COMPARATIVE ANALYSIS OF CRUDE AND AQUEOUS EXTRACT OF PIPER

BETEL L. AS AN ANTIBACTERIAL AGENT

GONZALES, DEVONE CLAIRE B., BIEN, MARIEL AGNES B., MARTINEZ, JAIME JEANNE P.,
MONILLA, JEWLLIAN VERA KENDRA N., BALBIN, JOHANNE RICHEL C., ESPEDIDO, ALFRED
P., DELA ROSA, ANNE KRIZZEA M.

University of Santo Tomas-Legazpi, Rawis, Legazpi City, Philippines, 4500

ABSTRACT
Bacteria are becoming resistant to antibacterial drug due to the excessive use of
antibiotics. Thus, resistance to antibiotics becomes more common and a greater need for
alternative antibacterial drug arises. Piper betel L. are known to have numerous curative and
healing health benefits such as treating diabetes, shedding weight, prevents carcinogens that
lead to cancer, cures headache and heals wounds. Therefore, the researchers investigated the
antibacterial effect of Piper betel leaves on one (1) gram negative bacterial strain, e. coli, and
one (1) gram positive bacterial strain, s. epidermidis. The study mainly aims to compare
aqueous and crude extract effectivity on gram positive and negative bacteria. The researchers
hypothesizes that there will be no significant difference. Piper betel leaves extract was tested
using Pour Plate Method and Kirby- Bauer Disk Diffusion Susceptibility test towards
Escherichia coli and Staphylococcus epidermidis. Each experiment was carried out in three
setups. Zero colonies of Escherichia coli and staphylococcus epidermidis on average were
observed in crude extract, aqueous extract and ceftriaxone. The crude extract demonstrated an
average of 6.5 mm zone of inhibition against Escherichia coli and an average of 5.7 mm zone
of inhibition against Staphylococcus epidermidis. The aqueous extract demonstrated an
average of 6.3 mm against both Escherichia coli and Staphylococcus epidermidis. Moreover,
based on the result of the experiment that the outcome of the aqueous extract from the piper
betel leaves is said to be more effective than the crude extract.
KEYWORDS: Piper betel, Antibacterial, gram positive, gram negative, aqueous extract,
INTRODUCTION cancer, cures headache and heals
wounds (India Today 2016).
Substantial number of plants
are widely known and considered In China, Bangladesh and
as medicinal and has profound India, the roots and leaves of Piper
components that are necessary in Betel are used to expel gas from
treating many illnesses. The Piper the stomach or intestines to relieve
betel L. or Buyo are glossy oblong distension, applied to relieve
to heart-shaped leaves belonging to headache, chewed as Betel quid for
Piperaceae family, it is mostly alleviating mouth sores, the leaf
cultivated in South and Southeast can also aid digestive problems and
Asian countries. Piper betel L. are treat cuts and wounds. In some
known to have numerous curative countries it is applied in medicine
and healing health benefits such as and boiled as it can lower blood
treating diabetes, shedding weight, pressure.
prevents carcinogens that lead to
According to the
researchers of Medicine in Manila
Central University, extract of Piper
betel L. mitigate symptoms of
those suffering from gastric
ailments. Chauhan E.K.,
Aishwarya J., Singh A. & Tiwari
A. (2016) also stated in their
review study that leaves of Piper
betel (Buyo) contain several
nutraceutical properties,
phytochemicals and antioxidants.
Piper betel L. also have anti
carcinogenic properties, proving
that even without the presence of
the Betel quid ingredients, itself
can be a very beneficial medicinal
plant.
Escherichia coli a gram-
negative rod-shaped bacteria of
genus Escherichia are found in
lower intestines of both people and
animals. Most type of strains are
harmless but strains such as E. coli
O157:H7, can cause serious
abdominal cramps, diarrhea and
vomiting. This strain of E. coli
produces a harmful toxin capable
of damaging the lining of the small
intestine causing bloody diarrhea.
Contaminated water or food and
human contact are potential
sources of exposure to E. coli, most
commonly in raw vegetables,
undercooked meat and fresh
products.
Staphylococcus epidermidis
a gram-positive bacterium
belonging to the family
Staphylococcaceae. This bacterium
resides in any number of the
human tissues and biofluids,
typically in the human skin,
mammary glands, placenta and
other gastro intestinal tracts. One
of the leading pathogens of
commonly acquired infections,
in different ways, providing a wider
susceptible to infections are drug
users, especially those who use
artificial appliances. These
organisms produce glycocalyx,
which plays a role in regulation of
endothelial vascular tissue,
including the modulation of red
blood cells volume in capillaries,
therefore S. epidermidis as a
biofilm bacteria has enhanced anti-
biotic resistance. S. epidermidis has
now became the main agent
causing infections in medical
devices due to its abundance to the
skin.
The study of Balaji Kaveti,
Lisa Tan, Sarnnia, Tan Sin Kuan,
and Mirza Baig (2011) entitled
“Antibacterial Activity of Piper
Betel Leaves”, they evaluated for
an antibacterial activity against
three Gram positive, two Gram
negative bacteria using Aqueous
and ethanol extract of the leaves.
The study will focus on the
reducing of E. coli and S.
epidermidis cells by analyzing the
effects produced and the results of
the study. The study mainly aims to
compare aqueous and crude extract
effectivity on gram positive and
negative bacteria. Specifically, the
researchers aims to determine the
(1) Length of zone of inhibition of
E. coli and S. epidermidis; and (2)
growth in the number of colonies
of E. coli and S. epidermidis (3) to
determine which form of extract is
more effective on both types of
bacteria. The researchers
hypothesizes that there will be no
significant difference, thus, this
study proposes a null hypothesis.
The study may benefit the society
knowledge about Piper betel L. aqueous
and crude extract in combating E. coli and
S. epidermdis. To the victims of food
poison, diarrhea, UTI, the study may
propose and suggest knowledge about
combatting these diseases. To the future
researchers who wish to fill the gap of the
study and to serve as a reference to their
study. To the medical field, it will propose
information about the methods and results
of the Piper betel L. on E. coli and S.
epidermidis.
METHODOLOGY
This study is a quantitative
research that uses a two- way
ANOVA approach, and a quasi-
experimental research. In this way,
the researchers can implement and
obtain the intended results of the
research. In conducting the study,
the researchers compares the mean
differences between the crude and
aqueous extract of piper betel leaf
against pathogenic bacteria such as
the Escherichia coli and
Staphylococcus epidermidis.
Materials
A bacterial sample of
Escherichia coli and
Staphylococcus epidermidis were
used as the source of bacterial
colonies that will be used in the
experiment. Betel leaves are the
source of the extract that is to be
tested in the study. Ceftriaxone
30ug Antibiotic Discs are the
antibiotics that will be used to
demonstrate the zone of inhibition
of E. coli bacteria against the
antibiotic and will serve as the
positive control of the study.
Nutrient agar will be the medium
used to support growth of bacteria.
Mueller Hinton Agar (MHA) will
be used as the standard medium for
the Bauer Kirby method for
antimicrobial susceptibility testing.
Mortar and pestle will be
used to crush the betel leaves. 
Analytical balance is a device
designed to measure small mass in
the sub-milligram range. Autoclave
is a device used to sterilize solids,
liquids, hollows, and instruments to
kill bacteria, spores and germs
resistant to boiling water and
powerful detergents.
Sterile water or saline
solution will be used for the
dipping of the cotton swab in the
streaking of the petri dish.
Beakers, Test tubes, and
Flasks will be used as the
containers of substances. Petri
dishes will be used for
demonstrating the effects of certain
substances against bacterial
colonies.
Pipettes will be used to
transfer a measurable amount of
liquid from one container to
another. 10 CC and 1 CC syringe
are needed to inject fluid into, or
withdraw fluid from, the body of
certain substances.
Incubator is a device used
to grow and maintain
microbiological cultures or cell
cultures.
A Colony counter will be
used to estimate density of
microorganisms by counting
individual colonies of E. coli on an
agar plate or Petri dish.
Sterilization of lab equipment
Materials were gathered for
sterilization to avoid any
contamination in the experiment.
Each were wrapped with paper and
 placed in Ziplocs to avoid melting
and/or deformation of any lab
equipment. The wrapped materials
were then placed in the metal box
of the autoclave. A significant
amount of distilled water was
poured into the autoclave as the
source of steam that will be used in
the sterilization process of
autoclave. The metal box was then
placed inside the autoclave with its
slides open. The autoclave was
then closed and locked. Screw
down the upper reservoir cover of
the autoclave and switch the timer
to fifteen minutes.
Preparation of Agar
The preparation of 28 grams of
McConkey Agar for cultivation of
E.coli, 19 grams of Müller-Hinton
Agar, 28 grams of Nutrient Agar
follows the same procedure:
Suspend 28 grams of Müller-
Hinton Agar in 1000 milliliters distilled
water; suspend 19 grams for the
Nutrient Agar in milliliters of distilled
water. Heat to boiling to completely
dissolve the medium. Autoclave at 121
˚C for 15 minutes for sterilization and
cool to room temperature. Pour the
cooled Agar into sterile petri dishes and
make sure to pour on a uniform depth;
for NA & MHA plates a 10 ml
measurement of Agar in each plates is
maintained.
For Mannitol Salt Agar in S.
epidermidis, According to
MicrobeOnline (2013), the
preparation of the Mannitol Salt
Agar will follow the subsequent
procedure:
Sterilize the agar by autoclaving at
121 ˚C for 15 minutes. (2) When the
agar has cooled down around 50-55˚C,
mix it well and distribute it to sterile
petri dishes. (3) Store the petri dishes in
plastic bags at 2-8˚C to avoid loss of
moisture.
Cultivation of the S. epidermidis &
E.coli
A cotton swab was used in
getting a small amount of bacterial
strain from the incubated plate and
in mixing the strain with sterile
water in a test tube. The dipped
cotton swab was then used in
streaking the strain into three (3)
petri dishes that contained Mueller-
Hinton Agar and another three (3)
petri dishes for Mannitol Salt Agar.
The agar plates were incubated for
24 hours.
Crude Extraction of Juice in Betel
leaves
By using the mortar and
pestle, the piper betel leaves were
crushed to draw out their juice. The
crushed leaves were then gathered
and squeezed in the cheesecloth.
The extracted juice is poured into
the glass jar covered with foil and
plastic. The contained extract was
then placed in the refrigerator for
storage.
Aqueous Extraction of juice in Betel
leaves
150 grams of piper betel
leaves were broken down into
smaller pieces using a blender with
300 grams of distilled water. The
extract were poured into the 1 liter
beaker and was heated on a hot
plate until its temperature has risen
to 100 degrees. The extract was
allowed to cool down and was then
filtered by using a strainer and a
filter paper.
Adding of S. epidermidis to MHA Agar
plates
A small amount of bacteria
strain from the incubated MHA
plate was mixed with distilled
water in a test tube. A sterile swab
was used in streaking the strain
into petri dishes that contained the
MHA. Bacterial strain is incubated
for 24 hours.
Pour Plate Method
Experimental Group (Piper Betel L.
Crude & Aqueous Extract)
Acquire 60 ml of heated NA and
combine with 18 grams of Piper Betel
Extract (Crude) and pour into 6 petri
dishes, amounting of 10 ml each, using
10 cc syringe for equal measurement.
Repeat and use Aqueous Extract. Each
experimental group requires 9 petri
dishes containing mixture of NA and
Piper Betel L. Extract.
Controlled Group (Tap Water &
Ceftriaxone)
For testing the Ceftriaxone, 9 mg
of Ceftriaxone is suspended in 18 ml of
sterile water and combined with 60 ml of
NA, heat the mixture to even the
concentration of substance. After
heating, transfer the substance into 6
petri dishes by using a 10 cc syringe, 10
ml of mixture for each plates. Allow the
heated substance to cool down for each
petri plates.
For combining Tap Water with
NA, 20 ml of Tap water is added into
60ml of NA. Heat the mixture again to
even the concentration, after heating
pour it into 6 petri dishes evenly with 10
ml syringe and let it solidify.
Kirby- Bauer Susceptibility Test
Heated MHA Agar is poured into 8 petri
dishes with 10 ml of MHA Agar each.
Each petri dish will have a set of three
disks of filter papers. One set of filtered
paper will be dipped with crude extract.
Another set will be dipped with aqueous
extract. And the other set will be dipped
with tap water. The last agar plate will be
placed with three ceftriaxone discs for the
demonstration of their antibacterial effect
against S. epidermidis and E.coli
Incubation of Contaminated Medium
The petri dishes will be left
incubated overnight for bacteria to
grow in a controlled environment.
The results will demonstrate the
effects of each substance against E.
coli bacteria.
Observation of the Three (3) Controlled
Setups
The experimental and
control groups had three (3) petri
dishes each which contained ten
(10) milliliters of Nutrient Agar
mixed with Piper betel leaf
extracts, ten (10) milliliters of
nutrient agar mixed with
ceftriaxone powder and ten (10)
milliliters of plain nutrient agar.
Analyzing the Result
Samples will then be
observed and analyzed by using the
colony counter to determine the
growth of the number of colonies
of bacteria developed and calipers
or ruler to measure the zone of
inhibition of E. coli. The data
collected will vary on the
observation by categories: (a)
Length of zone of inhibition of E.
coli; and (b) growth in the number
of colonies of E. coli
Sample
 The researcher will be difference in terms of the
having three trials for every antibacterial activity of the two
experimental group set-up and one kinds of extract. As indicated in the
controlled group. A table, 0 colonies of Escherichia
Staphylococcus epidermidis coli and Staphylococcus
bacteria sample and an Escherichia epidermidis on average were
coli bacteria sample were used in observed in crude extract, aqueous
the study as the pathogenic bacteria extract and ceftriaxone. In tap
that will be tested upon. water, which serves as the negative
control and ceftriaxone as the
Statistical and Data analysis
positive control, there is an average
Each experiment was carried out in of 98.7 colonies of Escherichia
three setups. Thus, it was computed using coli present and an average of
mean. The clear zone around the discs 2837.7 colonies of Staphylococcus
were measured in mm. The analysis will epidermidis.
be two-way ANOVA wherein it is being
The crude extract
utilized in Microsoft Excel. Tap water and
demonstrated an average of 6.5
piper betel leaves extract will be the
mm zone of inhibition against
independent variable that will react with
Escherichia coli and an average of
the Escherichia coli and Staphylococcus
5.7 mm zone of inhibition against
epidermidis which will be the dependent
Staphylococcus epidermidis. The
variable.
RESULTS AND DISCUSSION NUMBER OF
COLONIES (cfu)
Table 1 shows the effects of piper T1 T2 T3
betel leaves extract towards Escherichia CRUDE
coli and Staphylococcus epidermidis using 0 cfu 0 cfu 0 cfu
EXTRACT
Pour Plate Method and Kirby- Bauer Disk AQUEOUS
0 cfu 0 cfu 0 cfu
Diffusion Susceptibility test. The number EXTRACT
of colonies and zone of inhibition of 77 87
TAP WATER 132 cfu
certain bacterial cultures were emphasized cfu cfu
as the basis of determining the significant CEFTRIAXONE 0 cfu 0 cfu 0 cfu
aqueous
Table 1. Antibacterial activity of leaf extract on E. coli and S.
epidermis Number of Colonies (cfu) Zone of inhibition (mm)

Escherichia Staphylococcus
Escherichia coli Staphylococcus epidermis
coli epidermis
Crude 0 0 0
0 cfu 0 cfu 0 cfu 5.0mm 7.0mm 7.5mm 5.0mm 6.0mm 6.0mm
extract cfu cfu cfu
Aqueous 0 0 0
0 cfu 0 cfu 0 cfu 4.9mm 6.0mm 7.9mm 4.0mm 7.0mm 8.0mm
extract cfu cfu cfu
13
77 87 1656 2987 3870
Tap water 2 0mm 0mm 0mm 0mm 0mm 0mm
cfu cfu cfu cfu cfu
cfu
0 0 0
Ceftriaxone 0 cfu 0 cfu 0 cfu 12mm 13mm 12.5mm 22mm 21mm 22mm
cfu cfu cfu
 extract demonstrated an average of Three trials were conducted
6.3 mm against both Escherichia for crude and aqueous extract. This
coli and Staphylococcus figure shows crude and aqueous
epidermidis. In tap water, 0 mm of extract from betel leaf displaying
zone of inhibition were recorded antibacterial activity by no sign of
for both Escherichia coli and E.coli growing on (a) Crude extract
Staphylococcus epidermidis as it is with E. coli 1 (b) Crude extract
proven that it has no effect on the with E. coli 2 (c) Crude extract
two pathogenic bacteria, with E. coli 3 (d) Aqueous extract
with E. coli 1 (e) Aqueous extract
with E. coli and (f) Aqueous
extract with E. coli 3.
Table 3. Length of zone of inhibition in
e. coli
ZONE OF INHIBITION
(a) (b)
Escherichia T1 T2 T3
coli and CRUDE
5.0mm 7.0mm 7.5mm
EXTRACT
AQUEOUS
4.9mm 6.0mm 7.9mm
EXTRACT
TAP WATER 0mm 0mm 0mm
CEFTRIAXONE 12mm 13mm 12.5mm
Staphylococcus epidermidis.
Three trials for each experimental
Table 2. Number of colonies in e. coli group- crude extract, aqueous extract and
ceftriaxone, were tested. The crude extract
attained an average of 6.5 mm zone of
As indicated in table 2, the inhibition. The aqueous extract attained an
crude extract, aqueous extract, and average of 6.3 mm zone of inhibition. The
ceftriaxone attained an average of 0 ceftriaxone antibiotic attained an average
(f)
bacterial colonies of Escherichia of 12.5 mm zone of inhibition. The tap
coli which proves that the three water had no effect on the bacteria
substances are effective against the resulting to an average of 0 mm zone of
bacteria. The tap water attained an inhibition against Escherichia coli.
average of 98.7 bacterial colonies
of Escherichia coli. This shows that Table 4. . Number of colonies in s.
tap water has no significant effect epidermidis
against Escherichia coli, which NUMBER OF
serves as the negative control of COLONIES (cfu)
the set ups. T1 T2 T3
CRUDE
0 cfu 0 cfu 0 cfu
EXTRACT
AQUEOUS
0 cfu 0 cfu 0 cfu
EXTRACT
1656 2987 3870
TAP WATER
(b) cfu cfu cfu
CEFTRIAXONE 0 cfu 0 cfu 0 cfu
 The table shows similar results
with table 1. The crude extract, aqueous
extract, and ceftriaxone attained an
average of 0 bacterial colonies of
staphylococcus epidermidis. The tap water
attained an average of 2837.7
staphylococcus epidermidis colonies,
having no significant effect against the
bacteria.
aqueous extract attained an average
of 6.3 mm zone of inhibition. The
ceftriaxone attained the greatest
zone of inhibition having an
average of 21.7 mm zone of
inhibition. The tap water, having
no significant effect on the
bacteria, attained an average of 0
mm zone of inhibition.

Table 5. Length of zone of inhibition in

s. epidermidis
LENGTH OF ZONE OF
INHIBITION
T1 T2 T3
CRUDE 5.0m 6.0m 6.0m
EXTRACT m m m
AQUEOUS 4.0m 7.0m 8.0m
EXTRACT m m m
TAP WATER 0mm 0mm 0mm
CEFTRIAXONE 22mm 21mm 22mm

(a) (b)

(c) (d)

The crude extract attained an

average of 5.7 mm zone of inhibition
against staphylococcus epidermidis. The (f)

(e)
 Three trials were conducted The researcher used two
for aqueous and crude extract. This way ANOVA to determine if there
figure shows aqueous and crude is a difference in Kirby-Bauer Disk
extract from betel leaf displaying Diffusion Susceptibility Test and
antibacterial activity by no sign of Pour Plate Method between the
S. epidermidis growing on (a) variables aqueous extract, crude
Aqueous extract with s. extract, tap water and ceftriaxone.
epidermidis 1 (b) Aqueous extract
H0: There is no significant difference
with s. epidermidis 2 (c) Aqueous
among the experimental group on each
extract with s. epidermidis 3 (d)
tests.
Crude extract with s. epidermidis 1
(e) Crude extract with s. Alpha level used is 0.5.
epidermidis 2 (f) Crude extract
with s. epidermidis 3. Based on the results of
ANOVA, the researcher
determined that the F values are
Mueller Hinton Agar greater than the F critical value,
combined with both (a) crude and and P-value > 0.5 α
(b) aqueous extract showing To reject the null
antibacterial activity by the clear hypothesis: (a) If the F-value is
zones around the discs against E. larger than the f critical value, and,
coli. (b) If the p-value is smaller than
your chosen alpha level.
In conclusion, 260.4935;
22.75314; 28.84414 < 3.238872;
4.493998; 3.238872(b)
and the p-value:
8.69x10 ; 0.000209; 1.09x10-06 > 0.5 α.
-14

Therefore, the null hypothesis is rejected and

there is a significant difference in Kirby Bauer
Disk Diffusion Test.

Based on the ANOVA for

the Pour Plate method, the
(a)Mueller Hinton Agar researcher determined (b) that the F
combined with both (a) crude and values are greater than the F
(b) aqueous extract showing critical value, and P-value < 0.5 α.
antibacterial activity by the clear F < F crit
zones around the discs against S. = 20.80891; 18.10601;
epidermis. 18.10601 > 3.23887; 4.493998;
3.238872
--------------------MARIEL insert P-value < 0.5 α
here--------- Therefore, there is a
significant difference in Pour Plate
-----comparison rrl and study
Method
result-----------
CONCLUSION
Based on the experiment,
the result of the zone of inhibition
 of Escherichia coli and epidermis to test the most effective
Staphylococcus epidermis on piper antibacterial concentration.
betel leaves extract on both
The researcher would also
aqueous and crude are said to be
recommend to use distilled water for the
effective. Therefore, the aqueous
negative control of the experiment. Tap
and crude extract of piper betel leaf
water may contain contaminants. They
is an effective antibacterial agent
would also recommend to use a low level
on both gram positive and gram
antibiotics for the positive control of the
negative bacterial strain.
experiment.
Moreover, based on the result of
In addition, the researcher would
the experiment it is known that the
highly recommend to undergo
outcome of the aqueous extract from the
phytochemical test, to know the
piper betel leaves is said to be more
components of piper betel leaves in terms
effective than the crude extract. The results
of it being an antibacterial agent.
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 APPENDIX A
STATISTICAL ANALYSIS
Table 8. ANOVA: Two factor with replication of Kirby Bauer Disk Diffusion Test
Table 9. ANOVA: Two factor with replication of Pour Plate Method

APPENDIX B

METHODOLOGY

Figure 1 - 2. Sterilization of equipment.

The researchers sterilized the equipment needed using the autoclave before
performing an experiment. Sterilizing the equipment helps avoid contamination.
 Figure 3-4. Preparation of agar
Figure 3 shows the agar being boiled and stirred continuously to avoid the agar from
being burnt.

Figure 5-6. Cultivation of S. epidermidis and E.coli

Figure 5 and 6 shows the researcher taking bacterial strain from an incubated petri
plate using a cotton swab and mixing it with sterile water.

Figure 7-8. Incubation of Agar Plates streaked with Bacteria

 Figure 7 and 8 shows the researcher placing the petri plates streaked with bacteria in
the incubator and incubation takes place for 24 hours.

Figure 9. Crude extraction of Juice in Betel leaves

Figure 9 shows that the researchers use mortar, pestle and cheesecloth in order to get
the extracted juice from the leaves.

Figure 10-11. Aqueous extraction of Juice in Betel leaves

Figure 10 and 11 shows the crushed betel leaves with distilled water being boiled
while being stirred and poured out in the Erlenmeyer flask using a funnel and cheesecloth.