DATE COMPLETED SIGNATURE OF THE

Our Lady of Fatima University

PROPONENT
College of Medical Laboratory
Science
Valenzuela City, Philippines

RESEARCH PROPOSAL CAPSULE

Gono, Claire Leader
Nicolas, Rahayana Marie Secretary
Caralos, Angela Monique Treasurer
Proponents Naces, Alyza Christine Member
Kazaoka, Mariya Member
Manaloto, Jenny Rose Member
Last Name First Name Middle Name
Field of
Bachelor in Medical Laboratory Science
Specialization
Research Approach Quantitative Approach
Research Design True Experimental Research Design
Research
Research Technique Single Technique
Attributes
Research Domain Experimental
Delineated Factors Plant materials and test microorganisms
Project Title
The efficacy of Piper betle linn against methicillin resistant Staphylococcus aureus and vancomycin-
(as proposed by
resistant Enterococcus
the candidates)
Project Title
(as redirected
The efficacy of Piper betle linn against methicillin resistant Staphylococcus aureus and vancomycin-
by the MedTech
resistant Enterococcus
Research1
Professor)
ISI Journals
Reviewed Antibacterial Activity Test of Jatropha multifida L. sap against Staphylococcus aureus and
(Follow the Methicillin Resistant Staphylococcus aureus (MRSA) in vitro. (2021). Indian Journal of Forensic
APA Style)
Medicine & Toxicology. https://doi.org/10.37506/ijfmt.v15i3.15611

Baneres, R. (2009, January 21). Vancomycin: Uses, Interactions, Mechanism of Action | DrugBank
Online. DrugBank. https://go.drugbank.com/drugs/DB00512

Braun, T. (2018, September). Community-associated Methicillin-Resistant Staphylococcus. . .:

Medicine & Science in Sports & Exercise. LWW.
https://journals.lww.com/acsm-msse/Fulltext/2018/09000/Community_associated_Methicillin
_Resistant.10.aspx

BRAUN, T., & KAHANOV, L. (2018). Community-associated Methicillin-Resistant Staphylococcus

aureus Infection Rates and Management among Student-Athletes. Medicine & Science in Sports &
Exercise, 50(9), 1802–1809. https://doi.org/10.1249/mss.0000000000001649

Bush, L. M., & Vazquez-Pertejo, M. T. (2021, December 14). Staphylococcal Infections. MSD
Manual Professional Edition.
https://www.msdmanuals.com/professional/infectious-diseases/gram-positive-cocci/
staphylococcal-infections

Cẩm HàChế Thị. (2021, March 1). Influence of Piper betle L. extract on umbilical cord cells in
vitro and potential treating cutaneous wound. ScienceDirect.
https://www.sciencedirect.com/science/article/pii/S2405844021003534#bib14

Demetrio, L. V. J. (2015a, July 1). Antibacterial activities of ethanol extracts of Philippine

medicinal plants against multidrug-resistant bacteria. ScienceDirect.
https://www.sciencedirect.com/science/article/pii/S2221169115000854

Demetrio, L. V. J. (2015b, July 1). Antibacterial activities of ethanol extracts of Philippine

medicinal plants against multidrug-resistant bacteria. ScienceDirect.

Page 1 of 12
 https://www.sciencedirect.com/science/article/pii/S2221169115000854#bib2

Devi.S, D. (2017). Methicillin Resistant Staphylococcus aureus in Pus samples. International

Journal of Medical Science and Clinical Invention, 4(12).
https://doi.org/10.18535/ijmsci/v4i12.09

D.L.Jr., V. (2021). Antimicrobial efficacy and activity of ethanolic extract of Piper betle L. on
Staphylococcus aureus-infected wound in mice and clinical isolates of multiple drug-resistant
bacterial pathogens. Tropical Biomedicine, 38(2), 134–142.
https://doi.org/10.47665/tb.38.2.049

Elshafie, S. S., Landreau, P., & Popovic, N. (2018). Preoperative Screening and Decolonization
Protocol for Methicillin – Resistant Staphylococcus aureus, Methicillin – Resistant Staphylococcus
epidermidis and Methicillin Sensitive Staphylococcus aureus Prevents Orthopedic Surgical Site
Infections. Microbiology & Infectious Diseases, 2(2). https://doi.org/10.33425/2639-9458.1031

FactSheet: Methicillin Resistant Staphylococcus Aureus. (2003). New South Wales Public Health
Bulletin, 14(6), 123. https://doi.org/10.1071/nb03037

Hartini, Y. S., & Nugroho, L. H. (2020). Short Communication: Antibacterial effect of red betel
(Piper crocatum) extract in combination with vancomycin against Staphylococcus aureus.
Biodiversitas Journal of Biological Diversity, 21(7). https://doi.org/10.13057/biodiv/d210750

Muhammad, R. (2022, January 1). Prevalence of blaZ gene and antibiotics susceptibility test
profile of Î2-lactams resistant Staphylococcus aureus isolated from subclinical mastitis in
lactating Beetal goats. ScienceDirect.
https://www.sciencedirect.com/science/article/abs/pii/S1871141321004054

Pastuović, T. (2015). Methicillin-resistant Staphylococcus aureus in North-east Croatia. Acta

Medica Academica, 44(1), 10–17. https://doi.org/10.5644/ama2006-124.122

Sun, Z. L., He, J. M., Wang, S. Y., Ma, R., Khondkar, P., Kaatz, G. W., Gibbons, S., & Mu, Q. (2016).
Benzocyclohexane oxide derivatives and neolignans from Piper betle inhibit efflux-related
resistance in Staphylococcus aureus. RSC Advances, 6(49), 43518–43525.
https://doi.org/10.1039/c6ra10199b

Thompson, E. G. (2020, November 23). Vancomycin-Resistant Enterococci (VRE) | Michigan

Medicine. University of Michigan Health.
https://www.uofmhealth.org/health-library/tp23381spec

Valle, D. L., Cabrera, E. C., Puzon, J. J. M., & Rivera, W. L. (2016). Antimicrobial Activities of
Methanol, Ethanol and Supercritical CO2 Extracts of Philippine Piper betle L. on Clinical Isolates
of Gram Positive and Gram Negative Bacteria with Transferable Multiple Drug Resistance. PLOS
ONE, 11(1), e0146349. https://doi.org/10.1371/journal.pone.0146349

Valle, D. L., Puzon, J. J. M., Cabrera, E. C., & Rivera, W. L. (2016). Thin Layer Chromatography-
Bioautography and Gas Chromatography-Mass Spectrometry of Antimicrobial Leaf Extracts from
PhilippinePiper betleL. against Multidrug-Resistant Bacteria. Evidence-Based Complementary
and Alternative Medicine, 2016, 1–7. https://doi.org/10.1155/2016/4976791

VRE in Healthcare Settings | HAI | CDC. (2019, November 13). Centers for Disease Control
Prevention. https://www.cdc.gov/hai/organisms/vre/vre.html

Weiner, H. R. (2008). Methicillin-ResistantStaphylococcus aureuson Campus: A New Challenge to

College Health. Journal of American College Health, 56(4), 347–350.
https://doi.org/10.3200/jach.56.44.347-350

Proposal
Abstract
Piper betle linn has been utilized in alternative medicine for numerous therapeutic purposes in
several locations, including as an anti-infective agent. However, investigations in the literature focus

Page 2 of 12
 mostly on its effects on drug-resistant bacterial strains. The antibacterial properties of its ethanol and
methanol extracts are to be test on clinical isolates of different drug-resistant bacteria: methicillin
resistant staphylococcus aureus and vancomycin-resistant enterococcus as among the most difficult
strains to manage now. The minimum inhibitory concentration (MIC) and minimum bactericidal
concentrations (MBC) of the extracts for the test bacteria are going to be use along with the
conventional disc diffusion method and the broth microdilution method, respectively.

Introduction
1.0 Introduction
Review of
Literature Piper betle Linn which is locally known as “ikmo” is cultivated widely in most of the humid
(Synoptic and tropical climate of Southeast Asia, including Philippines. Their leaves are used as a folk medicine
Argumentative) because of their medicinal properties. They have a major stimulation effect on pancreatic lipase
function, promote digestion, help prevent pulmonary disease, and help treat a variety of diseases
including boils, abscesses, conjunctivitis, and constipation (M.S. Prabhu, 2010). Piper betle L.
Research extracts contain bio-compounds that have been shown to have positive effects in the treatment
Methodology of diabetes, conjunctivitis, mastitis, burns, sores, as well as having antibacterial, anti-
inflammatory, antioxidant and anti-cancer properties. However, scientists are continuing to
explore the mechanisms of Piper betle L. extracts in different use of it as medical plant. There
have been numerous documentations found in the medical literature concerning the significance
of traditional medicinal plants as alternatives to synthetic antibacterial and antifungal
medications (Sujogya, 2014). Most of these published works come from many countries that are
still practicing the use of herbal medicine for the treatment of various diseases for practical and
economic reasons. These studies are valuable resources for local medical scientists who seek to
explore and substantiate the antibacterial and antifungal activities of Philippine medicinal herbs,
particularly against MDR bacteria. Knowledge on the different antimicrobial assays and the
plants’ bioactive compounds are vital for the design of future studies (Nascimento, 2020). The
Philippines is one of the Asian countries with a diverse flora, and numerous species are believed
to possess curative properties including Piper betle L. However, most of these claims lack
scientific validation and still needs further additional studies.

As antibiotic resistance is a problem that continues to challenge the healthcare sector in a large
part of the world in both developing and developed countries. The spread of multidrug-resistant
(MDR) bacteria in hospital and community settings remains a widely unresolved problem and a
heavy burden to health services. Despite advances in antibiotic therapy, infectious complications
remain an important cause of mortality and morbidity among hospitalized patients. Although
medical practitioners can resort to second or third choice of drugs for treating these patients, the
use of these synthetic drugs may subject the patient to a higher risk, due to the possibility of the
drugs producing more harmful side effects. As address to this challenge, actions must be taken to
reduce this problem, such as controlling the use of antibiotics, understanding the genetic
mechanisms of resistance and developing new antibiotics and new therapeutic strategies.
Advances in identifying new sources of natural products with antimicrobial activities and
expanding antibiotic chemical diversity are providing chemical leads for new drugs (Nascimento,
2000). Resorting to herbal medicines as other choice can be sound unreliable for the treatments
but according to Cowan (2020), the practices of plant-based traditional medicine are founded on
hundreds of years of belief and observations, which predate the development of modern
medicine. Medicinal plants have been used for centuries as remedies for human diseases as they
contain components of therapeutic value. There are numerous plant natural products which have
antifungal, antibacterial and antiprotozoal activities that could be used either systemically or
locally.

During the second half of the 20th century, the acceptance of traditional medicine as an
alternative form of health care and the development of microbial resistance to the classical
antibiotics led researchers to investigate the antimicrobial activities of medicinal plants.
Antimicrobials of plant origin have enormous therapeutic potential (Cowan 2020). They are
effective in the treatment of infectious diseases while simultaneously mitigating many of the side
effects that are often associated with synthetic antimicrobials. Traditionally used medicinal
plants produce a variety of compounds of known therapeutic properties. One of the vital
activities possessed by these medicinal plants is antimicrobial.

Bacteria are unicellular microorganisms which have cell walls but lack organelles and an
organized nucleus. It may find inside and outside of our bodies which are sometimes harmless,
and some can even be helpful to humans, but most of it can cause infections and severe diseases.
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Some of the bacteria known leading causes of these infectious diseases in the Philippines are
Corynebacterium diphtheria, Neisseria meningitidis, Bordetella pertussis, Mycobacterium
tuberculosis and Salmonella typhi (Department of Health, 2021). Staphylococci are gram positive
aerobic organisms. Staphylococcus aureus is the most pathogenic, it is typically causing skin
infections and sometimes pneumonia endocarditis and osteomyelitis. It is commonly lead to
abscess formation and some strains elaborate toxins that cause gastroenteritis, scalded skin
syndrome and toxic shock syndrome. Treatment is usually with penicillinase resistant beta-
lactams, but because antibiotic resistance is common vancomycin or other newer antibiotics may
require since some strains are partially or totally resistant to all.

Another one knows organisms for being MDR are Enterococci. According to Centers for Disease
Control and Prevention (2019), Enterococci are bacteria that are normally present in the human
intestines and in the female genital tract, and are often found in the environment, like in soil and
water. These bacteria can cause infections and constantly finding new ways to avoid the effects
of the antibiotics used to treat the infections they cause. Antibiotic resistance occurs when the
bacteria no longer respond to the antibiotics designed to destroy them. If these bacteria develop
resistance to vancomycin, the need alternative antibiotic is used to treat it. As a result, these
bacteria enterococci become vancomycin-resistant enterococci (VRE).

Vancomycin-resistant enterococci (VRE) are a type of bacteria called enterococci that have
developed resistance to many antibiotics, especially vancomycin. Enterococci bacteria live in our
intestines and on our skin, usually without causing problems. But if they become resistant to
antibiotics, they can cause serious infections, especially in people who are ill or weak. These
infections can occur anywhere in the body. Some common sites include the intestines, the
urinary tract, and wounds. Vancomycin is a glycopeptide antibiotic used to treat severe but
susceptible bacterial infections such as MRSA (methicillin-resistant Staphylococcus aureus)
infections. A variety of dosage forms for example oral or injections exist for the treatment of
serious or severe infections caused by susceptible strains of methicillin-resistant (beta-lactam-
resistant) staphylococci. It is a branched tricyclic glycosylated nonribosomal peptide often
reserved as the "drug of last resort", used only after treatment with other antibiotics has failed.
Vancomycin has been shown to be active against most strains of the following microorganisms,
both in vitro and in clinical infections: Listeria monocytogenes, Streptococcus pyogenes,
Streptococcus pneumoniae (including penicillin-resistant strains), Streptococcus agalactiae,
Actinomyces species, and Lactobacillus species. The combination of vancomycin and an
aminoglycoside acts synergistically in vitro against many strains of Staphylococcus aureus,
Streptococcus bovis, enterococci, and the viridans group streptococci.

This study aimed to investigate the antibacterial activities of crude ethanol extracts of Piper betle
Linn against methicillin resistant staphylococcus aureus and vancomycin-resistant enterococcus.
This plant has traditionally known antibacterial and antifungal activities, with no further
extensive bioassay studies performed locally. To identify the plant supplementary potential
bioactive molecules of pharmaceutical importance in the management of MDR bacteria, in vitro
methods of evaluation on test organisms were performed using the plant extracts.

2.0 Background of the Study

2.1 Literature Review
According to BRAUN (2018), MRSA (methicillin-resistant Staphylococcus aureus) is gaining
popularity in the public, particularly among athletes. MRSA skin infections were found to be a
rising problem among Nebraska's student athletes, according to a 2007-2008 inquiry. Following
that, statewide surveillance was done for four school years (2008-2012) to estimate the
incidence of MRSA-like skin and soft tissue infections (SSTI) among student athletes. Following
the winter and fall sports seasons, high school athletic officials completed Internet-based
surveys.

As formulated by Weiner, H. Richard (2018) Staphylococcus aureus' exceptional ability to evolve

antibiotic resistance in tandem with the establishment of highly virulent and/or transmissible
strains has firmly entrenched the bacterium as a primary cause of human bacterial infections all
over the world. Methicillin-resistant Staphylococcus aureus (MRSA) was once virtually
exclusively found in hospitals and other health-care settings. Based on New South Wales Public
Health (2013), the bacteria called Staphylococcus aureus (or staph) are found on people's skin
and in their noses. Staph infections are normally innocuous, but they can occasionally lead to
significant disease and infection. Staph strains have developed resistance to the antibiotic
methicillin, as well as other medications formerly used to treat infections. Most antibiotics are
ineffective against methicillin-resistant Staphylococcus aureus (MRSA) infections. According to
Devi.S, D. the Methicillin-resistant Staphylococcus aureus is a multidrug-resistant bacterium that

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is spreading over the world, with prevalence rates ranging from 4.6 percent to 54.4 percent.
MRSA is divided into two types: HA MRSA (Healthcare-Associated Methicillin-Resistant
Staphylococcus aureus) and CA MRSA (Cancer-Associated Methicillin-Resistant Staphylococcus
aureus) (Community acquired methicillin resistant Staphylococcus aureus). HA MRSA is a
disease that is commonly acquired during extended or frequent hospitalizations, whereas CA
MRSA is a pathogen that affects healthy persons and is transmitted within the community.

As reported by Elshafie (2018), the rate of surgical site infections after orthopedic surgery is
quite high. Between December 2017 and October 2018, there was a 2% increase. The dominating
organism is MRSE. As a center, we want to realize our goal. We expected that the use of a
standard of excellence with an SSI rate of 0% would be beneficial. A preoperative MRSA and
MRSE screening, followed by Patients' infection rates would be reduced if a decontamination
regimen was used. It is possible to save money by having orthopedic surgery. Staphylococcus
aureus was added after the first incidence of Staphylococcus aureus SSI was diagnosed in the
month of October 2009. A multidisciplinary team was formed at the hospital with the goal of
achieving infection-free surgical sites a portion of the team effort entails empowering patients to
take part in an activity program of eradication Since the beginning of preoperative screening, the
overall infection rate fell from 2% to 0%.

In comparison to S. aureus, MRSA-related infections increased. In 2011, and 2012, aureus

infections were found in wound and aspirate samples (57.9% /34.9 percent and 35.2 percent
/16.3%, respectively). The majority of MRSA-related infection strains were resistant to a variety
of medications, including erythromycin and clindamycin, which had a susceptibility rate of less
than 10%. Vancomycin, teicoplanin, and linezolid were all effective against MRSA conforming to
Pastuović, T. (2015).

Macroscopic examination on the Nutrient Agar (NA) media revealed that S. Aureus created a 0.5
mm colony that was white in color, spherical in shape, with a fine border and an even surface.
Gram staining revealed a bacterium in the shape of a coccus, purple in color, and formed a small
cluster like grapes, indicating that it was a Gram-positive bacterium. The catalase test yielded a
positive result, which was indicated by the formation of gas bubbles, and the coagulation test
utilizing plasma in tubes yielded a positive result, namely the formation of plasma coagulation.
The outcome of a S. resistance test. S. aureus to methicillin revealed that S. aureus is susceptible
to the antibiotic isolate aureus Methicillin-resistant bacteria are those that are resistant to the
antibiotic methicillin. All the findings show that the examined bacteria were MRSA.

The number of pharmaceutical companies producing novel antimicrobial medications has

dropped dramatically during the last few decades. Because of the limited number of medications
that may be used therapeutically for this organism and the resistance associated with their use,
methicillin-resistant Staphylococcus aureus (MRSA) remains a source of worry. The
development of drugs that can inhibit bacterial resistance mechanisms, known as resistance-
modifying agents, is a supplement to overcoming resistance (RMAs). Synergism is a phenomenon
in which two or more different chemicals are combined to enhance their individual activity, and
this method is now widely known and can be directly applied to the development of new
medications (Sun, 2016).

In the Philippines, the leaves of the P. betle linn have long been employed to enhance wound
healing and cure wound infections according to D.L.Jr., V. (2021). The vivo effectiveness of an
ethanolic extract of Leaves of P. betle linn againts S. aureus wounds in mice infected with Staph
aureus Regarding its antibacterial activity against clinical MDR isolates pathogens caused by
bacteria. The wound healing efficacy of the P. betle linn ethanolic extract cream formulations on
S. aureus infected wounds in mice is very promising since it is comparable to the standard drug
mupirocin cream. The compositions of 2.5 percent and 5.0 percent as evidenced by the same
efficacy of mupirocin cream based on the various wound healing parameters measured includes
granulation tissue development, re epithelialization, and regarding the arrangement of collagen.

According to Valle (2016), The Gram-positive vancomycin-resistant Enterococcus (VRE) and

methicillin-resistant Staphylococcus aureus (MRSA) bacteria, the Gram-negative extended
spectrum -lactamase (ESL)-producing Enterobacteriaceae, carbapenem-resistant
Enterobacteriaceae (CRE), metallo-lactamase (ML)-producing Pseudomonas aeruginosa, and
Acine. All of these bacteria's resistance methods involve genetic components that code for
numerous drug resistances as well as the ability to insert additional resistance genes. At the
same time, they can be passed from one bacterium to the next. Enterococci have long been
recognized as a significant source of hospital-acquired infections and a major cause of
endocarditis. They have built-in resistance to a variety of antimicrobials from distinct classes.

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Until the discovery of VRE, vancomycin was the medication of choice against MDR enterococci. S.,
on the other hand, the bacterium Staphylococcus aureus is an opportunistic infection that causes
significant mortality and morbidity from both clinical and community settings.

Based on Valle (2016), The plant is said to be widely dispersed in Sri Lanka, Malaysia, Indonesia,
Thailand, China, and Philippines probably because it is classified as a tropical and subtropical
countries. The research has been demonstrated P. betle's potential as a multidrug-resistant
infection treatment (MDR) bacterium. The extricates demonstrated to be stronger against the
Gram-positive methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant
Enterococcus (VRE) than for the Gram-negative test bacteria. VRE separates were more
vulnerable to all the extricates than the MRSA segregates. For the most part, the ethanol
extricates demonstrated to be more powerful than the methanol extricates and supercritical CO2
extricates as appeared by their lower MICs for both the Gram-positive and Gram-negative MDRs.
P. betel leaf extract can use to treat gram positive and gram-negative bacteria. According to
Journal of medicinal plant studies (2016) “The maximum bacterial activity of Piper betle linn was
only observed in Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus.” This
means that Piper betle linn is more effective in other bacteria.

This study “Antibacterial activity of betle leaf… (2020)” supports that “P. betle extract shows
with the concentrations of 0.5%, 1%, 1.5%, 2%, 2.5%, and 3% did form clear zones around the
well.” Piper betle betle has possibility to use as an antibacterial against Staphylococcus aureus.
This study of Valle (2016)” was added that “VRE isolates were more susceptible to all the
extracts than the MRSA isolates”. Piper betle is more effective in VRE compared to MRSA.

Piper betle have potential antioxidant activity. According to Effect of Piper betle linn Extract on
the Growth of Staphylococcus Aureus Atcc 25923 (2020) “Green betel extract fractions (Piper
betle L.) have strong inhibitory activity against food pathogenic bacteria (Escherichia coli,
Salmonella typhimurium, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus cerius and
Listeria monocytogenes)” this means that piper betle shows a high potential to use against
bacteria. In additional the Green betel leaf shown an effect against the growth of S. aureus and E.
coli, which is indicated by the presence of clear zones formed on the culture media (Gloria et al.,
2021).

The most essential portion is the betle leaves, which have medical, religious, and ceremonial
value. Chewing betel leaves is said to help with foul breath, gum hardening, tooth conservation,
and breath sweetening. Traditional herbs have been known to utilize the infusion made from the
leaves as mouthwash and cough medication. Because of its antibacterial effectiveness against
food spoilage yeasts, oil from betel leaves has recently been determined to be a potential agent in
controlled and released food packaging technology as stated by Kaypetch & Thaweboon (2018).

According to Lawal (2018), for the treatment of infections caused by MDR enterococci,
vancomycin was the most commonly utilized antibiotic. Antimicrobial resistance is found in both
Gram-positive and Gram-negative bacteria around the world, and new attempts are being made
to standardize the descriptions and classifications of these bacteria so that surveillance data may
be collected reliably across countries. Multidrug-resistant infections are more difficult and
complicated to treat since they are resistant to numerous antibiotics, and they may require
multiple rounds of antibiotic therapy. MDRO infections can result in inadequate or delayed
antimicrobial treatment, which is linked to poorer patient outcomes. To make matters worse,
only a few new antibiotics have been approved for clinical use in the last 20 years, despite the
growing need for new antibiotics to combat the rising number of MDROs.

According to (Guha, 2017) Paan is the name given to the fresh leaves of the betle vine, which are
consumed by roughly 15-20 million people in India. It is grown in India using conventional
methods on approximately 55,000 acres, with an annual production value of over Rs 9000
million. The state of West Bengal contributes about 66 percent of total production, with over
20,000 ha comprising about 4-5 lakh Boroj and employing almost the same number of
agricultural families. During storage, transit, and glut season, there is a significant loss of leaves.
Furthermore, if the excess leaves are not properly disposed of, they may pollute the environment
and pose health risks. Waste can be reduced in a variety of ways, including the extraction of
essential oil from leftover betel leaves. This oil can be used to make medications, perfumes,
mouth fresheners, tonics, food additives, and other industrial products. The leaves are nutritious
and contain anticarcinogens, indicating that they could be used to develop a blood cancer
treatment.

According to Nalina & Rahim (2007), The crude extract of Piper betle L. has been proven to have

Page 6 of 12
anti-inflammatory properties. Leaves have antibacterial properties against S. mutans by
interfering with glucan adhesion, growth, and generation. The goal of this investigation was to
see how the extract affected the ultrastructure and acid production of S. mutans. In addition, the
extract's chemical components were identified.

2.2 Problem Statements

The researcher aims to determine the efficacy of Piper betle linn against methicillin-resistant
staphylococcus aureus and vancomycin-resistant enterococcus. Specifically, this sought to
answer the following questions.

1.1.1 What are indication that the ethanolic leaf extract of P. betle linn produces compounds that
can combat the bacterial defenses against MRSA and VRE?
1.1.2 What are the antimicrobial effectivity of leaf extract of P. bettle linn using methods of:
a. Minimum Inhibitory Concentration (MIC)
b. Minimum Bactericidal Concentration (MBC)
1.1.3 In what concentration does the extract of P. betle linn required to inhibit the growth and
affect death of the pathogens?

2.3 Significance Statement

The purpose of the study is to determine the efficacy of P. betle linn in methicillin-resistant
staphylococcus aureus and vancomycin enterococcus. The findings of this study will be beneficial
to the following:
a. Infected patients- this study will serve as valuable tool for the treatment of infected
patient with gram positive staphylococcus aureus and enterococcus by using P. betle linn.
b. Medical doctor- This study is beneficial to the doctor this will be their guide to use Piper
betle linn as a treatment in staphylococcus and enterococcus infection.
c. Medical technologist- This study will serve as a guide for medical technologist
undertaking similar studies.
d. Educators- this study will help to improve their knowledge about P. betle uses in
staphylococcus aureus and vancomycin enterococcus infection.
e. Students- This study will serve as a reference in doing research related to P. betle, staph.
Bacteria and enterococci bacteria.
f. Future researchers- This study could be an instrument for the future researcher in
conducting P. betle linn or staphylococcus and Enterococci infection. The study will serve as
valuable tool for identification.

2.4 Scope & Limitations

This research was conducted to determine the efficacy of P. betle against methicillin-resistant
staphylococcus aureus and vancomycin- resistance enterococcus. This covered P. betle leaf
extract to test the antibacterial effectivity of P. betle.

The research evaluates the (1) quantification of bacterial activity of P. betle. (2) effectiveness of
P. betle (3) potential response of P. betle against methicillin-resistant staphylococcus aureus and
vancomycin- resistance enterococcus. The P. betle for experimental purposes will purchase
either from Quiapo, Manila or Quezon Province. Evaluated by agar well diffusion method (Lubis,
R., et al, 2020). Piper betle extraction is going to be conducted at the Animal Care and
Experimentation Facilities (ACEF) of the Our Lady of Fatima University.

3.0 Research Methodology

3.1 Research Design
In this study researcher are going to use a True Experimental research design. All studies having
at least one experimentally altered independent variable and at least one dependent or outcome
variable are referred to as true experiments (Salkind, 2010). The researchers believe that by
using this kind of approach there is a possibility that can be altered to see whether it influences
the other variable, they will be able to determine if it has an influence towards it.

3.2 Ethical Consideration

The researchers will conduct an experiment in a test microorganism which covers a study that
only approved by the protocol which is strictly followed. Proper disposal and handling of
organisms will be practiced all throughout the research study.

3.3 Materials & Methods

Presented are the materials will be use by the researchers for the experimentation: extract,
ethanol, vancomycin, methicillin, test microorganisms, and minimum inhibitory concentration
(MIC) and minimum bactericidal concentration (MBC).

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 3.3.1 Plant extract of P. betle linn
It will be use as the main ingredient in the study because of its phytochemical components that
has ability to inhibit growth of Staphylococcus aureus and Enterococcus bacteria. The
researchers will gather 150 grams of powdered leaves of P. betle linn. And extract of the plant
material will be use for the susceptibility testing for bacterial growth and it will further compare
to other controls.
3.3.2 Ethanol
According to FDA, ethanol is the best option for plant extraction because it is efficient, effective
and safe to use. In this study, the researcher will be using ethanol for plant extraction and it will
be soaked under the ethanol for 24 hours. Ethanol is known as an effective chemical in
separating oils from the plant material that would result to large extraction.
3.3.3 Vancomycin and Methicillin
Vancomycin is an antibacterial drug that usually treats bacterial infections by inhibiting the
bacterial growth but Enterococcus bacteria are resistant to it due to altered peptidoglycan
terminus. Methicillin is an antibacterial drug but it is resistant to Staphylococcus aureus because
of the expression made by the organism which is the foreign PBP and PBP2a that makes it
resistant to the action of methicillin.
3.3.4 Test microorganisms
The gram-positive bacteria will be getting from Pamantasan ng Lungsod ng Maynila. All
microorganisms are going to be identified by automated biochemical test using Vitek MS
aspartate aminotransferase following minimum inhibitory concentration (MIC).
3.3.5 Minimum inhibitory concentration (MIC) and minimum bactericidal
concentration (MBC)
The MIC will be determined by selecting the lowest concentration of plant extract that
completely inhibited the growth of the organism in the well as detected by the unaided eye. To
determine the growth end points, that amount of the growth in the wells containing the plant
extracts is going to be compare with the amount of the growth in the growth-control well use in
the test. For the test to be considered valid, acceptable growth (2mm button or definite turbidity)
must occur in the growth-control well. While in MBC, it is going to be define as the lowest
concentration of the extract that did not permit any growth.

3.4 Data Analysis

The primary data will be collected from previous research studies determining the antimicrobial
effectivity of Piper betle against bacteria. The researcher will list an indicator in finding literature
based on the research topic and with the help of eligible studies. Then, the researcher will review
the methodology to evaluate how they process their outcomes, results and discussion.

In secondary data, the researchers will be using experimental research to examine the
methicillin-resistance staphylococcus aureus and vancomycin-resistant enterococcus that grown
and maintained on disc diffusion assay and this will be use as the test for antibacterial of Piper
betle that were able to purchase from Quiapo, Manila. (Mariano et al., 2017). The organisms that
grown in disc diffusion were transferred to the test tube with the help of a sterilized inoculating
loop. The suspension form in the test tube was immediately introduced to the sterilized petri
dish to assure the homogenous distribution of organisms and kept the petri dish at low
temperature for 4-6 hours. This experiment was conducted in triplicates then, the plates agar
incubates for 24 hours at the room temperature (Nahid et al., 2014).

Page 8 of 12
 Gathering and Preparation of Materials

Extraction of Piper bettle

Determination of plant
Collecting materials linn
constituents

Validation

Experimental Phase

Group 1 Group 2

Gram-positive bacteria Gram-positive bacteria

- Staphylococcus aureus - Enterococcus

Laboratory Test

Results

Research Analysis and Interpretation of Results

Flowchart

Page 9 of 12
 CURRICULUM VITAE

CLAIRE L. GONO
Ph4C Pkg6 Blk41 Bagong Silang, Caloocan City
Jjay3504@gmail.com

Personal Information
Birthday: January 18, 2001
Birthplace: Sta. Cruz, Manila
Religion: Catholic
Mother’s name: Marina L. Gono
Father’s name: Danilo O. Gono
Sibling/s’ name: Kyle Gono, Kenneth Gono

Education
School School Year
College Our Lady of Fatima University 2019-Present
Senior High School Our Lady of Fatima University 2017-2019
Junior High School Ramon Magsaysay (Cubao) High 2013-2017
School
Elementary Lucas R. Pascual Elementary 2007-2013
School

RAYAHANA MARIE NICOLAS

B5 L23 Diamond St. Citation Homes, Brgy. Bahay Pari Meycauyan City, Bulacan
rmnicolas1@student.fatima.edu.ph
Personal Information
Birthday: February 27, 2000
Birthplace: Sipocot, Bicol Camarines Sur
Religion: Catholic
Mother’s name: Eden M. Teves
Father’s name: Ronald M. Nicolas
Sibling/s’ name: Roden Victor M. Nicolas

Education
School School Year
College Our Lady of Fatima University 2019-Present
Senior High School Our Lady of Fatima University 2017-2019
Junior High School St. Mary’s Academy of Sto. Nino 2016-2017
Elementary Colegio De Sta. Cecilia 2004-2012

ANGELA MONIQUE CARALOS

4704 hermogina st. Francisco comp., Gen T. de Leon Valenzuela City
angelacaralos19@gmail.com

Personal Information
Birthday: December 19, 1999
Birthplace: Lying in Marulas, Valenzuela City
Religion: Roman Catholic
Mother’s name: Racquel G. Caralos
Father’s name: Randy S. Caralos
Sibling/s’ name: John Cedric G. Caralos, Shery G. Caralos

Education
School School Year
College Our Lady of Fatima University 2019-Present
Senior High School Our Lady of Fatima University 2017-2019
Junior High School Saint Marys Angels College of 2013-2017
Valenzuela
Page 10 of 12
 Elementary Gen. T. de Leon Elementary School 2008-2013

ALYZA CHRISTINE NACES

224 Sto. Domingo St. Brgy. Balingasa, Quezon City
alyza.naces7@gmail.com

Personal Information
Birthday: August 25, 2001
Birthplace: Quezon City
Religion: Roman Catholic
Mother’s name: Rener Naces
Father’s name: Winifred Naces
Sibling/s’ name: Laurence Naces

Education
School School Year
College Our Lady of Fatima University 2019-Present
Senior High School Our Lady of Fatima University 2017-2019
Junior High School Masambong High School 2013-2017
Elementary Balugo Elementary School 2007-2013

MARIYA KAZAOKA
9013 DRT Highway Tarcan Baliuag, Bulacan
kazaokamariya@gmail.com

Personal Information
Birthday: November 14, 1999
Birthplace: Warabi City, Japan
Religion: Muslim
Mother’s name: Carolina Peralta Velasco
Father’s name: Kazaoka Jubedo
Sibling/s’ name: Marian Kazaoka, Munir Kazaoka

Education
School School Year
College Our Lady of Fatima University 2019-Present
Senior High School Todo Shoyo High School, Japan 2017-2019
Junior High School District Public School Kasur, 2013-2017
Pakistan
Elementary District Public School Kasur, 2008-2013
Pakistan

JENNY ROSE MANALOTO

Blk 108 Lot 12 Brgy. Cristo Rey Capas, Tarlac
Jennymanalotosicat@gmail.com
Personal Information
Birthday: January 8, 2001
Birthplace: Cristo Rey Capas
Religion: lll
Mother’s name: Elvira Sicat Manaloto
Father’s name: Bonifacio Augustin Manaloto
Sibling/s’ name: Sherry Ann Manaloto, Jay Dester Manaloto, Francis John Manaloto, Rea Lynn Manaloto

Education
School School Year
College Our Lady of Fatima University 2019-Present
Senior High School Cristo Rey High School 2017-2019
Junior High School Cristo Rey High School 2013-2017
Elementary Cristo Rey Central Elementary 2008-2013
School

RECOMMENDING APPROVAL REVIEWED BY APPROVED BY

Page 11 of 12
 P
Justin Kim Vergara, RMT, PROF. JOSE JUREL M. NUEVO, RMT, MSMT, Ph.D.
Res 1 Laboratory Instructor Chair, Panel of Evaluators Dean of the College of Med. Lab. Science

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