BACTERIOSTATIC POTENTIAL OF BLACK MULBERRY AND WAX APPLE

CAVITE NATIONAL HIGH SCHOOL

Chief E. Martin St., Caridad, Cavite City

Bacteriostatic Potential of Black Mulberry (Morus nigra) leaves and Wax Apple
(Szyzygium samarangense) leaves extract against Salmonella Typhi

A Science Investigatory Project Research Plan

(Life Science Category)
presented to
the Faculty of Science Department
Cavite National High School

In partial fulfillment of the requirements

for the subject Research III of the
Science, Technology, and Engineering (STE)
Program

LUIS GABRIEL S. KALUGDAN

GRADE 9– ODL 29
2020
 BACTERIOSTATIC POTENTIAL OF BLACK MULBERRY AND WAX APPLE
CAVITE NATIONAL HIGH SCHOOL
Chief E. Martin St., Caridad, Cavite City

Bacteriostatic potential of Black mulberry (Morus nigra) leaves and Wax Apple
(Szyzygium samarangense) leaves extract against Salmonella Typhi

Luis Gabriel S. Kalugdan

Author

Jimmy M. Maquirang
Research Adviser

SCIENCE INVESTIGATORY PROJECT

RESEARCH PLAN

RATIONALE

Living life can be difficult, but it can be more difficult if one catches a disease.

The City of Cavite is a coastal area which makes Caviteños, especially the ones living

near the bay more prone to waterborne diseases. One example of these dangerous

diseases is typhoid fever. Typhoid fever can be caused by the bacteria called Salmonella

typhi which is commonly acquired in contaminated and stagnant waters (Mayo Clinic,

2018). Salmonella Typhi is a gram negative bacteria which stops the growth lympatic

tissues of the liver, spleen and small intestine of the infected person. It can spread into the

bloodstream and move to the small intestine to multiply (Ahirwar, et al., 2014)

Salmonella Typhi can be the cause of holes in the gut, internal bleeding, liver damage and

even death when left disregarded (Your Genome, 2015). Using antibiotics to treat cases

of typhoid fever can cause negative side effects. The most commonly prescribed

antibiotics in treating typhoid fever are Ciprofloxacin (Cipro), Azithromycin

(Zithromax), and Ceftriaxone. These antibiotics are known to have bad side effects.

Ciprofloxacin can cause nerve damage, tendon rupture, diarrhea, heartburn, stomach
 BACTERIOSTATIC POTENTIAL OF BLACK MULBERRY AND WAX APPLE
CAVITE NATIONAL HIGH SCHOOL
Chief E. Martin St., Caridad, Cavite City

pain, and even nerve damage (Medlineplus, 2020), Azithromycin can cause abdominal

pain, constipation, headache, and diarrhea. (Rxlist, 2020). Ceftriaxone can cause rash,

lump where the medicine was injected, sweating, blood clots, and diarrhea. (Rxlist,

2018). Antibiotic resistance can also be a problem in using antibiotics. Salmonella typhi

has already evolved to gain antibiotic resistant to Ciprofloxacin, ampicillin, and the ideal

drug of choice in the past, chloramphenicol. (Mayo Clinic, 2018).

According to the Department of Health in 2019, there were 16,888 cases of typhoid

fever in the Philippines with 30 deaths including the 1,160 cases in Region IV-A with 2

death. The most affected age group was between 4-10 years old with 51% of the cases

being male.

One of the known solutions in treating such diseases is using medicine which are

produced in a more natural way. Medicinal plants are the best source of natural medicine.

Around 66% to 79% of the population of developing countries use medicinal plants as

source of medicine (World Health Organization, 2011). Medicinal plants have plenty of

therapeutic properties, some plants can be used as a source of antimicrobial agents, and

can produce medicine without industrial processing (Palhares, et al., 2015). These plants

can be the source of obtaining new natural medicine which can be used to treat antibiotic

resistant bacteria.

The Moraceae family has a history on having plants that have high antimicrobial

activities. Examples of these plants are White mulberry (Morus alba) and Red mulberry

(Morus rubra).The aqueous and hydroethanolic extracts of White mulberry (Morus alba)

and Red mulberry (Morus rubra) has antimicrobial activities against Staphylococcus

aureus, Enterococcus feacalis, Staphylococcus epidermis, Escherichia coli, and

 BACTERIOSTATIC POTENTIAL OF BLACK MULBERRY AND WAX APPLE
CAVITE NATIONAL HIGH SCHOOL
Chief E. Martin St., Caridad, Cavite City

Salmonella Typhimurium bacteria (Thabti, et al., 2013). The ethanolic extract of the

White mulberry (Morus alba) stem has a natural chemical compound that has a high

antibacterial activity against Porphyromonas gingivalis and

Actinobacillus actinomycetemcomitans (Yiemwattana, Chaisomboon, & Jamdee, 2018).

The plant that will be used in the study is the Black mulberry (Morus nigra) which also

comes from the Moracceae family. It has therapeutic properties and can be used as a

natural medicine. It is also abundant in Cavite City. One example of its medicinal uses is

making molasses using the fruit to treat inflamed mouth sores during cancer treatment

(Demir Doğan, Can, & Meral, 2017). Its leaves also prevent kidney damage, liver tissue

damage and has antidiabetic properties (Hago, Mahrous, Moawad, Abdel-Wahab, &

Abdel-Sattar, 2019). The aqueous and ethyl extracts of Black mulberry (Morus nigra)

contain anti-diarrheal properties (Akhlaw, et al., 2016). Some studies prove that Black

mulberry (Morus alba) shows antibacterial activities against some bacteria. Black

mulberry (Morus nigra) is said to be rich in anthocyanins and flavonols which has a high

antibacterial activity against Escherichia coli, Pseudomonas aeruginosa and

Staphylococcus aureus (Chen, et al., 2017). The leaves of Black mulberry (Morus nigra)

are said to have a high antibacterial activity. Its aqueous extract has a high antibacterial

activity against E.coli and Salmonella Typhimurium (Thabti, et al., 2013). Its ethyl

acetate extract has an antibacterial activity against Streptococcus mutans, Escherichia

coli, Staphylococcus aureus, and Bacillus subtilis while its methanolic extract only

showed antibacterial activity against B. subtilis (Tahir, et al., 2017). Its ethanolic extract

has bactericidal activities against Bacillus cereus, Enterococcus faecalis, and E. coli.

(Souza, et al., 2018)

 BACTERIOSTATIC POTENTIAL OF BLACK MULBERRY AND WAX APPLE
CAVITE NATIONAL HIGH SCHOOL
Chief E. Martin St., Caridad, Cavite City

The Myrtaceae family that has plenty of plants that can be the source of new

natural medicine. Some lower classifications of Myrtaceae family has medicinal uses and

antibacterial properties. Examples of these plants are Guava (Psidium guajava) leaves

and Rose apple (Syzygium jambos). The extract of Guava (Psidium guajava) has high

antibacterial activity against gram positive bacteria, while having minimal antibacterial

activity against gram negative bacteria (Farhana, Hossain, & Mowlah, 2016). The

methanolic extract of Rose apple (Syzygium jambos) leaves has high antibacterial activity

against 2-gram positive bacteria which are Bacillus cereus and Staphylococcus aureus,

while showing minimal antibacterial activity against 2-gram negative bacteria which are

Aeromonas hydrophila and Alcaligenes faecalis (Mohanty, & Cock, 2010). The Wax

apple (Syzygium samarangense) is a lower classification of Myrtaceae family that can be

used as a source of natural medicine. It has plenty medicinal properties such as

antidiabetic, antioxidant, anti-inflammation, and anthinociceptive properties. Wax apple

(Syzygium samarangense) leaves contain a high percentage of terpenoids and flavonols

which gives it a high antibacterial activity against Klebsiella pneumonia Peseudomonas

aeruginosa and Cryptococcus neoformans (Khandaker, Sarwar, Mat, & Boyce, 2015)

With the use of Black mulberry (Morus nigra) and Wax apple (Syzygium

samarangense) leaves, the researcher aims to extract and determine the Bacteriostatic

Activity of the ethanolic extract against Salmonella typhi by measuring its zone of

inhibition with varying ratios.

RESEARCH QUESTIONS
 BACTERIOSTATIC POTENTIAL OF BLACK MULBERRY AND WAX APPLE
CAVITE NATIONAL HIGH SCHOOL
Chief E. Martin St., Caridad, Cavite City

This study aims to determine the bacteriostatic potential of Black mulberry

(Morus nigra) leaves and Wax Apple (Syzygium samarangense) against Salmonella

Typhi, it seeks to answer the following questions.

1. Does the pure extract of Black mulberry (Morus nigra) leaves have

bacteriostatic potential against Salmonella Typhi?

2. Does the pure extract of Wax Apple (Syzygium samarangense) leaves

have bacteriostatic potential against Salmonella Typhi?

3. Is it possible to obtain extract using dried leaves of Black mulberry

(Morus nigra)?

4. Is it possible to obtain extract using dried leaves of Wax Apple (Syzygium

samarangense)?

5. Which amongst the ratios exhibit the greatest bacteriostatic activity?

100:0? 0:100? 50:50? 75:25? 25:75?

HYPOTHESES AND ASSUMPTIONS

The aim of this study is to determine the bacteriostatic potential of Black

Mulberry (Morus nigra) leaves and Wax Apple (Szyzygium samarangense) leaves extract

against Salmonella Typhi.

Null Hypotheses

 Pure Black Mulberry (Morus nigra) leaves extract has no bacteriostatic

activity.

 Pure Wax Apple (Syzygium samarangense) leaves extract has no

bacteriostatic activity
 BACTERIOSTATIC POTENTIAL OF BLACK MULBERRY AND WAX APPLE
CAVITE NATIONAL HIGH SCHOOL
Chief E. Martin St., Caridad, Cavite City

 It is not possible to obtain extract using dried leaves of Black mulberry

(Morus nigra)

 It is not possible to obtain extract using dried leaves of Wax Apple

(Syzygium samarangense)

 All ratios of Black mulberry (Morus nigra) leaves and Wax Apple

Aternative Hypotheses

 Pure Wax Apple (Syzygium samarangense) leaves extract has

bacteriostatic activity

 Pure Black Mulberry (Morus nigra) leaves extract has bacteriostatic

activity.

 It is possible to obtain extract using dried leaves of Black mulberry

(Morus nigra)

 It is possible to obtain extract using dried leaves of Wax Apple (Syzygium

samarangense)

 All ratios of Black mulberry (Morus nigra) leaves and Wax Apple

(Syzygium samarangense) leaves extract has bacteriostatic activity

PROCEDURE

a. Project Design
BACTERIOSTATIC POTENTIAL OF BLACK MULBERRY AND WAX APPLE
CAVITE NATIONAL HIGH SCHOOL
Chief E. Martin St., Caridad, Cavite City

The aim of this study is to determine the bacteriostatic potential of Black

mulberry (Morus nigra) dried leaves and Wax Apple (Syzygium samarangense)

dried leaves extract against Salmonella Typhi. In order to test the Black mulberry

(Morus nigra) leaves and Wax Apple (Syzygium samarangense) leaves against

Salmonella Typhi, the researcher must perform the experiment based on the

procedure.

First, gather the needed materials and prepare it for ethanolic extraction

(Make sure that the Black mulberry leaves and Wax apple leaves are clean and

dried). Then, extract the dried Black mulberry (Morus nigra) and Wax Apple

(Syzygium samarangense) leaves by rotary evaporation. Next, test the

bacteriostatic activity of the solutions by disc diffusion method. After that, collect

the data. Finally, analyze the data.

b. Procedural Flowchart

Ethanolic extraction
Gathering of Preparation of of Black Mulberry
materials materials leaves and Wax
Apple leaves

Bacteriostatic
Data Analysis Data Collection testing using disc
diffusion method

Figure 1. The Flowchart of the Study

 BACTERIOSTATIC POTENTIAL OF BLACK MULBERRY AND WAX APPLE
CAVITE NATIONAL HIGH SCHOOL
Chief E. Martin St., Caridad, Cavite City

DATA ANALYSIS PROCEDURE

The extracts will be divided into 5 setups with different ratio. Setup A will be

pure Black mulberry (Morus nigra) leaves extract, setup B will be pure Wax Apple

(Syzygium samarangense) leaves extract, setup C will be 50% Black mulberry (Morus

nigra) leaves and 50% Wax Apple (Syzygium samarangense) leaves extract, setup D will

be 75% Black mulberry (Morus nigra) leaves and 25% Wax Apple (Syzygium

samarangense) leaves extract, setup E will be 25% Black mulberry (Morus nigra) leaves

and 75% Wax Apple (Syzygium samarangense) leaves extract. The diameter of inhibition

of zone will be recorded.

Percentage of Percentage of Diameter of Zone

Setups Black mulberry Wax Apple of Inhibition

(mm)

A 100 0

B 0 100

C 50 50

D 75 25

E 25 75

RISK ASSESSMENT

The anticipated risk of this study is improper use of lab apparatus. Improper

handling of lab apparatus may cause fire, cuts, spills of dangerous solutions and

electrocution. To avoid this risk, wear protective equipments, report all actions to

instructor and be very cautious.

 BACTERIOSTATIC POTENTIAL OF BLACK MULBERRY AND WAX APPLE
CAVITE NATIONAL HIGH SCHOOL
Chief E. Martin St., Caridad, Cavite City

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Chief E. Martin St., Caridad, Cavite City

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Chief E. Martin St., Caridad, Cavite City

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Chief E. Martin St., Caridad, Cavite City

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