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ANTIBACTERIAL ACTIVITY OF THE METHANOLIC FLOWER EXTRACT OF

CARICA PAPAYA (PAPAYA) AGAINST ESCHERICHIA COLI

By

Jairah Mapa Berboso

Chapter 1

Introduction of the study

Chapter One consists of five parts: (1) Background and Theoretical Framework of the

study, (2) Statement of the Problem and the Hypothesis, (3) Significance of the study,

(4) Definition of Terms and (5) Scope and Limitation of the Study.

Part One, Background of the Study and Theoretical Framework of the Study, gives

the reasons and basis for the choice of the problem, and identifies the variables used in

the study, and presents the theoretical framework upon which the study will be anchor.

Part Two, Statement of the Problem and the Hypothesis, presents the main and

specific problems to be answered by the researcher and hypothesis to be tested.

Part Three, Significance of the Study, cites the benefits that may be derived from the

result of the investigation

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Part Four, Definition of Terms, gives the conceptual and operational meaning of the

important terms that will be use in the study.

Part Five, Scope and Limitation of the Study, specifies the limits and coverage of

the study.

Background of the study

Antibacterial agents are a group of materials that fight against pathogenic bacteria.

Thus, by killing or reducing the metabolic activity of bacteria, their pathogenic effect in

the biological environments will be minimized. Furthermore, these materials can prevent

bacterial plaque accumulation in the oral environment and therefore, can reduce the

prevalence of plaque-related diseases such as caries. However, it should be noted that

antibacterial agents do not necessarily possess antiplaque accumulation properties.

Today, the manufacture and use of biomaterials with antibacterial effects in medical

and dental treatment plans is rapidly progressing. Manufacturing products containing

antibacterial agents, or coatings of the material with antibacterial properties, has

become an interesting topic for research in the field of dentistry. Various types of

implants, restorative materials, adhesives, denture-based materials, and tissue

conditioners are being produced with antibacterial properties, and analyzed in

laboratories; some have even been marketed.

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The idea of adding antibacterial agents to the restorative materials such as resin-based

composites or glass ionomers is not a new concept. However, introducing

nanotechnology in the medical and dentistry fields has proven to be a promising

concept. Previous efforts were made to produce resin-based composites with

antibacterial properties by adding chlorhexidine; however, they had short-term effects.

Furthermore, the addition of chlorhexidine and antibiotics to the resin-based composites

compromised their mechanical properties such that as these compounds came out of

the composites and their mechanical properties deteriorated.

In addition, these organic antibacterial materials were highly susceptible to damage

upon exposure to heat.

Numerous studies are being conducted to overcome these problems. Antibacterial

agents can be added to either the fillers or resin matrix of composites. One thing that

must be considered for all these innovative factors is that their integration into the

composite material should not cause a decrease in the mechanical or esthetic

properties. (Maryam Pirmoradian, Tabassom Hooshmand, in Applications of

Nanocomposite Materials in Dentistry, 2019).

Methanol also called methyl alcohol and wood spirit, amongst other names is an

organic chemical and the simplest aliphatic alcohol, with the formula CH3OH (a methyl

group linked to a hydroxyl group, often abbreviated as MeOH). It is a light, volatile,

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colourless, flammable liquid with a distinctive alcoholic odour similar to that

of ethanol (potable alcohol).[17] A polar solvent, methanol acquired the name wood

alcohol because it was once produced chiefly by the destructive distillation of wood.

Today, methanol is mainly produced industrially by hydrogenation of carbon monoxide.

[18]

Methanol consists of a methyl group linked to a polar hydroxyl group. With more than

20 million tons produced annually, it is used as a precursor to other commodity

chemicals, including formaldehyde, acetic acid, methyl tert-butyl ether, methyl

benzoate, anisole, peroxyacids, as well as a host of more specialised chemicals . (  IUPAC

Recommendations and Preferred Names, 2013, Cambridge: The Royal Society of

Chemistry, 2014.)

Papaya (Carica Papaya L.) has become a popular fruit due to its fast growth, high yield,

long fruiting period and high nutrient value as well. Papaya can be a highly profitable

crop now. Papaya is a common man’s fruit is also known as a melon tree, papaw or

papita. Papaya is an excellent source of vitamin ‘A’ and has a good amount of vitamin

‘C’, minerals (Fe, Ca and P) and carbohydrates. (agrifarming.in, 2020).

The Papaya flowers has been used to several ailments by our forefathers. After the

consumption of Papaya flowers, a significant change has been seen in the levels of
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insulin in diabetic patients. Papaya flowers can stabilize high blood pressure; prevent

heart disease and many other ailments. After the consumption of the Papaya Flowers,

patients noticed a remarkable improvements in themselves. These flowers have Vitamin

A, C, E and folate with anti-oxidant properties that prevent cholesterol and oxidation.

(agrifarming.in, 2020).

In addition, Escherichia coli or E. coli is a lactose fermenting facultative organism which

is the most common bacteria that infects the urinary tract (Foxman and Brown, 2003).

Empirical treatment, mechanisms of resistance are varied and resistance to multiple

antibiotics problem (Kim, et al., 2008 cited in Llanes, et al., 2012).

On the other hand, there is a worldwide increase in the antimicrobial resistance E.

coli. in Latin America. It is responsible for higher prevalence of bloodstream and

pneumonia. In the Asia and Pacific regions, urinary tract infections by this

microorganism prevail with high reports of antimicrobial resistance (Dapeng, 2007, cited

in Llanes, et al. 2012).

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Figure 1. Shows the diagrammatic representation of the relationship

between the independent variable, and dependent variable.

Independent Variable

Different Concentrations of Carica

Papaya (Papaya) flower extract

Dependent Variable
Treatment A
Mean Zones of
Inhibition in
50%- Carica Papaya flower extract
Escherichia coli

Treatment B

75%- Carica Papaya flower extract

Treatment C

100%- Carica Papaya flower

extract
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Statement of the Problem

This study will determine the antibacterial activity of the methanolic flower

extracts of Carica Papaya against Escherichia Coli.

Specifically, this study will seek to answer the following questions:

1. What are the zones of inhibition against Escherichia Coli when treated by different

concentrations of Carica Papaya flower extracts such as:

(a) 50% extract

(b) 75% extract

(c) 100% extract

(d) Chloramphenicol antibiotic- positive control

2. Which concentration yield the highest zone of inhibition against Escherichia Coli?

3. Is there a significant difference on the mean zone of inhibition of Escherichia Coli

when treated by:

(a) 50% extract

(b) 75% extract

(c) 100% extract

(d) Chloramphenicol antibiotic- positive control

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Hypothesis of the Study

1. There is no significant difference on the mean zone of inhibition of Escherichia Coli

when treated by:

(a) 50% extract

(b) 75% extract

(c) 100% extract

(d) Chloramphenicol antibiotic- positive control

Significance of the Study

Presently, The antimicrobial resistance (AMR) crisis is the increasing global

incidence of infectious diseases affecting the human population, which are

untreatable with any known antimicrobial agent. This crisis will have a devastating cost

on human society as both debilitating and lethal diseases increase in frequency and

scope.(Carolyn Anne Michael, Dale Dominey-Howes, Maurizio Labbate, 2014).

This study will be conducted to determine the antibacterial activity of methanolic flower

extract of Carica Papaya (Papaya) against escherichia coli.

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This study will be a great help to the field of science, specifically medicine, and to the

Food and Drug Administration (DFA) for it may be used as a medicine that may

eliminate antibiotic resistant bacteria that brings several diseases.

This study may be significant to human health for it can help eliminating these

antibiotic resistant bacteria, preventing severe and incurable diseases to occur thus

decreasing the percentage of morbidity, mortality, and the cost for health assistance.

It may also help the researcher in improving his skills and knowledge for upcoming

challenges, especially in search for scientific excellence, and to gain more experiences

in doing researches.

Definition of Terms

Methanol. Is an organic chemical and the simplest aliphatic alcohol, with the formula

CH₃OH. It is a light, volatile, colourless, flammable liquid with a distinctive alcoholic

odour similar to that of ethanol. A polar solvent, methanol acquired the name wood

alcohol because it was once produced chiefly by the destructive distillation of wood.

Today, methanol is mainly produced industrially by hydrogenation of carbon monoxide.

(IUPAC Recommendations and Preferred Names, 2013, The Royal Society of Chemistry,

2014).
In this study, it is to determine the methanolic flower extract of carica papaya against

escherichia coli.

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Antibiotics. Are powerful medicines that fight certain infections and can save lives

when used properly. They either stop bacteria from reproducing or destroy them

(MedicalNewsToday, 2019).

In this study, it refers to methanolic flower extract’s acid.

Antimicrobial. is defined as a natural or synthetic substance that kills or inhibits the

growth of microorganisms such as bacteria, fungi and algae (ScienceDirect, 2019).

In this study, it refers to the ability of Escherichia coli to resist the effects of

antimicrobial agents.

Concentration. an increasing of strength (as of a solute or a gas in a mixture) or a

purifying by partial or total removal of diluents, solvents, admixed gases, extraneous

material, or waste (as by evaporation or diffusion) (Meriam-Webster Online Dictionary,

2019).
In this study, it refers to the amount of solvent and extract of a Carica Papaya flower in

a treatment.

Escherichia coli. It is a type of bacteria that normally lives in your intestines. It’s also

found in the gut of some animals. Most types of E. coli are harmless and even help

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keep your digestive tract healthy. But some strains can cause diarrhea if you eat

contaminated food or drink fouled water. (WebMD, 2019).

In this study, it will serve as the subjects where the antibacterial activity of the Carica

Papaya flower crude methanolic extract will be determined.

Extract. It is a substance made by extracting a part of a raw material, often by using

solvent such as ethanol or water. To withdraw by expression, distillation, or other

mechanical or chemical process. (Definitions.net, 2019).

Inoculum. It is a substance introduced into the body to create or increase the body’s

resistance or immunity to a disease. Material used in inoculation. (YourDictionary,

2019).

In this study, it refers to Escherichia coli, which is the bacteria to be killed.

Carica Papaya (Papaya) flower. The papaya flowers are tubular, trumpet-shaped

blossoms emerging from the axils of the papaya leaf. Growing to between one and two

inches (2.5 - 5 cm) long, they are waxy and sweetly perfumed. Their color ranges from

a pale ivory to golden yellow. (HerbaZest, 2020).

In this study, it refers to the main subject because it has an antibacterial activity of

methanolic extract to kill Escherichia coli.

Resistant. It refers to the act or an instance of resisting or the capacity to resist

(Medical Dictionary Online, 2018).

In this study, it refers to the property of Escherichia coli to remain unaffected by

an antibiotic and antibacterial agent/s.

Susceptible. It means lacking immunity or resistance and thus at risk of infection

(Medical Dictionary Online, 2019).

In this study, it refers to lack of resistance of Escherichia coli to different treatments will

be considered.

Zone of inhibition. It is the area without bacterial growth surrounding an

antimicrobial-impregnated disk in an antimicrobial sensitivity test (Medical Dictionary

Online, 2018).

In this study, it will be determined for each concentration of the crude methanolic

extract of Carica Papaya’s flower.

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Scope and Limitation of the Study

This study will be only limited on the antibacterial activity of the methanolic flower

extract of Carica Papaya (Papaya) against Escherichia Coli. The antibacterial ptoperty

must be based only on the zone of inhibition. The study will be conducted either in a

microbiology laboratory or medical laboratory of West Visayas State University or

University of San Agustin. The Papaya flower will be taken at Baras Trapiche Oton, Iloilo

and the bacteria that will be used in the study is Escherichia coli ATCC 25922 that will

be taken at University of San Agustin Laboratory. There will be 3 treatments (A,B,C)

that will be compose of the following: Treatment A- 50% Carica Papaya flower extract,

Treatment B- 75% Carica Papaya flower extract, Treatment C- 100% Carica Papaya

flower extract, positive control (Chloramphenicol antibiotic) with three replications each.
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Chapter 2

Review of Literature and Related Studies

This chapter presents and discusses literature to substantiate the researcher’s

conceptual framework and various related studies.

Chapter Two has five major parts: (1) Antimicrobial and Antibiotic-Resistance, (2)

Papaya flower, (3) Methanol, (4) Escherichia coli, (5) Kirby-Bauer Method, and (6)

Summary.

Antimicrobial and Antibiotic Resistance, gives its definitions, importance for the study,

its history and big effect on people’s lives, and related studies for the study.

Papaya flower, presents the main reason why this subject was choosen, its

definition, its chemical components and related studies for the study.

Methanol, cites the benefits on human health, its definitions, its importance in killing the

designed bacteria and related studies for the study.

Escherichia coli, specifies its definition, reasons why it is one of the bacteria to be killed,

and how worst it is in human health.

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Summary, consists of all the summarized informations in every subject that will be used

in the study.

Antimicrobial and Antibiotic-Resistance

Antimicrobial agents have been used for the last 70 years to treat infectious

diseases. Since the 1940s, these drugs have greatly reduced illness and death from

infectious diseases. However, these drugs have been used so widely and for so long

that the bacteria and fungi develop the ability to defeat the drugs designed to kill them.

That means the germs are not killed and continue to grow. Some microorganisms may

develop resistance to a single antimicrobial agent, while others are often referred to as

multidrug-resistant or MDR strains. In some cases, the microorganisms have become so

resistant that no available antibiotics are effective against them. Also, antimicrobial drug

resistance occurs everywhere in the world. Hospitals and other healthcare facilities are

battling drug-resistant organisms that widely. Drug-resistance infections also spread in

the community at a very large state, like skin and soft tissues infections (Data retrieved

from Center for Disease Control and Prevention on December 23, 2019).

Penicillin was the first antibiotic, wherin it was discovered in 1929 by Sir Alexander

Fleming, who observed inhibition of staphylococci on an agar plate contaminated by a

Penicillium mold. Fleming was searching for potential antibacterial compounds. He

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noticed that a patch of the mold Penicillium notatum had grown on a plate containing

the bacterium Staphylococcus and that around the mould there was a zone no

Staphylococcus could grow. After more research, he was able to show that culture

broth of the mould that prevented the growth of the Staphylococcus even when diluted

up to 800 times. He named the active substance penicillin. Unfortunately, he was

unable to isolate it (Todar, 2019).

In 1939, Ernst Chain and Howard Florey developed a way to isolate penicillin and

used it to treat bacterial infections during Second World War. The new drug came into

clinical usage in 1946 and made a huge impact on public health. Their discovery and

development revolutionized modern medicine and gave the way for the development of

more natural antibiotic (Sciencehistory.org, 2019).

In 1946, penicillin became generally available for treatment of bacterial infections,

especially those caused by staphylococci and streptococci. Penicillin was effective

against all sorts of infections cause by these two Gram-positive bacteria. It exhibited an

unbelievable ability to kill these bacterial pathogens without harming the host. Human

diseases such as pneumonia, skin infections and wound infections are cause by these

bacteria (Gaynes, 2017).

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After Several years, evidence began to accumulate that bacteria could pass genes for

drug resistance between strains and even between species. In any case, genes for

antibiotic resistance can be exchanged between strains and species of bacteria by

means of the processes of horizontal gene transmission (HGT) (Todar, 2019).

Multiple drug resistant organisms are resistant to treatment with several

antimicrobial agents. Some of the most important types of multiple drug resistant

organisms that have been encountered are MRSA or methicillin/oxacillin-resistant

Staphylococcus aureus, VRE or vancomycin-resistant enterococci, ESBLs or extended-

spectrum beta-lactamases (which are resistant to cephalosporin and monobactam) and

PRSP or penicillin-resistant Streptococcus pneumonia (Silpi Basak et. al., 2016).

Antimicrobial drug resistance occurs everywhere in the world. Hospitals and other

healthcare facilities are battling drug-resistant organisms that spread widely. Drug-

resistance infections also spread in the community at a very large state, like drug-

resistant pneumonias, sexually transmitted diseases (STDs), and skin and soft tissue

infections (Data retrieved from Center for Disease Control and Prevention on November

14, 2019).

People infected with drug-resistant organisms are more likely to have longer and

more expensive hospital stays, and may be more likely to die as a result of infection.
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They require treatment with second- or third-choice of drugs that may be less effective,

more toxic, and more expensive when the chosen drug for treating the infection doesn’t

work. It only means that patients with an antimicrobial-resistant infection may suffer

and pay more for (Data retrieved from Center for Disease Control and Prevention on

November 14, 2019).

Carica Papaya (Papaya) flower

The papaya flowers are tubular, trumpet-shaped blossoms emerging from the axils

of the papaya leaf. Growing to between one and two inches (2.5 - 5 cm) long, they

are waxy and sweetly perfumed. Their color ranges from a pale ivory to golden yellow.

Because of their bright color, papaya tree flowers are very often used as colorful food

garnishes or festive decorations. In Southeast Asia, flower arrangements are sold

throughout local food markets, and are either candied, or boiled and eaten as a

vegetable. Meanwhile, a warm papaya flower decoction is said to cure bronchitis,

congestion, and respiratory illness when taken orally. The papaya flower is easily the

most complex part of any papaya tree. It is characterized not only by its shape and

appearance, but also by the presence or absence of stamens, pistils, and ovaries.

The female papaya flower contains a reproductive part known as a pistil. It is a long

canal containing a five-lobed stigma and egg-shaped ovary. The ovary does not exist in
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a male papaya flower, which is a non-pistillate blossom with 10 functional stamens -

thin, wispy plant parts that carry grains of pollen. The third type of papaya flower is

unique and multifaceted. Hermaphroditic by nature, it contains both male and female

parts and it is capable of self-pollination. These 'perfect flowers', as they have come to

be known, develop between 5 - 10 stamens and contain ovaries of varying shapes, from

smooth and bulbous to oblong, stretched, or with sporadic folds and ridges.

Although the above mentioned types are the most common, it is also important to note

that under certain climatic and environmental conditions, the papaya flower may

experience sex changes. It have been recorded that some male papaya flowers have

become hermaphroditic in extremely warm weather. Ensuring healthy reproduction of

the entire plant is one of the reasons why papaya flowers are so important. While male

papaya flowers cannot bear fruit, their pollen is transferred by wind, by insects, or by

hand in order to fertilize the female ovule. From the ovaries of a successfully-pollinated

papaya flower spring forth the fleshy tropical fruit, much treasured on the commercial

market. These fruits contain viable papaya seeds, which are later used to grow new

trees. To further complicate the issue of flower type and reproduction, the sex of a

papaya flower cannot be determined until nearly five to six months post-planting. For

this reason, self-reproducing hermaphroditic plants are highly sought after for home

gardening. To achieve bisexual plants and higher crop yields, it is not uncommon for
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commercial farmers to plant three or five papaya seeds per mound, removing unwanted

plants along the way. Although there is no real difference in the taste or quality of fruits

grown from different types of papaya flowers, they often yield fruits of varying shapes:

the female papaya flower usually produces round fruits, while hermaphrodite flowers

develop pear-shaped or elongated fruits. Their respective demands are determined by

commerce and nation-specific preferences. Over the years, a number of creative food

and medicinal preparations have paid homage to the unsung Carica papaya flower.

More than just a colorful plant ornament, the papaya flower is one of the most

economically-significant parts of the papaya plant, responsible for fruit health and plant

reproduction. (HerbaZest, 2020).

Methanol

methanol (CH3OH), also called methyl alcohol, wood alcohol, or wood spirit, the

simplest of a long series of organic compounds called alcohols, consisting of a methyl

group (CH3) linked with a hydroxy group (OH). Methanol was formerly produced by the

destructive distillation of wood. The modern method of preparing methanol is based on

the direct combination of carbon monoxide gas and hydrogen in the presence of

a catalyst. Increasingly, syngas, a mixture of hydrogen and carbon monoxide derived

from biomass, is used for methanol production.

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Pure methanol is an important material in chemical synthesis. Its derivatives are used in

great quantities for building up a vast number of compounds, among them many

important synthetic dyestuffs, resins, pharmaceuticals, and perfumes. Large quantities

are converted to dimethylaniline for dyestuffs and to formaldehyde for synthetic resins.

It is also used in automotive antifreezes, in rocket fuels, and as a general solvent.

Methanol is also a high-octane, clean-burning fuel that is a potentially important

substitute for gasoline in automotive vehicles. The methanol derived from wood is used

chiefly for rendering industrial ethyl alcohol unfit to drink. Methanol is a colourless liquid

that boils at 64.96 °C (148.93 °F) and solidifies at −93.9 °C (−137 °F). It forms

explosive mixtures with air and burns with a nonluminous flame. It is completely

miscible in water. Methanol has an odour that is similar to ethyl alcohol, the intoxicant

of alcoholic beverages, but is a dangerous poison; many cases of blindness or death

have been caused by drinking mixtures containing it. (Melissa Petruzzello, 2022).

Escherichia coli

Escherichia coli is a Gram-negative bacteria. Figure 1 shows that it is a facultative

anaerobic, rod-shaped bacterium that is commonly found in the lower intestine of

warm-blooded organisms (endotherms). Most E. coli strains are harmless, but some
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serotypes can cause serious food poisoning in humans, and are occasionally responsible

for product recalls due to food contamination. The harmless strains are part of the

normal flora of the gut, and can benefit their hosts by producing vitamin K2 and by

preventing the establishment of pathogenic bacteria within the intestine (Percival, et

al., 2019).

Figure 1

Microscopic Image of Escherichia coli

E. coli normally colonizes an infant’s gastrointestinal tract within 40 hours of birth,

arriving with food or water or with the individuals handling the child. In the bowel, it
adheres to the mucus of the large intestine. It is the primary facultative anaerobe of the

human gastrointestinal tract. As long as these bacteria do not acquire genetic elements

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encoding for virulence factors, they remain benign commensals intestine (Percival, et

al., 2019).

E. coli is a good indicator of the presence of fecel contamination and possible

disease-causing bacteria or viruses in water. The test for E. coli is far less expensive

and more reliable than other bacterial tests (Missouri Department of Natural Resources,

2015).

Kirby-Bauer Method

Kirby-Bauer Method testing is also known as KB testing or disk diffusion antibiotic

sensitivity testing. It is a test which uses antibiotic-impregnated wafers to test whether

particular bacteria are susceptible to specific antibiotics. A known quantity of bacteria is

grown on agar plates in the presence of thin wafers containing relevant antibiotics. If

the bacteria are susceptible to a particular antibiotic, an area of clearing surrounds the

wafer where bacteria are not capable of growing and it is called a zone of inhibition

(Data retrieved from shsmanual.ucsc.edu on February 4, 2020).

 The rate of antibiotic diffusion will be used to estimate the bacteria’s sensitivity to

that particular antibiotic. In general, larger zones correlate with smaller minimum

inhibitory concentration (MIC) of antibiotic for those bacteria. This will be used to

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choose appropriate antibiotics to combat a particular infection (Data retrieved from

shsmanual.ucsc.edu on February 4, 2020).

The purpose of the Kirby-Bauer Dish Diffusion Susceptibility Test is to determine

the sensitivity or resistance of pathogenic aerobic and facultative anaerobic bacteria to

various antimicrobial compounds in order to assist a physician in selecting treatment

option for his or her patients. The pathogenic organisms is grown on Mueller-Hinton

agar in presence of various antimicrobial impregnated filter disks. The presence or

absence of growth around the disks is an indirect measure of the ability of that

compound to inhibit that organism (Data retrieved from American Society for

Microbiology on June 30, 2016).

Summary

Antimicrobial agents have been used for the last 70 years to treat infectious

diseases. Since the 1940s, these drugs have greatly reduced illness and death from

infectious diseases. However, these drugs have been used so widely and for so long
that the microorganisms designed to kill have adapted to them, making the drugs less

effective.

The papaya flowers are tubular, trumpet-shaped blossoms emerging from the axils

of the papaya leaf. Growing to between one and two inches (2.5 - 5 cm) long, they

are waxy and sweetly perfumed. Their color ranges from a pale ivory to golden yellow.

methanol (CH3OH), also called methyl alcohol, wood alcohol, or wood spirit, the

simplest of a long series of organic compounds called alcohols, consisting of a methyl

group (CH3) linked with a hydroxy group (OH). Methanol was formerly produced by the

destructive distillation of wood. The modern method of preparing methanol is based on

the direct combination of carbon monoxide gas and hydrogen in the presence of

a catalyst. Increasingly, syngas, a mixture of hydrogen and carbon monoxide derived

from biomass, is used for methanol production.

Escherichia coli is a Gram-negative bacteria. Figure 1 shows that it is a facultative

anaerobic, rod-shaped bacterium that is commonly found in the lower intestine of

warm-blooded organisms (endotherms). Most E. coli strains are harmless, but some

serotypes can cause serious food poisoning in humans, and are occasionally responsible

for product recalls due to food contamination.

Kirby-Bauer Method testing is also known as KB testing or disk diffusion antibiotic

sensitivity testing. It is a test which uses antibiotic-impregnated wafers to test whether

particular bacteria are susceptible to specific antibiotics.

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Chapter 3