1

Chapter I

Introduction

Every human wanted to be safe and free from different kinds of illnesses. That is

why most of them buy varieties of disinfecting agents such as alcohols for them just to be

sanitized not knowing that the place where they live for years itself comprises of hundreds

of different kinds of bacteria.

According to the Society for General Microbiology (2018) bacteria are single celled

microbes which can be found in every habitat on earth and some of it can divide every 20

minutes with the right temperature and nutrients. That is the reason why symptoms appear

quickly after an infection. Most contaminated objects in home found over 340 different

bacteria on 30 different objects (Charles River Laboratories International, Inc., 2016) but

not all of it are harmful. Staphylococcus aureus, Streptococcus pyogenes, Salmonella typhi,

and Escherichia coli are the dominant household germs that can make an individual sick

(Healthline Media, 2018).

Staphylococcus aureus is the leading cause of skin and soft tissue infections such

as abscesses or boils, furuncles, and cellulitis. Although most staph infections are not that

serious, it can still cause serious infections such as bloodstream infections, pneumonia, or

bone and joint infections (Minnesota Department of Health, 2017). While Streptococcus

pyogenes also known as flesh-eating bacteria, ranges from mild illnesses such as strep

throat and impetigo to more serious diseases such as scarlet fever, glomerulonephritis, and

necrotizing fasciitis yet if untreated, it will lead to rheumatic fever (Microbewiki, 2011).

However, Salmonella typhi is a common bacterial disease that affects the intestinal tract.
 2

Typically, bacteria live in human and animal intestines and are shed through feces. Most

frequently humans become infested through polluted water or food. Symptoms include

nausea, vomiting, abdominal cramps, diarrhea, fever, chills, headache, and blood in the

stool (Mayo Foundation for Medical Education and Research, 2018). On the other hand,

Escherichia coli refers to a wide range of bacteria that can cause various diseases, including

pneumonia, urinary tract infections, and diarrhea. Some strains of E. coli infection can

include nausea, vomiting, and fever while for susceptible individuals, certain types of E.

coli infection can lead to kidney failure (Medical News Today, 2017)

Commercial disinfectant spray contains denatured ethanol, which can affect the

eyes, mucous membranes, and can affect the central nervous system if inhaled or ingested.

It also contains ammonium hydroxide, which is thought to be a respiratory toxin, and is

linked to serious health conditions such as bronchitis, pulmonary edema, emphysema, and

cancer (Centers for Disease Control and Prevention, 2016).

With this instance, the researchers examined some possible alternatives for

disinfection purposes which can kill all known bacteria in every household without

instigating negative effects to human health and gratefully found some results on Piper

betle.

P. betle (Piper betle) leaves have long been in use in the Indian local system of

medicine for its antioxidant and antimicrobial properties. The antimicrobial activity of

ethanol extract of Piper betel leaves was assessed against human pathogenic bacteria. As

seen in its phytochemical screening, the leaves were found to contain carbohydrate, protein,

polyphenolic compounds, flavonoid, alkaloids and total antioxidant. The extract

 3

established strong antimicrobial activity against all bacterial strains tested. (Datta, Singh,

and Ghoshdastidar, 2010).

Out of this material, the researchers were prompted to venture on a study entitled

“Betel Leaves Extract as Antibacterial Disinfectant Spray” which will give housekeepers

and office workers an alternative disinfecting agent that will surely not harm their health,

especially the kids who are way more sensitive and prone to diseases and illnesses.

Due to the dominance of bacteria such as E. coli, Streptococcus pyogenes,

Staphylococcus aureus, and Salmonella in every household and even in hotels, offices, and

other establishments which can be killed by commercial disinfectant sprayers but

instigating negative effects to human health, the researchers thought of conducting a study

entitled “Betel Leaves Extract as Antibacterial Disinfectant Spray”.

One of the beneficiaries of this study is the office workers. Office workers are a

group of individuals employed in a certain organization that are responsible in typing or

filing documents, correspondence, reports, statements and other material and their

workstation usually has a computer, laptop, keyboard and telephone. With the use of betel

leaves extract as disinfectant spray, their health is safe because it does not contain harmful

chemicals.

Family members are persons in a household which comprises of spouses, parents,

brothers, sisters, sons, and daughters. Grandparents, aunts, uncles, cousins, nephews,

nieces, and siblings-in-law are also included if talking about an extended family. They are

also a beneficiary of this study because they are exposed to the residues of commercial

disinfectants which can make them prone to diseases.

 4

Children are human being between the stages of birth and puberty. They are one of

the beneficiaries because they are the primary victims since Nurseries and Child Care

Centers across the globe condone the practice of spraying toys with commercial

disinfectant allowing toys to air dry and returning them immediately for use not realizing

that leaving disinfectant residue on toys and surfaces is a chemical hazard and can cause

serious health risks to every individual’s health.

This study can also function as a prior knowledge for the future researchers.

Enhancements of the study through new sightings and invention of a new disinfectant and

plant extracts with antibacterial properties in their locality can be undertaken. Moreover, it

will be beneficial to those researchers with similar studies as their basis and reference

because they can utilize the results to upkeep and defend their proposals.

This study mainly focuses on the formulation of a disinfectant spray using betel

leaves extract. It was conducted at Regional Science High School for Region I, Bangar, La

Union from June 2018 to February 2019. It aimed to investigate and determine the

effectiveness of the betel leaves extract as antibacterial agent in making disinfectant spray.

It was only delimited with the use of betel leaves extract, isopropyl alcohol, and distilled

water as the substances used in the making of treatments. Likewise, five treatments were

prepared including the control and betel leaves extract alone and the amount of the

substances used was varied in treatments 1, 2, 3 and 4. Furthermore, the treatments were

only subjected to antimicrobial analysis using E. coli and S. aureus since it was the only

available cultured bacteria in the testing laboratory of DOST La Union.

 5

This study aimed to determine and investigate the effectiveness of betel leaves

extract as antibacterial disinfectant spray. In which it sought to answer the following

questions; What is the phytochemical analysis of the Betel leaves extract?, Which of the

treatments yielded the best result in terms of zone of inhibition and bacterial sensitivity?,

and Is there a significant difference in terms of zone of inhibition and bacterial sensitivity?
 6

INPUT PROCESS OUTPUT

Phytochemical Anti-bacterial
Betel Leaves
and
analysis
Extract Anti-microbial
Tests for: properties of
1. Flavonoid Betel Leaves
2. Alkaloids
3. Polyphenolic
compounds

FEEDBACK
Figure 1. Phytochemical Analysis of Betel Leaves Extract
 7

INPUT PROCESS OUTPUT

Piper betle leaves Effectivity test of the Betel (Piper
extract in the Piper betle (Betel) betle) leaves
following
concentrations: leaves extract in extract as
1. 25% Piper betle different Antibacterial
(Betel) leaves
2. 50% Piper betle concentrations and the Disinfectant
(Betel) leaves
3. 75% Piper betle control treatment, Spray
(Betel) leaves
against the test
Test organism –
common bacteria organism, common
bacteria

FEEDBACK
Figure 2. Effectivity test of the Betel (Piper betle) leaves Extract
 8

Antibacterial Agent. Are naturally obtained from fungal sources, semi-synthetic members

which are chemically altered natural product and or synthetic.

Betel Leaves. Piper betel is one of the most vital therapeutic plants where its leaves have

been used for numerous medicinal purposes.

Disinfectant Spray. A chemical that destroys vegetative forms of harmful microorganisms

especially on inanimate objects but that may be less effective in destroying spores

Escherichia coli (E. Coli). Are a large and diverse group of bacteria normally live in the

environment, foods and intestines of people and animals. E. coli is referred to as the best

or most studied free-living organism. It is the most prevalent infecting organism in the

family of gram-negative bacteria.

Ethanol Extraction. The process of transferring a substance from any matrix into ethanol.

Staphylococcus aureus. Staphylococcus aureus is a type of bacteria. It is found in grape-

like (staphylo-) clusters. In humans S. aureus is one of the normal microbiotas that is

existing in the upper respiratory tract.

Chapter II

Methodology
 9

This chapter comprises the Experimental Design Diagram, the materials used, the

general procedure, and the flowchart of this study.

This study used Experimental Design in determining the antibacterial activity of Betel

leaves extract as disinfectant spray. This design was used to establish cause-effect

relationship among a group of variables in the study.

A. Experimental Design Diagram

Title: Betel Leaves Extract as Antibacterial Disinfectant Spray

Hypothesis:
1. There is no significant difference among the treatments in terms of:
a) Zone of Inhibition; and
b) Bacterial Sensitivity.
Independent Variable: Dependent Variable:
Amount of Betel Leaves Extract Zone of Inhibition
Bacterial Sensitivity

Control Variable: Extraneous Variable:

Commercial Disinfectant Spray Source of Bacteria
Room Temperature

Treatments:
𝐓𝟎 – Commercial Disinfectant Spray
𝐓𝟏 – 25ml BLE + 15ml isopropyl alcohol + 10ml distilled water
𝐓𝟐 – 50ml BLE + 15ml isopropyl alcohol + 10ml distilled water
𝐓𝟑 – 75ml BLE + 15ml isopropyl alcohol + 10ml distilled water
𝐓𝟒 – 100ml BLE

Trials: 1
 10

B. General Procedure

1. Materials

Seven hundred grams of betel leaves was gathered and extracted using ethanol

extraction and 250ml of isopropyl alcohol was acquired. On the other hand, graduated

cylinder was used to measure the volume of distilled water, isopropyl alcohol, and betel

leaves extract. Moreover, 150ml beakers and stirring rod was used for mixing purposes.

And lastly, surgical gloves, masks, and cotton buds was purchased to avoid

contamination while making the process.

2. Treatments

This study was delimited into five treatments namely; Commercial disinfectant

spray labelled as T0 . T1 was prepared by mixing together 25ml betel leaves extract, 15ml

isopropyl alcohol, and 10ml distilled water. However, T2 was set by combining 50ml betel

leaves extract, 15ml isopropyl alcohol, and 10ml distilled water. Meanwhile, T3 was

established by combining 75ml betel leaves extract, 15ml isopropyl alcohol, and 10ml

distilled water. Lastly, T4 was prepared by having 100ml betel leaves extract.
 11

3. Experimental Procedure
PROCESS FLOW

Phase 1: Preparation and

Procurement of Raw Materials

Collecting and Phytochemical Preparation of

Verifying Analysis: Betel extract: Ethanol
the Variety of Plant extract
Leaves Extract
samples

Phase 2: Laboratory Analysis

and Procedures

Preparation of
Treatments
Phase 3: Experimental
Testing

Bacterial Zone of
Sensitivity Inhibition
Figure 3. Flowchart
 12

4. Data Gathering

The manner of data collection of this study is through the analysis of betel leaves extract

in terms of zone of inhibition, bacterial sensitivity and phytochemical analysis. The

gathered data was then used in order to test the hypotheses of causal relationships among

variables.

The researchers requested permission to the research adviser as well as the school

administration before the conduct of phytochemical analysis. The assigned date of data

collection was scheduled September 2018.

After receiving the approval, the researchers went to LORMA Colleges, San Fernando,

La Union to give the plant sample and subjected to phytochemical analysis. The

phytochemical analysis is the extraction, screening and identification of the medicinally

active substances found in plants. Substances such as flavonoids, alkaloids, carotenoids,

tannin, antioxidants and phenolic compounds are bioactive constituents that are found from

the plant. The process lasted for seven days and after knowing the results, the researchers

made the treatments in the RSHS Senior High School Laboratory.

The researchers requested permission again from the research adviser and school

administrator for the conduction of antimicrobial analysis of the treatments to collect data

for the zone of inhibition and bacterial sensitivity. The zone of inhibition, also called Kirby-

Bauer Test, is a qualitative method used clinically to measure antibiotic resistance and

industrially test the ability of solids and textiles to inhibit microbial growth. This is used to

test in a quick and easy way to measure and compare levels of inhibitory activity.
 13

5. Statistical Tool

Analysis of Variance (ANOVA) was used to interpret the result of the antimicrobial

analysis of the experimentation. According to Hindle (2013), ANOVA is the statistical tool

used to compare the differences of means among more than 2 groups or treatments. Since

this study have five different treatments, the use of this tool was essential to determine the

comparison of the results of the treatments.

6. Ethical Consideration

The researchers ensured that all the details of the experimentation were stated

unmistakably to show veracity or accuracy and to avoid confusion. Utmost honesty and

transparency were done in all communication in connection to the research and any kind

of misleading information as well as presentation of biased data was avoided. The

researchers presented results that were valid and reliable to manifest the trustworthiness of

this research. Furthermore, the works of authors that were used in the research book was

properly cited using the American Psychological Association (APA) referencing system.
 14

Chapter III
Results and Discussions
This chapter presents the results, interpretation and discussion of the data gathered

in the methodology.

A. Phytochemical Analysis of Betel Leaves Ethanol Extract

Table 1: Result of Phytochemical Analysis of Bethel Leaves Ethanol Extract

Type of Test Result

Mayer’s Test (-)
Hager’s Test (-)
Wagner’s Test (-)
Dragendorff’s Test (-)
Molisch Test (-)
Benedict’s Test (-)
Fehling’s Test (-)
Lead Acetate test (+)
Alkaline Reagent Test (+)
Ferric Chloride Test (+++)
Froth Test (+)
Gelatin Test (+)
Spot Test (+)
The Phytochemical Analysis result indicated that Piper Betle were positive for

ample amounts of Tannins and Phenolic compounds, Flavonoids, Glycosides, Saponins,

and Volatile Oils.

According to Shekhar (2009), Tannins are polyphenols that are obtained from

various parts of different plants belonging to multiple species. These substances serve as a
 15

barricade for micro-organisms like bacteria and fungi. The Betel also contains flavonoids

which are well known antibacterial agent against a wide range of pathogenic

microorganism (Xie, et.al, 2015). Phenolics are as well present in Betel. As stated by

Gyawali and Ibrahim, the hydroxyl (-OH) group in phenolic compounds may cause

bacterial inhibition. The number and position of binary bonds existing in phenolic

compounds play an important role in relation to antimicrobial effectiveness.

Saponins comprise a triterpenoid and a steroidal aglycone to which one or more

sugar cables are attached. These chemical structures determine their biological properties

as natural nonionic detergents which have cytotoxic, hemolytic, molluscicidal, anti-

inflammatory, antifungal, antiyeast, antibacterial, and antiviral activities (Hindawi, 2012).

Glycosides, according to the New World Encyclopedia, play numerous important

roles in living organisms. Many plants store important chemicals in the form of inactive

glycosides and if these chemicals are needed, the glycosides are brought in contact with

water and an enzyme, wherein the sugar part is broken off, making the chemical accessible

for use. Plant glycosides are often used as suppositories. In humans, even

animals, poisons are habitually assured to sugar molecules in order to eradicate them from

the physique. On the other hand, Volatile oils are mixture of hydrocarbon terpenes,

sesquiterpenes and polyterpenes and their oxygenated derivatives obtained from various

parts of the plant. Its oil is responsible for the essence or odour of the plant and they are

also known as essential oils (Kaur, n.d.).

 16

A. Diameter of Zone of Inhibition

Table 2. Mean Diameter of Zone of Inhibition

Test T0 T1 T2 T3 T4
Organisms

E. coli 8.17 8.1 10.42 13.15 13.34

S. aureus 8.6 18.84 23.92 24.36 25.12
Total 16.77 26.94 34.34 37.71 38.46
Mean 8.385 13.47 17.17 18.855 19.23

Table 1 presented the mean diameter of zone of inhibition of the five treatments

against E. coli and S. aureus. It showed that in T0, the treatment is inactive in E. coli and S.

aureus since it has only a diameter of 8.17 mm and 8.6 mm. On the other hand, T1 was

found inactive in E. coli with a diameter of 8.1 mm and active in S. aureus with a diameter

of 18.84. T2 also has a positive result and was proven partially active in E. coli with a

diameter of 10.42 and very active in S. aureus with a diameter of 23.92. For T3, it was

found that E. coli was partially active with a diameter of 13.15 and very active in S. aureus.

Lastly, T4 yielded the best result with a diameter of 13.34 in E. coli interpreted as partially

active and a diameter of 38.46 in S. aureus inferred as very active.

Based on the results of the phytochemical analysis, the presence of antibacterial

properties like Tannins and Phenolic Compounds, Flavonoids, Glycosides, Saponins, and

Volatile Oils are confirmed to be in the betel leaves. Hence, these components helped in

inhibiting the growth of microorganisms such as E. coli and S. aureus. Therefore, it can be

inferred that T4 had the greater inhibitory activity compared to other treatments since it is

a 100% pure concentration of betel leaves extract.

 17

B. Analysis of Variance of Zone of Inhibition

Table 3. Analysis of Variance in terms of Zone of Inhibition

ANOVA
Source of
SS Df MS F P-value F crit
Variation
Between Groups 163.98586 4 40.996465 0.729196 0.608973 11.39193
Within Groups 281.1075 5 56.2215

Total 445.0934 9

Using the statistical tool ANOVA (Analysis of Variance) where the level of

significance is 0.01, it was found out that the calculated Fstat (0.729) is lower than the Fcrit

(11.392). Therefore, there is no significant difference among the treatments in terms of

zone of inhibition.

Based on the result of the microbial analysis, all the treatments have a positive

result. Since T1, T2, T3 and T4 contain different concentrations of betel leaves extract which

is proven to have antibacterial properties on its phytochemical analysis, it can be

manifested that the results are accurate and precise.

 18

Chapter IV
Conclusions and Recommendations

Phytochemical analysis showed that the Betel leaves extract contains ample

amounts of Tannins and Phenolic compounds, Flavonoids, Glycosides, Saponins, and

Volatile oils.

Antimicrobial susceptibility analysis revealed that Piper betle possessed

antibacterial activity against Staphylococcus aureus and Escherichia coli. Based on the

findings, it resulted that Betel leaves extract can be used as a disinfectant spray. T4 (pure

Betel leaves extract) yielded the best result in terms of zone of inhibition. The result of the

ANOVA test indicates that there is no significant difference among the treatments, thus H0

is accepted.

It is highly recommended that further experimentation should be conducted

regarding the Piper betle’s contents. The antibacterial property of the disinfectant spray

should also be tested against different types of bacteria. The stem and other parts of the

Piper betle should also be subjected to experimentation in order to know if they have the

same component as that of its leaves. It is recommended that the result of this study should

be disseminated to the areas where Betel plant is abundant and presented to the households

and workplaces.
 19

Bibliography

Websites

Centers for Disease Control and Prevention. (2016, September 18). Infection Control.
Retrieved June 26, 2018, from
https://www.cdc.gov/infectioncontrol/guidelines/disinfection/disinfection-
methods/chemical.html
Datta, A., Ghoshdastidar, S., & Singh, M. (2011). Antimicrobial Property of Piper betel
Leaf against Clinical Isolates of Bacteria.
Kaur, M. (n.d.). Volatile Oil: Properties, Classification and Extraction. Retrieved October
12, 2018, from http://www.yourarticlelibrary.com/biology/volatile-oil/volatile-oil-
properties-classification-and-extraction/49811
Macé, S., Hansen, L. T., & Rupasinghe, H. V. (2017, June). Anti-Bacterial Activity of
Phenolic Compounds against Streptococcus pyogenes. Retrieved June 26, 2018,
from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5590061/
Mayo Clinic. (2018, September 07). Salmonella infection. Retrieved from
https://www.mayoclinic.org/diseases-conditions/salmonella/symptoms-
causes/syc-20355329
Microbiology Society. (n.d.). Retrieved June 24, 2018, from
https://microbiologyonline.org/about-microbiology/introducing-microbes/bacteria
Microchem Laboratory. (n.d.). Zone of Inhibition Test for Antimicrobial Activity.
Retrieved October 12, 2018, from https://microchemlab.com/test/zone-inhibition-
test-antimicrobial-activity
Minnesota Department of Health. (2010, February). Causes and Symptoms of
Staphylococcus aureus. Retrieved June 24, 2018, from
http://www.health.state.mn.us/divs/idepc/diseases/staph/basics.html
New World Encyclopedia. (2008, September 22). Glycoside. Retrieved October 12, 2018,
from http://www.newworldencyclopedia.org/entry/Glycoside
Nordqvist, C. (2017, December 11). E. coli infection: Symptoms, causes, and treatment.
Retrieved June 25, 2018, from
https://www.medicalnewstoday.com/articles/68511.php
NSF International, & Charles River Accugenix. (n.d.). Microbial Hotspots and Diversity
on Common Household Surfaces. Retrieved from
https://www.criver.com/sites/default/files/resources/MicrobialHotspotsandDiversi
tyonCommonHouseholdSurfaces.pdf
Savage, G. (2016). Saponin. Retrieved June 25, 2018, from
https://www.sciencedirect.com/topics/agricultural-and-biological-
sciences/saponin
 20

Shekhar. (2009, January 10). PHYTOCHEMISTRY: Tannins. Retrieved October 12, 2018,
from https://shekhar-alk.blogspot.com/2009/01/tannins.html
Todar, K. (2013). Streptococcus pyogenes. Retrieved June 24, 2018, from
https://microbewiki.kenyon.edu/index.php/Streptococcus_pyogenes
Weatherspoon, D. (2015, August 15). The 9 Dirtiest Spots in Your Home. Retrieved from
https://www.healthline.com/health/germy-places#how-they-spread
 21

Appendices

Appendix A

Appendix Table 1: Mean Diameter of Zone of Inhibition

Test T0 T1 T2 T3 T4
Organisms

E. coli 8.17 8.1 10.42 13.15 13.34

S. aureus 8.6 18.84 23.92 24.36 25.12

Total 16.77 26.94 34.34 37.71 38.46

Mean 8.385 13.47 17.17 18.855 19.23

Appendix Table 2: Analysis of Variance in Terms of Zone of Inhibition

SUMMARY
Groups Count Sum Average Variance
T0 2 16.77 8.385 0.09245
T1 2 26.94 13.47 57.6738
T2 2 34.34 17.17 91.125
T3 2 37.51 18.755 62.83205
T4 2 38.46 19.23 69.3842

ANOVA
Source of
SS Df MS F P-value F crit
Variation
Between Groups 163.98586 4 40.996465 0.729196 0.608973 11.39193
Within Groups 281.1075 5 56.2215

Total 445.0934 9

H0: There is no significant difference between the treatments in terms of zone of inhibition.
 22

H1: There is a significant difference between the treatments in terms of zone of inhibition.

dfbetween = k-1 dfwithin = N-k α = 0.01

dfbetween = 5-1 dfwithin = 10-5

dfbetween = 4 dfwithin = 5

Fcrit=11.25862

SSbetween=

(16.772 +26.942 +34.342 +37.512 +38.462 ) (154.02)2

SSbetween= -
2 10

SSTbetween= 163.98586

SSWithin=
(16.772 +26.942 +34.342 +37.512 +38.462 )
SSWithin = (8.172+ 8.12 + 10.422 + 13.152 +13.342…+25.122)- 2

SSWithin= 281.1075

SStotal = SSWithin + SSbetween = 281.1075+163.98586

= 445.0934

𝑆𝑆
MSBetween = 𝑑𝑓𝐵𝑒𝑡𝑤𝑒𝑒𝑛
𝐵𝑒𝑡𝑤𝑒𝑒𝑛

163.98586
= 4

MSBetween = 40.9964

𝑆𝑆
MSWithin = 𝑑𝑓𝑊𝑖𝑡ℎ𝑖𝑛
𝑊𝑖𝑡ℎ𝑖𝑛

281.1075
= 5
 23

MSWithin = 56.2215
𝑀𝑆𝐵𝑒𝑡𝑤𝑒𝑒𝑛
F= 𝑀𝑆𝑊𝑖𝑡ℎ𝑖𝑛

40.9964
F= 56.2215
F= 0.729196
Fstat<Fcrit
0.729196<11.25862
Therefore, accept Ho.
 24

Appendix B

Communication Letters

Division of La Union
REGIONAL SCIENCE HIGH SCHOOL FOR REGION I
Bangar, La Union

October 1, 2018

The Principal
Regional Science High School for Region I
Ma. Cristina East, Bangar, La Union

Thru:
SHERILYN G. DE CASTRO
Laboratory Custodian
SHS RSHS for Region 1

Sir:
Good day!
In partial fulfilment of the requirements in our subject Research IIA (Practical Research II), we are
pleased to inform you that the undersigned is currently conducting a research study entitled: “Betel
Leaves (Piper betle) Extract as Antibacterial Disinfectant Spray”
In this connection, we would like to request for your assistance and full cooperation by allowing
the researchers to conduct the experimentation of the above-mentioned research study in the SHS
laboratory. Furthermore, I would like to ask for your permission to allow us to utilize the laboratory
and borrow the following Laboratory Apparatuses on October 2, 2018:
1. 4 Graduated Cylinder for the measurement of the Betel Extract and the other liquid materials
2. Stirring Rod for the mixture of the Betel Extract and the other liquid materials
3. 7 150-ml Beakers as a container of the different treatments

Rest assured that the laboratory apparatuses will be returned and taken care of properly. Your
approval to conduct this study will be greatly appreciated. Thank you for your time.
 25

Republic of the Philippines

DEPARTMENT OF EDUCATION
Division of La Union
REGIONAL SCIENCE HIGH SCHOOL FOR REGION I
Bangar, La Union

Letter of Request for Phytochemical Analysis

September 20, 2018
Dean Marianne Menecio
College of Pharmacy
Lorma Colleges
San Fernando City, La Union

Madam,
Greetings!
The undersigned are Grade 12 learners from Regional Science High School for Region 1
in Ma. Cristina East, Bangar La Union. Presently, we are conducting an experiment on a
study entitled “Betel Leaves Extract as Antibacterial Disinfectant Spray” in partial
fulfilment of the requirements of our subject Research IIA, since quantitative research is
part of our curriculum. Thus we are requesting for the Phytochemical Analysis of:
Sample: Betel Leaves: 500g
Client’s Names: Deryll Jayne Maqui
Abigail D. Monis
Zion Joy A. Yadao
The report analysis shall be used as supporting documents of the aforementioned research
study. Thank you for your favourable response to our request.

Respectfully yours,
 26

Republic of the Philippines

DEPARTMENT OF EDUCATION
Division of La Union
REGIONAL SCIENCE HIGH SCHOOL FOR REGION I
Bangar, La Union

Letter of Request for Ethanol Extraction

September 20, 2018
Dean Marianne Menecio
College of Pharmacy
Lorma Colleges
San Fernando City, La Union

Madam,
Greetings!
The undersigned are Grade 12 learners from Regional Science High School for Region 1
in Ma. Cristina East, Bangar La Union. Presently, we are conducting an experiment on a
study entitled “Betel Leaves Extract as Antibacterial Disinfectant Spray” in partial
fulfilment of the requirements of our subject Research IIA, since quantitative research is
part of our curriculum. The above-mentioned research will include the formulation of a
spray using the extract of betel leaves. With this, we are requesting for an Ethanol
Extraction of the plant sample of betel leaves for the researchers to be able to formulate the
antibacterial spray. Thank you for your favourable response to our request.

Respectfully yours,

Appendix C
27
 28

Appendix C

Test Result
29
30
31
32
33
 34

Appendix D

Plagiarism Check
35
36
 37

Appendix E

Plates

Plate no. 1: Gathering the Leaves

 38

Plate no. 2: Laboratory Materials

 39

Plate no. 3: Betel Leaves Extract

 40

Plate no. 4: Labelling the Containers

 41

Plate no 5: Measuring the Amount of the Isopropyl Alcohol

 42

Plate no 6: Transferring the measured amount of Isopropyl Alcohol

 43

Plate no 7: Measuring the Amount of Betel Leaves Extract

 44

Plate no. 8: Measured Amount of Betel Leaves Extract for Treatment 1

 45

Plate no. 9: Pouring the Measured Amount

 46

Plate no. 10: Mixing the Treatments

 47

Plate no 11: The Treatments

 48

Plate no. 12: Transferring the treatments to the Container

 49

Plate no. 13: The treatments; Ready for Data Gathering

 50

Appendix F

DATA BOOK

Activity Date Planned Date Accomplished Description

Choosing a topic and The researchers

identifying a June 12, 2018 June 15, 2018 decided to venture a
problem study about betel
leaves extract as
antibacterial agent in
making disinfectant
spray.

Collecting The researchers

background June 15, 2018 June 18, 2018 browsed the internet
information and read books to
look for details for
the background of
the study.

Before pursuing the

study, the
Title Defense June 20, 2018 June 20, 2018 researchers need an
approval from the
Research adviser
through conducting
a title defense.

Making the write-up After collecting the

of Chapter I June 21, 2018 June 28, 2018 information needed,
the researchers
started finishing the
Chapter I of the
study.

The researchers
were able to come
Formulating the July 2, 2018 July 4, 2018 up a final research
Research Design design.
 51

Identifying the Before data

materials and the gathering, the
Data Gathering July 26, 2018 August 18, 2018 researchers
Tools and identified what are
Procedures. the materials, the
data gathering
procedures and tools
to be used.

Finalizing the August 23, 2018 August 26, 2018 The researchers did
completing the the Methodology to
write-up of Chapter be reflected on the
II data gathering.

Gathering of
Materials September 18, September 24,2018 The researchers
2018 gathered plant
samples and
procured the other
materials in making
the treatments

The researchers
went to LORMA
Data Gathering September October 17,2018 Colleges, San
24,2018 Fernando, La Union
to give the plant
sample for the
phytochemical
analysis. After that,
the researchers went
to DOST La Union,
for the antimicrobial
analysis of the
treatments

After data gathering,

Transcribing of Data October 22, 2018 October 27,2018 the researchers
recorded the data
that they’ve
collected.
 52

After transcribing
the data that have
been collected, the
researchers used
Computing of Data November 11, November 13, ANOVA to compute
2018 2018 for the significant
differences of the
treatments.

Analyzing and After computing the

Interpreting the November 24, December 10,2018 data, the researchers
Results 2018 interpreted and
discussed the results
.

Finalizing and The researchers

completing the January 15, 2019 January 23,2019 looked for related
write-up of literatures that will
Chapter III support the results of
the data.

The researchers put

Drawing all the results of the
Conclusions and February 3, 2019 February 7,2019 study and summed it
Recommendations all up and stated the
suggestions from
others in the
recommendations.

The researchers took

Making the all the sources in the
Bibliography February 8, 2019 February 8,2019 coming up of the
study and put it all
under the
Bibliography.

After making the

Making the February 8, 2019 February 8,2019 bibliography, the
Appendices researchers compiled
all the
communication
 53

letters, plates and

data book.

After the making of

the manuscript, the
Finalizing the
February 19, 2019 February 20,2019 researchers checked
Manuscript
everything up for
errors and mistakes.

Final Oral Defense February 26,2019 February 26,2019 After finalizing the
manuscript, the
researchers defended
the study to the
panelists.
 54

CURRICULUM VITAE

Personal Information:

Name: ABIGAIL D. MONIS

Age: 18 years old
Gender: Female
Civil Status: Single
Date of Birth: January 8, 2001
Place of Birth: Cauayan, Isabela
Current Address: Poblacion, Sudipen, La Union

Educational Background:

Secondary: Senior High School

Regional Science High School for Region I
Junior High School (2017)
Regional Science High School for Region I
Bangar, La Union

Primary: Saint Christopher Academy

Bangar, La Union (2013)
 55

Personal Information:

Name: DERYLL JAYNE N. MAQUI

Age: 18 years old
Gender: Female
Civil Status: Single
Date of Birth: September 11, 2000
Place of Birth: Reyna Regente, Bangar, La Union
Current Address: Reyna Regente, Bangar, La Union

Educational Background:

Secondary: Senior High School

Regional Science High School for Region I
Junior High School (2017)
Regional Science High School for Region I
Bangar, La Union

Primary: RUS Elementary School

Bangar, La Union (2013)
 56

Personal Information:

Name: ZION JOY A. YADAO

Age: 17 years old
Gender: Female
Civil Status: Single
Date of Birth: March 1, 2001
Place of Birth: San Fernando, La Union
Current Address: Castro, Sudipen, La Union

Educational Background:

Secondary: Senior High School

Regional Science High School for Region I
Junior High School (2017)
Regional Science High School for Region I
Bangar, La Union

Primary: Saint Christopher Academy

Bangar, La Union (2013)