) was measured, the three replicates using
a 75% concentration showed an average inhibition of 15.67 millimeters, indicating 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.
Table 5
Kruskal Wallis H Test with the corresponding significance level of the experimental groups
and control groups and their interpretations
Asymp. Sig. Interpretation
75% concentration .368 Retain null hypothesis

Ciprofloxacin 1.000 Retain null hypothesis
100% concentration .368 Retain null hypothesis
Distilled water 1.000 Retain null hypothesis
Note. Significance level is 0.050.
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.
This implies that there is no significant difference between the experimental group
and the control group. As both groups have exceeded the 0.05 significance level. Which
accepts Ho2, indicating that there is no significant difference in the antibacterial activity of
Manila palm (Adonidia merrillii) fruit extract between the experimental and control groups.
According to Tenny and Abdelgawad (2022), if the p value is higher than the
significance level, the null hypothesis is not refuted, and the results are not statistically
significant. 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
The main objective of this study was to determine the antibacterial activity of Manila palm
(Adonidia merrillii) fruit extract against Escherichia coli and Staphylococcus aureus.
Furthermore, it aimed to determine whether there is a significant difference in the zone of
inhibition in each experimental group using the following concentrations of the fruit's
ethanolic crude extract: 100% and 75%.
The fruit of Manila palm was collected in an area near 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 study’s objectives, the researchers assigned the microorganisms of
interest to 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 it was subjected to the formulation were
all the same for the two microorganisms. This is to determine which 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) were
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
15 millimeters, indicating that the replicates also exhibited antimicrobial efficacy up to a
susceptible level. The two concentrations from all the replicates of Escherichia coli (ATCC
25922), on the other hand, showed no antibacterial inhibition at 6 millimetres, 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 no statistically
significant difference in pain score between the two different concentrations, namely 100%
and 75% concentrations having both a .368 significance level.
Conclusion
From the findings of the study, it can be inferred that from the two concentrations of
the crude extract, the 75% and 100% concentrations of Manila palm (Adonidia merrillii)
fruit’s ethanolic crude extract, only Staphylococcus aureus (ATCC 25923) exhibited
antimicrobial activity as being susceptible to the ethanolic crude extract. It did not exhibit
antimicrobial efficacy against Escherichia coli (ATCC 25922). Furthermore, both hypotheses
are accepted. Hence, there is no significant difference in the antibacterial activity of Manila
palm (Adonidia merrillii) fruit extract with the different concentrations of 75% and 100%.
And there is no significant difference in the antibacterial activity of Manila palm (Adonidia
merrillii) fruit extract between the experimental and control groups.
Recommendations
Based on the findings of the study, the following recommendations are suggested:
1. Utilize other microorganisms in order to determine the antibacterial activity of Manila
palm (Adonidia merrillii) fruit’s ethanolic crude extract.
2. For the pharmaceutical industry, conduct further research: invest in scientific studies to
determine the efficacy, safety, and appropriate dosage of Manila palm fruit ethanolic
extract against different strains of Escherichia coli. To use the other parts of the Manila
palm (Adonidia merrillii), such as the bark, flowers, leaves, stem, and roots, to create an
extract that kills microorganisms other than bacteria.
3. For Community, examine, research, and assess the various components of the manila
palm and note its various features to gain a deeper understanding of the plant.
4. For Patients, look into the plant's potential as a treatment for various bacterial infections.
Review the literature on the efficacy of the plant's other parts.
5. For the Medical Laboratory Science Students, review available scientific literature,
investigate the possibility of combining Manila palm fruit ethanolic extract with other
antimicrobial agents to see if there are any synergistic effects, and emphasize good
laboratory practices such as proper handling, preparation, and storage of Manila palm
fruit ethanolic extract.
6. For the future researchers, conduct a comprehensive literature review, consider exploring
different extraction techniques to determine the optimal method that yields consistent
results, and perform in vitro studies to assess the antimicrobial activity of Manila palm
fruit ethanolic extract against E. coli. and S. aureus, and consider adding more trials (up
to three trials) and concentrations (25% and 50%) of the Manila palm (Adonidia
merrillii) fruit’s ethanolic crude extract.
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a//nd Antimicrobial Agents. Journal of Pharmacy & Bioallied Sciences, 11(1), 1–11.
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Alrajhi, M., AL-Rasheedi, M., Eltom, S. E. M., Alhazmi, Y., Mustafa, M. M., & Ali, Al. M.
(2019). Antibacterial activity of date palm cake extracts (Phoenix dactylifera). Cogent
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APPENDICES
APPENDIX A
APPROVAL FOR IMPLEMENTATION

APPENDIX B
COMPLETION REPORT
APPENDIX C
CERTIFICATION OF PLANT
APPENDIX D
LETTER OF CONSENT FOR LABORATORY USE
May 24, 2023
Dr. Amelda C. Libres, RMT, DODT

Dean, College of Medical Laboratory Science
Dear Dr. Libres,
Good day!
It is with honor to write a letter to ask for your permission to be able to use and have access
to any of the MLS laboratory rooms. This is in accordance with performing our research
study's data gathering procedure. During the course of the procedure, we will be assisted by
Sir Rogin Artem Alamban.
We are third year students from Block 3A of the College of Medical Laboratory Science of
Liceo de Cagayan University conducting a study entitled "Antibacterial Activity of Manila
Palm (Adonidia merrillii (Becc.) Becc.) Fruit Extract against Escherichia coli and
Staphylococcus aureus”, which will be beneficial to the the medical and pharmaceutical field
for developing medications against these bacterial infections.
We are hoping for your kind consideration.
Thank You and Godspeed!
Sincerely,
Alan Louise Y. Bualat Umaynah E. Ahmed Maria BJ Theresa A. Aque

Researcher Researcher Researcher
Noted by: Recommending Approval:
Marilou O. Honculada, RMT, MA, MSMLS Amelda C. Libres, RMT, DODT

Research adviser Dean, College of Medical Laboratory Science
College of Medical Laboratory Science
APPENDIX E
LETTER OF CONSENT FOR LABORATORY USE
May 24, 2023

Mercedita S. Aquino RPh, MSPharm, PhD

Dean, College of Pharmacy
Thru: Jereyca P. Memoracion, RPh, CPh

Laboratory Coordinator
Dear Dr. Aquino,
We are presently Third Year students of Liceo de Cagayan University, under the College of
Medical Laboratory Science. We are currently conducting experimental research entitled
“ANTIBACTERIAL ACTIVITY OF MANILA PALM (Adonidia merrillii (Becc.) Becc.)
FRUIT EXTRACT AGAINST Escherichia coli AND Staphylococcus aureus", as part of our
requirement for the school year 2022-2023, and assisted by our research adviser, Mrs.
Marilou O. Honculada.
We would like to ask for your authorization to utilize the pharmaceutical laboratory of the
College of Pharmacy, as we conduct our experimental research. Our course of action is to
Conduct a Phytochemical Analysis with our variables: Manila Palm (Adonidia merrillii
(Becc.) Becc.) using the rotary evaporator machine and other necessary and available
equipment for the optimal operation of our data gathering procedure.
We guarantee that this research is fully intended for educational purposes, and we will do our
best to safely and discretely perform this activity, and strongly agree to your department
regulations and conditions.
It is our pleasure to be able to accomplish this experimental research with your approval. You
may contact us at the email abualat81913@liceo.edu.ph or by phone at 09163711997. We
will patiently wait for your response and wholeheartedly ask for your consideration.
Sincerely,
Alan Louise Y. Bualat Umaynah E. Ahmed Maria BJ Theresa A. Aque

Researcher Researcher Researcher
Noted by: Recommending approval:
______________________________________ _____________________________
Ms. Marilou O. Honculada, RMT, MA, MSMLS Dr. Amelda C. Libres, DODT, RMT
Research Adviser Dean, College of Medical
College of Medical Laboratory Science Laboratory Science
Approved By:
_____________________________________
Mercedita S. Aquino RPh, MSPharm, PhD
Dean, College of Pharmacy
APPENDIX F
DOCUMENTATION
Plate 1. Obtaining of the Manila

Palm fruit sample
Plate 2. Cleaning the Manila Palm

Fruit with distilled water
Plate 3. Air-drying of the plant’s Plate 4. Pulverized the air-dried Manila

fruit palm fruit through mortar and pestle
Plate 6. Rotary evaporation was utilized

to separate the ethanol and extract
Plate 5. Maceration of the pulverized

fruit in 100% ethanol
Plate 7. Phytochemical screening test of the Manila palm ethanolic fruit extract
done in the laboratory of College of Pharmacy, Liceo de Cagayan University
Plate 8. Preparation of the Mueller-Hinton agar plates
Plate 9. Dilution of the fruit extract Plate 10. Preparation of standardized

into 75% and 100% concentration inoculum
Plate 11. Inoculation of E. coli and S. aureus in Mueller Hinton Agar plates
Plate 12. Disc impregnation with Plate 13. Discs were placed into the
the different concentrations Mueller Hinton Agar plates with E. coli
Plate 14. Incubation and Measurement
Plate 15. S. aureus of
zone of inhibition
Trial 1 Trial 2
Trial 3
Plate 16. Escherichia coli Agar plates
Trial 1 Trial 2
Trial 3
Plate 17. Staphylococcus aureus Agar plates
APPENDIX G
DESCRIPTIVE STATISTICS FOR THE ZONES OF INHIBITION OF ADONIDIA
MERRILLI EXTRACT IN MILLIMETERS (MM) AGAINST STAPHYLOCOCCUS
AUREUS AND ESCHERICHIA COLI
Descriptive Statistics
N Mean Std. Deviation Minimum Maximum

CIPROF 3 29.67 1.155 29 31
EX100 3 6.00 .000 6 6
EX75 3 6.00 .000 6 6
Descriptive Statistics
N Mean Std. Deviation Minimum Maximum
CIPROF 3 28.00 .000 28 28
EX100 3 15.00 1.000 14 16
EX75 3 15.67 2.309 13 17
APPENDIX H
KRUSKAL WALLIS H TEST
Test Statisticsa,b
CIPROF EX100 EX75

Kruskal-Wallis H 2.000 .000 .000
df 2 2 2
Asymp. Sig. Test Statistics

.368
a,b
1.000 1.000
a. Kruskal Wallis Test

CIPROF EX100 EX75
Kruskal-Wallis H
b. Grouping Variable: REP
.000 2.000 2.000
df 2 2 2
Asymp. Sig. 1.000 .368 .368
a. Kruskal Wallis Test
b. Grouping Variable: REP

APPENDIX I
SUMMARY OF THE HYPOTHESIS TEST
Hypothesis Test Summary

Null Hypothesis Test Sig. Decision
1 The distribution of Independent-Samples 1.000 Retain the null
CIPROF is the same Kruskal-Wallis Test hypothesis.
across categories of
Replicate
2 The distribution of Independent-Samples .368 Retain the null
EX100 is the same Kruskal-Wallis Test hypothesis.
Replicate
3 The distribution of Independent-Samples .368 Retain the null
EX75 is the same Kruskal-Wallis Test hypothesis.
Replicate
Asymptotic significances are displayed. The significance level is .050.
Hypothesis Test Summary
Null Hypothesis Test Sig. Decision

1 The distribution of CIPROF is Independent-Samples .368 Retain the null
the same across categories Kruskal-Wallis Test hypothesis.
of REP.
2 The distribution of EX100 is Independent-Samples 1.000 Retain the null

of REP.
3 The distribution of EX75 is Independent-Samples 1.000 Retain the null

of REP.
Asymptotic significances are displayed. The significance level is .050.
APPENDIX J
CURRICULUM VITAE
Name: Alan Louise Y. Bualat
Address: P-4 Lower Loboc Oroquieta City
Email Address: abualat81913@liceo.edu.ph
ORCID no: 20200081913
Name of Father: Alan D. Bualat
Name of Mother: Jessica P. Bualat
School Graduated:
Elementary: Firm Foundation Christian Academy

Junior High School: Misamis Occidental National High School
Senior High School: Misamis Occidental National High School
College: Liceo de Cagayan University (Present)
Name: Umaynah E. Ahmed
Address: Blk 4 Lot 21, Alta Tierra Napolan, Pagadian City
Email Address: uahmed01568@liceo.edu.ph
ORCID no: 20200101568
Name of Father: Saeed N. Ahmed
Name of Mother: Umayyah E. Tampogao
School Graduated:
Elementary: Pagadian Montessori Center Inc.

Junior High School: Pagadian City Science High School
Senior High School: Pagadian City Science High School
Name: Maria BJ Theresa A. Aque
Address: P-1, Libertad, Quezon, Bukidnon
Email Address: maque17546@liceo.edu.ph
ORCID no: 20200117546
Name of Father: Buenaventura R. Aque Jr.
Name of Mother: Annabelle A. Aque
School Graduated:
Elementary: Libertad Elementary School

Junior High School: Quezon National High School
Senior High School: Central Mindanao University

Antibacterial Activity of Manila Palm (Adonidia Merrillii

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ANTIBACTERIAL ACTIVITY OF MANILA PALM (Adonidia merrillii

(Becc.) Becc.) FRUIT EXTRACT AGAINST Escherichia coli AND

Maria BJ Theresa A. Aque

Alan Louise Y. Bualat