REPUBLIC OF THE PHILIPPINES

DEPARTMENT OF EDUCATION

REGION V (BICOL)

SCHOOL DIVISION OFFICE

TABACO NATIONAL HIGH SCHOOL

TABACO CITY

COMPARATIVE STUDY OF ANTIBIOTIC EFFECT OF GINGER

(Zingiber Officinale), GARLIC (Allium Sativium), AND

MALUNGGAY (Moringa Oleifera), EXTRACT COCKTAIL VERSUS

INDIVIDUALLY PREPARED EXTRACTS

ANGELO, DESIREE NELLY D.

CANTES, JOSE IGNATIUS C.

HONRA, LEO MATTHEW C.

LIM, EMERALD JADE

NABOR, GRACIELLA ALLYSA JEANEN

RESEARCHERS

MRS. MAY ANN GAMEZ-GUAB

RESEARCH ADVISER
ACKNOWLEDGEMENT

First and foremost, we want to thank God for his grace and blessings, without which this

research would not have been possible.

We would like to convey our heartfelt gratitude to Mrs. May Ann Gamez-Guab, our

research adviser, for allowing us to conduct our research and providing vital guidance throughout

the process. Her sincerity and motivation have left a lasting impression on us. Working and

studying under her supervision was a great honor and privilege. We are appreciative for

everything she has done for us.

We would also like to express our gratitude to our friends Thea Marie Buama, John Paul

Belardo, and Stephanie Don for their help and support.

I would like to thank the woman who served as an inspiration to me and continuously

motivated me when I had no drive to continue my educational endeavors.

Last but not least, we want to express our gratitude to our parents for their sacrifices and

unwavering support during our journey.

ABSTRACT

Escherichia coli (E. coli) is known to be the cause of some epidemic diseases such as urinary tract

infection (UTI), diarrhea, and other clinical infections. Using commercial antibacterial drugs can

inhibit E. coli’s spread, but they may have side effects once consumed. Garlic (Zingiber

officinale), Malunggay (Moringa oleifera), and Garlic (Allium sativum) are plants found in

several Asian countries which are known to have various uses in different fields including

medicine. With this, the researchers came up with a study focuses on investigating the

antibacterial properties present in Ginger, Malunggay, and Garlic. The researchers also aim to

develop an effective and cost-efficient antibacterial from these sources. The researchers will test

the antibacterial properties of Ginger, Malunggay, and Garlic by using the disk diffusion test with

Escherichia coli (E. coli) as the test bacteria. This was the chosen method by the researchers as it

was the method used by Khan and his co-researchers in their study to test the antimicrobial

activity of soybean oil extract of dried ginger powder. A total of three (3) trials of antimicrobial

sensitivity test shall be done to the garlic extract, ginger extract, malunggay extract, and antibiotic

cocktail---the combination of the previously mentioned three extracts---to determine their

antibacterial activity by measuring their zones of inhibition (mm).

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TABLE OF CONTENTS

Abstract 1

Introduction 3

Material and Methods 7

References 10

Definition of Terms 12

Appendix 13

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1.0 INTRODUCTION

Escherichia coli (E. coli) is a type of bacteria that is abundant almost everywhere and its presence

in water has been a long-time problem locally and globally. Many individuals have contracted

diseases due to this pathogen, with urinary tract infection (UTI) being the closely linked disease

towards E. coli. Researchers have continuously conducted research studies to develop a solution

for the diseases that E. coli inflicts towards different people, but because of the complexity of the

pathogen and its numerous sub-pathotypes, the development of an efficient antibacterial for E.

coli becomes harder and harder. In addition to that, commercial antibacterial drugs may cause

negative side effects towards the ones who took it in. Therefore, the researchers aim to develop

and create a natural antibacterial which can not only inhibit and put a stop to the diseases which

E. coli may bring to different individuals but is also easily accessible to them while at the same

time, not having adverse effects towards them.

According to Madappa et al. (2019), E. coli has been the primary cause of some epidemic

diseases such as cholecystitis, bacteremia, cholangitis, urinary tract infection (UTI), diarrhea, and

other clinical infections such as neonatal meningitis and pneumonia. The said pathogen also

caused death and fatality to the people who are infested with it. In the study conducted by Luby et

al. (2015), 59% of the drinking water sample in Bangladesh that they measured was contaminated

with E. coli, which caused the increased prevalence of diarrhea among children under the age of 5

years. Although E. coli is said to be common and most studied, this is still known to be a complex

human pathogen. With it contributing to an estimated 40 000 annual deaths in the USA, and the

existence of various pathotypes, the development of medicine against E. coli is increasingly

challenging (Vila, July 2016). With these being said, it is of paramount importance to find an

immediate source of antibacterial to put a halt on these problems and to ensure that there will

be no further

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threat on people’s health and emphasizing the knowledge of major pathogen sources and their

significant impacts on our resources is a must.

Microbial inhibition is the best possible way to prevent the growth and spread of E. coli.

This technique is widely used in the field of medicine and biotechnology. Microbial inhibition

can be also used once an organism is already infested with pathogenic bacteria. The use of

antibacterial drugs helps inhibit the growth of pathogenic microorganisms, specifically E. coli.

However, using commercial antibacterial drugs causes some negative effects to the ones who

consume it. According to the University of Illinois-Chicago, Drug Information Group (2019),

nausea, vomiting, cramps, allergies, and diarrhea are some of the negative side effects of

commercial antibiotics. In September 2018, the Center for Disease and Control Prevention

reported 2 million infections and 23,000 deaths in the US alone which is primarily caused by the

resistance of bacteria to commercial antibiotics. The information act as evidence for worldwide

concern that Escherichia coli is broadly recognized as an important and threatening pathogen and

commercial antibiotics are becoming less safe due to its side effects. Thus, the findings prove the

necessity to look for alternative microbial inhibition techniques which offer lesser drawbacks and

more benefits.

Ginger (Zingiber officinale) is a flowering plant which is native to Southeast Asia. It is

widely used as folk medicine and spice. Garlic (Allium sativum) is effective against several

gram-negative, gram-positive, and acid-fast bacteria, including Staphylococcus, Salmonella,

Vibrio, Mycobacteria, and Proteus species. It is well known in Iran and various parts of this plant

have long been used in traditional folk medicines of Iran and some other cultures. In the study of

Liu, Qing et al (2016), they have tested the antibacterial and antifungal activities of different

spices, including ginger and garlic. Ginger showed antibacterial activities against three

anaerobic Gram-negative bacteria namely

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Porphyromonas gingivalis , Porphyromonas endodontalis, and Prevotella intermedia ,

while the garlic showed the highest antimicrobial activities in its dried powder form. Malunggay

(Moringa oleifera) belonging to the family of Moringaceae is an effective remedy for

malnutrition. Malunggay is rich in nutrition owing to the presence of a variety of essential

phytochemicals present in its leaves, pods, and seeds. In fact, malunggay is said to provide 7

times more vitamin C than oranges, 10 times more vitamin A than carrots, 17 times more calcium

than milk, 9 times more protein than yogurt, 15 times more potassium than bananas and 25 times

more iron than spinach (Kumar et. al, 2016). Using these natural sources of antibiotics may help

in inhibiting the growth of E. coli because of their various antibacterial and antimicrobial

properties.

With the given information, the researchers aim to investigate and test the antibacterial

properties of Ginger (Zingiber officinale), Malunggay (Moringa oleifera), and Garlic (Allium

sativum). The present study concentrates on determining the capability of the Ginger Malunggay,

and Garlic as alternative natural antibiotics. The study also aims to test if the antibiotic cocktail,

wherein the previously mentioned natural extracts are combined altogether, can become a more

effective antibacterial when compared to the individually prepared extracts.

The hypotheses of the researchers are the following:

1.) The antibiotic cocktail has a significant effect on the inhibition of the growth of

E. coli when compared to the individually prepared extracts.

2.) The individually prepared extracts have various significant effects on the inhibition

of the growth of E. coli when compared to the antibiotic cocktail.

3.) Both the antibiotic cocktail and the individually prepared extracts have no

significant effects on the inhibition of the growth of E. coli.

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 4.) If the antibiotic cocktail is used, then it will inhibit the growth of the E. coli.

5.) If the individually prepared extracts are used, then they will inhibit the growth of the

E. coli.

The specific objectives are:

1.) to compare the antibiotic effect of different extracts namely:

Ginger (Zingiber officinale), Malunggay (Moringa oleifera), and Garlic (Allium

sativum) through disk diffusion test using E. coli as bacteria; and

2.) to develop a cost-efficient and effective antibacterial by using natural sources.

The study’s main focus only limited to testing the antibiotic effects of the mentioned

natural extracts namely ginger, malunggay, garlic, and the extract cocktail against the E. coli

bacteria, to achieve the main goal of the researchers which was to develop a cost- effective

natural antibacterial. A sample population of E. Coli WT is used to determine the possible

antibiotic effects of the aforementioned natural extracts.

The results and the findings gathered from conducting the research study will provide

numerous benefits to the following: the community, the field of medicine, and the field of

research & future researchers. The findings of this study are beneficial towards the community

both globally and locally. By testing and investigating the antibiotic effects of ginger, malunggay,

garlic, and the extract cocktail, we can be a step closer towards a natural and highly efficient

antibiotic which can cure diseases caused by the E. coli bacteria and is easily accessible to all. In

the field of medicine, conducting this study can help advance the development and creation of

medicine possessing great efficiency which can potentially solve not just the diseases caused by

the mentioned bacteria, but also its sub-pathotypes and possibly more. Similar to the benefits in

the field of medicine, the field of research and the future researchers may also benefit from this

by further developing

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existing studies and gaining reinforcement towards future studies with similar variables and

goals.

2.0 MATERIALS & METHODS

RESEARCH DESIGN

This study is designed as a quantitative study aiming to compare the antibiotic effect of

Malunggay, Garlic and Ginger extract cocktail versus their individually prepared extracts at a

laboratory in Camalig, Albay. Among the quantitative research methods, experimental research

was used in this study. The researchers utilized probability sampling because it is the most

statistically valid option in dealing with a very small and accessible population.

RESEARCH SETTING

The research study was carried out in Regional Animal Disease Diagnostic Laboratory in

Camalig, Albay. The rationale behind this choice is from the sense that RADDL has the Region’s

state of the art facilities and complete laboratory materials and bacterial cultures. Hence, there is a

probability that obtaining all relevant results and data will be achieved when the research is

conducted in the place.

POPULATION AND SAMPLING

The population that can possibly be used in this research study offers a wide choice between a lot

of pathogenic bacteria, however the researchers narrowed the number of samples to the most

common pathogenic bacteria that can be found. The researchers chose E. Coli WT as their sample

bacteria. The antibiotic effect of Malunggay, Ginger and Garlic extract versus their individually

prepared extracts will be measured using E. coli WT as the main sample.

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RESEARCH MATERIALS AND METHODS

COLLECTION OF MATERIALS

The experimentation will start with the collection of the materials to be used in the study,

such as garlic, malunggay, ginger, and the E. coli. The garlic, malunggay, and ginger will be

bought from the marketplace in Tabaco City. These will then be stored in a refrigerator to prevent

it from getting ruined.

The bacteria that will be used in the study are the Escherichia coli. The researchers

gathered these bacteria from Regional Animal Disease Diagnostic Laboratory in Camalig, Albay.

This is also the place where they will conduct their experiment.

Sample Preparation

The researchers will prepare the samples by using a blender. In order to test the

antibacterial properties of Garlic (Allium sativum), Ginger (Zingiber officinale), and Malunggay

(Moringa oleifera), each sample will be homogenized using a blender then decanted in a

container with cheesecloth. Each container will contain 30ml of each sample plus 10ml of

antibiotic cocktail.

PREPARATION OF CULTURE

In a test tube, the researchers will prepare and sterilize 10mL of Nutrient Broth. Then, a

loopful of E. coli WT will be transferred to the sterilized NB then will be incubated at 35 C for

24hrs.

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PREPARATION OF 1.5 X 10^8 CELLS

The researchers will transfer 50 microliters of E. coli WT cells from the tube that was

incubated for 24hrs to a tube containing 10 mL of sterilized distilled water to prepare

E. coli WT cells with counts of approximately 1.5x10^8 colony forming unit/ml.

TESTING OF TURBIDITY

The turbidity of the tube will be compared to the turbidity of the 0.5 McFarland standard.

Upon seeing that the turbidity does not yet match, the researchers will transfer another 50

microliters of E. coli WT cells from the tube that was incubated for 24 hrs. to the tube containing

10 mL of sterilized distilled water until it matches the 0.5 turbidity of the McFarland standard.

DATA COLLECTION

- Disk Diffusion Sensitivity Test/ Kirby Bauer Test will be used to collect data. From the

tube with 1.5x10^8 cells/ml of E. coli WT, the researchers will submerge three sterile

applicators until the tips get soaked. The applicators will be carefully lifted and pressed

against the side of the tube to dislodge some liquid.

DATA ANALYSIS

- The results will be analyzed based on the zone of inhibition of the E. coli WT in the

various experimental set-ups. The effectivity of the extracts will be determined according

to the area of the zone of inhibition of E. coli WT that they are applied to.

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References:

 Blumenthal, K. et al. (2019, January 12). Antibiotic allergy. The Lancet volume 393,

p183-198. Retrieved on March 6, 2022, from

https://www.thelancet.com/pdfs/journals/lancet/PIIS0140-6736(18)32218-9.pdf

 Centers for Disease Control and Prevention (2018, September 19). Antibiotic Resistance:

A Global Threat. Retrieved on March 6, 2022, from

https://www.cdc.gov/features/antibiotic-resistance-global/index.html

 Dresdeb, D. (2018, March 3) Top seven safe, effective natural antibiotics.

Retrieved on March 6, 2022, from

https://www.medicalnewstoday.com/articles/321108.php

 Gopalakrishnan L., Doriya K., Kumar, D.S. (2016). Moringa oleifera: a review on

nutritive importance and its medicinal application Food Sci Hum Wellness, 5 (2016), pp.

49-56. Retrieved on March 6, 2022, from

https://www.sciencedirect.com/science/article/pii/S2213453016300362

 Harding, M (2018, March 9). Antibiotics. Retrieved on March 6, 2022, from

https://patient.info/infections/antibiotics-leaflet

 Liu, Q. et al. (June 16, 2016). “Antibacterial and Antifungal Activities of Spices.”

International journal of molecular sciences vol. 18,6 1283. Retrieved on March 6,

2022, from doi:10.3390/ijms18061283

 Luby, S. et al. (2015, November 4). Microbiological Contamination of Drinking Water

Associated with Subsequent Child Diarrhea. Retrieved on March 6, 2022, from

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4703288/

 NA (2017, December 10). "Zingiber officinale". Germplasm Resources Information

Network (GRIN). Agricultural Research Service (ARS), United States Department of

Agriculture (USDA). Retrieved on March 6, 2022, from https://npgsweb.ars-

grin.gov/gringlobal/taxonomydetail.aspx?id=42254
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 NA (2017, May 26). "Allium sativum L". Kewscience; Plants of the World Online; Royal

Botanic Gardens, Kew, England. Retrieved on March 6, 2022, from

http://powo.science.kew.org/taxon/urn:lsid:ipni.org:names:528796-1#bibliography

 Nejad, A. et al. (2017 November 7). Antibacterial Effect of Garlic Aqueous Extract on

Staphylococcus Aureus in Hamburger. Retrieved on March 6, 2022, from

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4332239/

 Madappa, T. et al. (2019, February 11). Escherichia coli (E coli) Infections.

Retrieved on March 6, 2022, from

https://emedicine.medscape.com/article/217485-overview

 University of Illinois-Chicago, Drug Information Group (2019, April 19). Side Effects of

Antibiotics: What They Are and How to Manage Them. Retrieved on March 6, 2022,

from https://www.healthline.com/health/infection/antibiotic-side-

effects

 Vila, J. et al. (July 2016). Escherichia coli: an old friend with new tidings, FEMS

Microbiology Reviews, Volume 40, Issue 4, Pages 437–463. Retrieved on March 6,

2022, from https://doi.org/10.1093/femsre/fuw005

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DEFINITION OF TERMS:

E. Coli - E. coli is a strain of bacteria. E. coli is a Gram-negative, facultatively anaerobic, rod-

shaped coliform bacterium of the genus Escherichia that is commonly found in warm-blooded

organisms' lower intestine.

Antibiotic- An antibiotic is a type of antimicrobial substance active against bacteria.

Zone of Inhibition- This is an area of media where bacteria are unable to grow, due to presence

of a drug that impedes their growth

Nutrient Broth- is a liquid medium used for the cultivation of a wide variety of organisms from

clinical specimens and other materials.

Pathogens- an organism causing disease to its host, with the severity of the disease

symptoms referred to as virulence.

Kirby Bauer Test- is a test that determines the sensitivity or resistance of pathogenic

bacteria to various antimicrobial compounds in order to assist physicians in selecting

treatment options their patients.

Microbiological culture- A microbiological culture, or microbial culture, is a method of

multiplying microbial organisms by letting them reproduce in predetermined culture medium

under controlled laboratory conditions.

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APPENDIX:

a.) A photo of the Laboratory analysis for the research study.

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