Antibacterial Activity of Red Santan (Ixora coccinea) Leaves Extract Against

Escherichia Coli Bacteria of Street Foods Samples in Tayabas City

A Research Study
Presented to
The Junior High School Faculty of
Luis Palad Integrated High School
City of Tayabas, Quezon Province

In Partial Fulfilment
Of the Requirements in
RESEARCH 3

by:
Baasis, Franceska Ysabelle D.

Ching, Heaven Ashley

Jamilano, Andre Jaster S.
Odasco, Daniel Mico V.

Reyes, Princess Angela N.

Saavedra, Marc Emil S.
Shike, Hasheem Aktar P.

9 – Integrity/Wisdom
Proponents

Ms. Ladessa R. Padua

Research Adviser

September 2022
 CHAPTER 1

THE PROBLEM AND ITS SETTING

Introduction

Street food business is becoming a common business in the informal economy of

trade in the Philippines. Due to lack of employment opportunity, unemployed men and

women have forged innovative avenues for selling variety of goods such as food thus

making street food vending a thriving business activity (Milgram, 2019).

However, R. Papa, C.M. Recto, and P. Campa's street food vendors were lack of

training in handling and serving food and are unaware of hygienic practices (Hilario, 2015).

There is a total of 80 E. coli in Metro Manila, Philippines, E. coli was discovered in main

industrial sites. Coli isolated from irrigation water, soil, vegetables, and the feces of a cat,

carabao, chicken, dog, and goat were divided into separate phylogroups based on the

presence and lack of genetic markers (Zara & Vital, 2022).

Recent studies have documented the risks of ingesting polluted foods from street

vendors that are contaminated with pathogenic bacteria and coliform bacteria, such as

Escherichia coli, Salmonella spp., Staphylococcus aureus, Bacillus cereus, Clostridium

perfringens, and Vibrio cholera (Elobeid, et al. 2014).

Escherichia coli contamination in meat and warm-blooded animals are most

commonly found in broiler chicken meat, intestinal contents, and chicken carcasses.

Escherichia coli is a significant sign of fecal contamination when found in meat (Yulistiani,

et al. 2019).
 From the study of Canini, et al. (2013), pig’s blood or dried chicken blood

(Betamax) are classified as medium-to-high risk of transmitting pathogenic bacteria and

the presence of Escherichia coli is +(3). The chicken intestine is classified as no-to-low

risk of transmitting pathogenic bacteria and the presence of Escherichia coli is +(2).

According to World Health Organization (2019), it is estimated over 200 diseases

are originated from eating foods that is polluted in bacteria, viruses, parasites or chemical

substances. The Department of Health (DOH) has reported over 32,000 cases of food- and

water-borne illness in the Philippines alone as of September this year. WHO Global Burden

of Foodborne Diseases report, diarrheagenic E. coli is thought to be responsible for >300

million illnesses and close to 200,000 deaths worldwide every year (Winstead, et al. 2019).

Additionally, according to the World Health Organization (2015), the first

estimates of the worldwide burden of foodborne infections were about one in ten people

become ill each year from eating contaminated food, and 420 000 die as a result. Children

under the age of five are especially vulnerable, with 125 000 children dying from foodborne

infections each year. The largest incidence of foodborne infections is seen in the WHO

African and South-East Asia Regions.

The researchers will use santan (Ixora coccinea) leaves extract due to numerous

studies about santan (Ixora coccinea) leaves, as antibacterial activity against Escherichia

coli bacteria of the street food samples.

The objectives of this study will be reduced or eliminate the Escherichia coli

bacteria in street food samples, reduce the risk of having a foodborne disease or food
poisoning, and help avoid foods polluted with bacteria, viruses, and other chemical

substance.

Background of the Study

Several food products sold on the street in the Philippines have been discovered to

be contaminated with E. coli may pose a risk to consumers (Mesias, 2018).

In fact, thirty-three students were hospitalized for food poisoning in a Pangasinan

school after consuming “isaw” burgers purchased from the vendors outside the school

(Cardinoza, 2017). Another related incident happens in the Camarines Sur when two

children died after eating grilled chicken intestine, also known as "isaw" and grilled blood,

also known as "betamax" (Amo, 2018).

Contaminated chicken meat by Escherchia coli is a significant source of foodborne

infection. Throughout the world, Escherchia coli infects poultry products (Parvin, et al.

2020). In accordance with the U.S. Food and Drug Administration 2019, Escherichia coli

does affect and can pollute meat products and food crops.

Microbiological results from the DOST investigation showed the presence of

coliforms like Escherichia coli, Enterobacter aerogenes, and E. Cloacae can be found in

fried and grilled animal by-products. This indicates that the bacteria load in the chosen

food samples, which ranged from 240 to 2,400 per gram of food, was quite high (Tacio,

2021).

According to the World Health Organization (2018), consuming food with Shiga

toxin-producing E. coli (STEC) such as raw or undercooked ground meat products, raw

milk, and infected raw vegetables and sprouts can generate severe foodborne diseases and
severe food poisoning in humans. As mentioned by Metella in 2016, food scientists at the

University of Alberta have discovered that cooking ground beef at 71 degrees Celsius—

the level of heat long recommended by Health Canada—does not always eliminate all

strains of Escherichia coli.

To solve this kind of specific problem for the community, the researcher’s ideas

propose using santan (Ixora coccinea) leaf extract as an antibacterial against Escherichia

coli of street food samples.

The study of Marimuthu, et al. (2014) proves that the extract of santan (Ixora

Coccinea) leaf and stem presents good antimicrobial activity. In addition, the study of

Lozada, et al. (2021) showed that the highest antibacterial activity on S. aureus was seen

in santan (I. coccinea) leaf extract at a concentration of 75% among all the plant extracts

used.

The purpose of this research study will utilize santan (Ixora coccinea) leaves extract

as an antibacterial activity against Escherichia coli of the street food samples.

Statement of the Problem

This research study will determine the antibacterial activity of the red santan (Ixora

coccinea) leaves extract against Escherichia coli bacteria of the street food samples.

Specifically, this research study are planning to answer the following questions:

1. What is the content of the red santan (Ixora coccinea) leaves as an antibacterial against

Escherichia coli?
2. What is the effectivity of the red santan (Ixora coccinea) leaves extract into the street

food samples in terms of:

2.1. 25% plant extract concentration

2.2. 50% plant extract concentration

2.3. 75% plant extract concentration

2.4. 100% plant extract concentration

3. How much does the Escherichia coli of the street food samples will reduce before and

after applying the red santan (Ixora Coccinea) leaves extract?

4. Are there any significant differences before and after applying the different plant

extract concentration of red santan (Ixora Coccinea) leaves extract to the Escherichia

coli activity of street food samples in terms of:

4.1. Isaw-Isaw

4.2. Betamax

4.3. Chicken Barbeque

Hypothesis

The researcher’s hypothesis predicts that the Escherichia coli bacteria will be

reduced after applying the red santan (Ixora Coccinea) leaves extract, and there would be

a significant difference before and after applying the red santan (Ixora Coccinea) leaves

extract to the Escherichia coli of the street food samples.

The researcher’s hypothesis is supported by the study of Marimuthu, et al. 2014,

entitled, "Antimicrobial activity and phytochemical screening of various parts of Ixora

coccinea" and the study of Lozada, et al. 2021, entitled "Antibacterial Activity of Santan

(Ixora coccinea) Leaf, Cacao (Theobroma cacao) Pod Husk, and Betel Palm (Areca

catechu) Seed Extracts Against Staphylococcus aureus."

The researchers expect that the Escherichia coli bacteria in the street food samples

will be reduced by the application of red santan (Ixora coccinea) leaves extract, due to

scientific reports and existing studies about red santan (Ixora coccinea).

Significance of the Study

Several food products sold on the street in the Philippines have been discovered to

be contaminated with E. coli may pose a risk to consumers (Mesias, 2018). Escherichia

coli contamination in meat and warm-blooded animals is most commonly found in broiler

chicken meat, intestinal contents, and chicken carcasses. Escherichia coli is a significant

sign of fecal contamination when found in meat (Yulistiani, et al. 2019).

This research study will use red santan (Ixora coccinea) leaves extract as an

antibacterial activity against Escherichia coli bacteria of the street food samples.

Specifically, this research study will be beneficial to the following group of individuals:

To the community, this study will lessen the dangerous cases and chances of

humans consuming street food samples with a strain of Escherichia coli in it.

To the street food vendors, this study will be very beneficial and important to the

food vendors, especially if they sell street foods that might have Escherichia coli bacteria.

The street food vendors will be benefit by this experimentation as it will give protection
and safety of their products against strains of Escherichia coli and any existing particulate

matter.

To the consumers, this study will benefit the consumers, as this study will help

them to be knowledgeable enough about the street food they consume. This study will give

safety for the consumer to consume street foods against Escherichia coli bacteria.

To the reasearchers, this study will be beneficial to the researchers for enhancing

their knowledge about this study and in improving their knowledge towards creating

quantitative research. Furthermore, the researchers will also improve their understanding

on how and what measurement should be use in this study.

To the future researchers, this study will serve as their guide similar to their future

research, and it will be a good recommendation in creating quantitative research and

expanding their insight towards quantitative research. This research study will also be a

support to the future researchers to have a new finding or to continue this kind of study to

find more antibacterial activities against Escherichia coli bacteria.

Conceptual Framework

INPUT

• Problem Stated and Opportunities

• Related Literatures and Studies

PROCESS
• Gathering Equipment and Materials
• Safety Procedures in Extraction and Conduction
• Preparation of Santan (Ixora coccinea) leaves extract
• Detection of the Escherichia Coli and Antibacterial Content
of Santan (Ixora coccinea) Leaves Extract
• Application of the Different Plant Concentration to the
Street Food Samples
• Detection of Escherichia coli After the Conduction

OUTPUT
• Antibacterial Activity of Red Santan (Ixora coccinea) Leaves
Extract Against Escherichia Coli Bacteria of Street Foods
Samples In Tayabas City

Figure 1: Conceptual Framework of Antibacterial Activity of Red Santan (Ixora

coccinea) Leaves Extract Against Escherichia Coli Bacteria of Street Foods Samples

In Tayabas City
 The purpose of this study will be the use of red santan (Ixora coccinea) leaf extract

as an antibacterial against Escherichia coli bacteria found in street food samples of Tayabas

City.

This study will use the Input-Process-Output (IPO) paradigm, it will represent the

structure of the research study. As illustrated in the conceptual framework above, the input

of this study has to discuss the stated problem, as well as the relevant literature and studies.

The process of the research study will have six phases for the researchers to perform

the study. The six phases of this study are: (1) gathering equipment and materials, (2) safety

procedures in extraction and conduction, (3) preparation of red santan (Ixora coccinea)

leaves extract, (4) detection of the Escherichia coli and antibacterial content of red santan

(Ixora coccinea) leaves extract, (5) application of the different plant concentrations to the

street, and (6) the detection of Escherichia coli after the conduction.

The output of this study will be the production of red santan (Ixora coccinea) leaves

extract as an antibacterial activity against Escherichia coli bacteria found in street food

samples of Tayabas City.

Scope and Limitations of the Study

The purpose of this research study will be the use of red santan (Ixora coccinea)

leaves extract as an antibacterial activity against Escherichia coli bacteria of street food

samples in Tayabas City. The researcher will choose the chicken-based products sold by

street food vendors, such as isaw-isaw, betamax, and chicken barbeque, as samples of street

food products. The reason why the researcher chose chicken-based products as samples is
because many studies indicate that Escherchia coli bacteria have a high prevalence in

chickens, and chicken-based products are one of the most popular eats in the street foods

of the Philippines. The objectives of this research study will: (1) reduce or eliminate the

Escherichia coli bacteria in street food samples; (2) reduce the risk of having a foodborne

disease or food poisoning; and (3) avoid foods polluted with bacteria, viruses, and other

chemical substances. The researchers will use red santan (Ixora coccinea) leaves extract as

an antibacterial agent for the Escherichia coli of the street food samples. The research’s

independent variables are the (1) 25% plant extract concentration, (2) 50% plant extract

concentration, (3) 75% plant extract concentration, and (4) 100% plant extract

concentration. The dependent variables of the research study will be the growth of

Escherichia coli of the street food samples which is isaw-isaw, betamax, and chicken

barbeque. The detection of Escherichia coli and the determination of the antibacterial

content of red santan (Ixora coccinea) will be brought in the laboratory of UP Los Baños

located in the towns of Los Baños and Bay in the province of Laguna. The extraction of

the red santan (Ixora coccinea) leaves and the conduction of the study will be conducted

on Quezon Province, Tayabas City, in the school of Luis Palad Integrated High School.

The duration of this research study is only 10 months, where it will start on

September 1, 2022 and end on June 30, 2023. The samples of this research study will only

be 10 sticks per uncooked street food samples: (1) 10 sticks for uncooked isaw-isaw, (2)

10 sticks for uncooked betamax, and (3) 10 sticks for uncooked chicken barbeque. This

research study will only be limited for the researchers to strictly follow the correct

procedures of the extraction and conduction of the study. The process of the research study

will be divided into 6 phases which are: (1) gathering equipment and materials, (2) safety
procedures in extraction and conduction, (3) preparation of red santan (Ixora coccinea)

leaves extract, (4) detection of the escherichia coli and antibacterial content of santan

(Ixora coccinea) leaves extract, (5) application of the different plant concentration to the

street food samples, and (6) detection of escherichia coli after the conduction. Tests that

will be only used in this research study are: (1) chemical test, to determine the antibacterial

content of red santan (Ixora coccinea) leaves extract, (2) Kirby Bauer test, to determine the

effectivity of the red santan (Ixora coccinea) leaves extract to the Escherichia coli of the

street food samples, (3) PCR-based detection of Escherichia coli, to efficiently detect the

presence of the E. coli bacteria before and after the appliance of the various concentrations

of santan leaf extract on the street food samples, and (4) ANOVA test, to determine the

significance difference of the different plant extract concentration and the significance

difference of before and after applying the red santan (Ixora coccinea) leaves extract to the

Escherichia coli bacteria of the street food samples.

Definition of Terms

Antibacterial. A substance that destroys bacteria or inhibits their growth or reproduction

(Stoppler, 2021).

Escherichia coli. Also known as E. coli. A gram-negative bacterium that normally lives in

your intestines (Felson, 2020). It is also classified as the species of coliform that best

indicates fecal pollution and the presence of pathogens (New York State Department of

Health, 2017).
Foodborne diseases/Food poisoning. Also known as foodborne illness, is any illness

caused by the consumption of contaminated foods or beverages (Boslaugh, 2016).

Red santan (Ixora coccinea). It is a grown as an ornamental plant that belongs from the

rubbicea family (Kharat AR et al. 2013). It contains major phytoconstituents like alkaloids,

glycosides, flavonoids, tannins, saponins, etc. (Nair, et al. 2019).

Street food samples. Ready-to-eat food sold on the street is a global phenomenon that is

more common in urban areas and serves as an important dietary source for people in low

and middle income countries (World Health Organization, 2019).

 CHAPTER 2

REVIEW OF RELATED LITERATURES AND STUDIES

Escherichia coli

According to Mayo Clinic 2020, Escherichia coli (E. coli) bacteria typically live in

the intestines of healthy humans and animals. The chicken's intestinal microbiota naturally

contains Escherichia coli. Very immature chicks may have it due to vertical transfer or

infection from the hatchery, transportation, or farm environment (Mourand, et al. 2020).

The GIT of chicken is where harmful and hazardous bacteria including Salmonella,

Escherichia coli, and Clostridium perfringens are found. (Pan & Yu, 2013). Escherichia

coli's primary and secondary habitats are warm-blooded animals' intestines and the

environment. In poultry like chicken and turkey, both habitats are both present. (Stromberg,

et al. 2017).

A significant source of foodborne infection with Escherichia coli is chicken meat

that has been infected with this bacterium. Throughout the world, the harmful bacteria

Escherchia coli infects poultry products. (Parvin, et al. 2020).

Animal intestines can become contaminated during animal slaughter, which could

lead to sickness in humans (Liur & Veerman, 2021). Additionally, in accordance with the

U.S. Food and Drug Administration (2019), Escherichia coli does affect and can pollute

meat products and food crops.

E. coli isolates from chicken feces have ExPEC-associated genes, have ExPEC-

related in vitro phenotypes, and can result in ExPEC-related infections in animal models,
which raises the possibility that they could be harmful to poultry and consumers

(Stromberg, et al. 2017).

Important pathogens known as extraintestinal pathogenic Escherichia coli (ExPEC)

strains to cause a wide range of illnesses in both people and animals. ExPEC-associated

virulence genes are found in some E. coli isolates from chicken feces, making them

potentially pathogenic. These isolates may spread to humans through handling and/or

consumption of tainted meat. The true extraintestinal pathogenic potential of fecal E. coli

from chickens is a complicated organism. Here, we evaluated the presence of fecal E. coli

(Stromberg, et al. 2017).

In experimental animal infection models, a number of fecal ExPEC isolates from

chickens were similar to avian and human clinical ExPEC isolates in their ability to cause

one or more ExPEC-related diseases. Furthermore, certain isolates that were labeled as

non-ExPEC were able to induce ExPEC-associated diseases in animal models; hence,

additional research is required to understand their virulence mechanisms. Escherichia, as

evidenced by these results (Stromberg, et al. 2017).

A risk to poultry and consumers' health may exist because E. coli isolates from

chicken feces have ExPEC-associated genes, show ExPEC-related in vitro phenotypes, and

can result in ExPEC-related illnesses in animal models (Stromberg, 2017).

According to the World Health Organization (2018), consuming food with Shiga

toxin-producing E. coli (STEC) such as raw or undercooked ground meat products, raw

milk, and infected raw vegetables and sprouts can generate severe foodborne diseases and

severe food poisoning to humans.

 According to World Health Organization (2019), it is estimated over 200 diseases

are originated from eating foods that is polluted in bacteria, viruses, parasites or chemical

substances. The Department of Health (DOH) has reported over 32,000 cases of food- and

water-borne illness in the Philippines alone as of September this year. WHO Global Burden

of Foodborne Diseases report, diarrheagenic E. coli is thought to be responsible for >300

million illnesses and close to 200,000 deaths worldwide every year (Winstead, et al. 2019).

Several food products sold on the street in the Philippines have been discovered to

be contaminated with E. coli may pose a risk to consumers (Mesias, 2018).

Microbiological results from the DOST investigation showed the presence of

coliforms like Escherichia coli, Enterobacter aerogenes, and E. Cloacae can be found in

fried and grilled animal by-products. This indicates that the bacteria load in the chosen

food samples, which ranged from 240 to 2,400 per gram of food, was quite high (Tacio,

2021).

An important public health issue is the contamination of poultry flesh with

foodborne microorganisms, which can lead to food poisoning [2]. Salmonella species and

Escherichia coli (E. coli) are the two most significant enterobacterial family

(Enterobacteriaceae) foodborne pathogens that cause foodborne illness and are spread

through poultry meat, resulting in health issues around the world. (Bintsis, 2017).

Each of the 40 samples (chicken flesh, intestinal contents, and rinse water) were

taken at the slaughterhouse in Surabaya's traditional markets for a total of 120 samples.

Salmonella spp. were checked for in the samples. in chicken meat, intestinal contents, and
rinse water, as well as E. coli. Salmonella spp. and Escherichia coli were isolated and

identified using the standard culture-based method (Yulistiani, 2019).

Both E. coli O157:H7 and Salmonella Typhimurium are foodborne pathogens that

are frequently identified in contaminated chicken flesh. (Ma, et al. 2022).

According to Argente, et al. (2020) study, the majority (71%) of the pork barbecue

sampled from those sold by respondents was safe or had no E. coli contamination. Grilled

pork intestine and coagulated chicken blood showed opposite results. Both exceeded the

value recommended by the International Commission on Microbiological Specifications

for Foods (1998), making them generally unsafe for consumption.

According to this study, E. coli was isolated from 77.50% of the chicken meat,

65.00% of the intestinal contents, and 75.00% of the rinse water, respectively. Salmonella

spp. prevalence and E. More coliforms are found in chicken flesh than in digestive fluids

or rinse water. Moreover, Escherichia coli in chicken meat originates from sources other

than intestinal waste and washing water (Yulistiani, 2019).

From the study of Canini, et al. (2013). Pig’s blood or dried chicken blood

(Betamax) are classified as medium-to-high risk of transmitting pathogenic bacteria and

the presence of its Escherichia coli is +(3). The chicken intestine is classified as no-to-low

risk of transmitting pathogenic bacteria and the presence of its Escherichia coli is +(2).

There cases were thirty-three students were hospitalized for food poisoning in a

Pangasinan school after consuming “isaw” burgers purchased from the vendors outside the

school (Cardinoza, 2017). Another related incident happens in the Camarines Sur when
two children died after eating grilled chicken intestine, also known as "isaw," and grilled

blood, also known as "betamax" (Amo, 2018).

Red Santan (Ixora coccinea) leaves

Santan (Ixora coccinea) (also known as Ixora grandiflora Bot and Ixora bandhuca

Roxbg) is grown as an ornamental plant. It is a multibranched, glabrous, evergreen shrub

that grows to 1-2 m in height but can reach up to 3.6 m in height, with many brilliant

crimson blooms in thick, senssile corymbiform cymes. Coriaceous leaves range in length

from 2 to 15 cm and are oblong, sessile or sub-sessile, and obtuse. It is a genus of flowering

plants in the Rubiaceae family with roughly 500 species whose names vary according on

the color of the blooms. Numerous ethnic groups in Africa, Asia, and Europe have used

the Ixora plant and its various components such as flowers, leaves, roots, barks, and fruits

(Kharat, et al. 2013).

Existing research indicates that plants, particularly their leaves and seeds, have

potent antibacterial capabilities that combat germs found in human mouths, as well as

antioxidant, anti-inflammatory, anti-carcinogenic, immune-modulatory, vasodilatory, and

analgesic effects (Baliga & Poruthukaran, 2012).

Three of the plants that are locally available in the Philippines include Santan (I.

coccinea), Cacao (T. cacao), and Betel Palm (A. catechu) (Lozada, et al. 2021).

Santan (Ixora coccinea) contains alkaloids, glycosides, flavonoids, tannins,

saponins, steroids, terpenoids, and other major phytoconstituents. In the oil of Ixora

coccinea flower, 54 compounds have been identified as representing 99.97% of all

compounds are present. Triterpenes account for 62.60% of the oil's composition, with

monoterpenes accounting for 31.73%. 3.35% sesquiterpenes and 2.29% ester. The

anticancer activity of Ixora coccinea leaves was discovered. Camptothecin, a known

alkaloid, is primarily responsible. RP-HPLC analysis confirmed the presence of

camptothecin. The average Camptothecin content in mature and young leaves was 2.8%,

paving the way for new discoveries. The chemical analysis of the roots results in the

isolation of six phyto-constituents, namely: 9, phthalate, - amyrin, kaempferol-7-

oglucoside, kaempferitrin, and 12-octadecadienoic acid and quercetin. (Nair, Jadhav, &

Bakare, 2018).

In both in vitro and in vivo studies, santan leaf extract has been shown to be

effective against Staphylococcus aureus, while its methanolic leaf extracts are effective

antioxidants, anti-inflammatory agents, fibroblast proliferative, and stimulates Smad

mediated collagen production at 2.5% with wound contraction rate greater than gentamicin

sulfate (0.01% w/w) (Rescober, et al. 2022).

Plant secondary metabolites, through their defensive systems, may provide a

possible countermeasure to these bacterial survival processes. Using AutoDock modeling

software and Biovia Discovery Studio, nine chosen plant secondary metabolites from Ixora

coccinea, Mimosa pudica, and Origanum vulgare in the Philippines were molecularly

docked against the RND efflux pump system, AcrAB-TolC of E. coli. All of the identified

metabolites had negative binding energies, indicating significant ligand-receptor affinity

and stability, particularly the secondary metabolites of I. coccinea (Segui, et al. 2022).

Methanolic extracts of Santan leaf, flower, and stem shown antibacterial activity. (Mani,

et al. 2014).
Related Studies

According to the study entitled "Antioxidant and antimicrobial activities of Ixora

coccinea root and quantification of phenolic compounds using HPLC" conducted in 2020

by Muhammad. Ixora coccinea Lynn. (Syn. I. grandiflora), sometimes called the flame of

wood, jungle geranium, and Vethi, is a stunning shrub with a number of therapeutic

benefits. It has a wide range of biological and therapeutic effects, including

hepatoprotective (Baliga & Kurian, 2012; Elumalai et al., 2012), chemoprotective (Latha

and Panikkar, (2001), Latha et al., 2004; Sun and Peng, 2008); antimicrobial (Latha et al.,

1995; Annapurna et al., 2003; Sharma & Smita, 2010); antioxidant (Versiani et al., 2012).

Based on the study entitled "Antioxidant and antimicrobial activities of Ixora

coccinea root and quantification of phenolic compounds using HPLC" conducted in 2020

by Muhammad. Alkaloids, flavonoids, tannins, steroids, terpenoids, and saponins are only

a few of the bioactive substances found in this plant (Latha & Panikkar, 2001; Maniyar et

al., 2010; Ikram et al., 2013). Recently, three triterpenoids isolated from I were studied by

our team for their antioxidant and cytotoxic properties. Coccinia blossoms (Ikram et al.,

2015). We have further assessed I's antioxidant activity. Using conventional and

electrochemical techniques, coccinea root extract (ICME) and fractions (ICPEF, ICCFF,

and ICEAF) were studied. All I have antimicrobial properties. samples of cocaine roots

were tested against 4 bacterial strains as well (Muhammad, 2020).

As stated in the study entitled "Antibacterial Activity of Ixora coccinea against

Selected Bacterial Pathogens Isolated from Clinical Samples" conducted in 2022 by

Benerjee. Overuse of several antibacterial medications typically results in the development

of drug-resistant bacterial strains, which makes it difficult to control the spread of

infectious diseases and even causes side effects (Tomasz & Tomin, 1986). Due to the

abundance of organic chemicals and secondary metabolites found in plants, medicinal

plants have been employed in Ayurveda for centuries to treat a variety of diseases. These

serve as powerful bioactive chemicals that can be used as bacteriostatic, bactericidal, and

chemotherapeutic agents (Bohra & Purohit, 1998).

The ge-nus Ixora, one of the low-growing evergreen perennial shrubs found across

Southeast Asia, has more than 400 species. This well-known flowering shrub is a member

of the Rubiaceae family. According to pharmacological studies, the leaves have

antibacterial, antidiarrheal, and antinociceptive properties (Annapurna et al., 2003;

Agashikar et al.,2010; Ratnasooriya et al., 2005). Flowers have anti-inflammatory,

antioxidant, and wound-healing properties (Saha et al., 2008; Udupa et al. 1999).

According to the current investigation, ethanolic and methanolic extracts of flowers and

leaves exhibit better antibacterial activity against clinical samples than aqueous extracts.

According to the study entitled "Antibacterial Activity of Ixora coccinea against

Selected Bacterial Pathogens Isolated from Clinical Samples" conducted in 2022 by

Benerjee.

Ixora coccinea's leaves and flowers were extracted using ethanol and methanol, and

both showed greater antibacterial activity against Staphylococcus aureus than they did

against Pseudomonasaeruginosa. Therefore, according to the results of the current

investigation, these plant parts exhibit more antistaphylococcal than antipseudomonal

activity. The study concludes that this plant may be utilized to treat pus infections and
wounds brought on by hardy bacteria. Further research into these plants' potential is also

necessary in order to create an alternative therapy for diseases brought on by bacteria

resistant to antibiotics (Benerjee, 2022).

According to the study entitled "Prevalence and characterization of Escherichia coli

isolated from the Upper Oconee Watershed in Northeast Georgia" conducted in 2018 by

Cho.

Water pollution by fecal, pathogenic, or antimicrobial-resistant (AR) bacteria is a

serious environmental and public health concern since surface waters are significant

supplies of water for drinking, industrial, agricultural, and recreational applications. The

abundance of these bacteria in surface water across a watershed is, however, mostly

unknown. In this work, Escherichia coli was characterized for possible pathogenicity and

AR in the Upper Oconee Watershed, a mixed-use watershed in Athens, Georgia, USA. E.

Colony counts were used to count the number of e. coli, enrichment and direct plating were

used to culture the coli, and phylogroups, diarrheagenic pathotypes, and antibiotic

susceptibility were used to describe the e. coli. 99.3% (455/458) of the total samples tested

positive for E, according to the analysis. coli that produced 496 isolates. E.

As stated in the study entitled "Prevalence and characterization of Escherichia coli

isolated from the Upper Oconee Watershed in Northeast Georgia" conducted in 2018 by

Cho. The United States Environmental Protection Agency (U.S. EPA) criterion for

recreational water (235 CFU/100 ml based on a one-time sample) was exceeded by coli

levels as high as 1.2104 CFU/100 ml. The most common phylogroups among the isolates

were B1 (30.8%; 153/496) and B2 (31.7%; 157/496). pathogenic enteric E. E. coli (EPEC)

(19/496) and E. coli that produce Shiga toxin. Only one pathotype known to cause diarrhea,
E. coli (STEC) (1/496), was found. 6.9% (34/496) of the isolates had AR, with 15 of them

being multidrug-resistant (MDR; resistance to two or more classes of antimicrobials).

Ampicillin (32.4%; 11/34), streptomycin (23.5%; 8/34), sulfisoxazole (23.5%; 8/34), and

nalidixic acid (14.7%; 5/34) were the next most often discovered drugs to show resistance,

with tetracycline resistance being the most common (76.5%; 26/34). The study’s findings

revealed that E. The Upper Oconee Watershed has high levels of coli, which may be

indicative of widespread fecal contamination. the existence of AR E that is pathogenic. The

presence of coli in the watershed suggests that ambient water can operate as a reservoir for

resistant bacteria that can be consumed by humans through recreational activities and

drinking (Cho, 2018).

Based on the study "Escherichia coli as a Multifaceted Pathogenic and Versatile

Bacterium" conducted in 2020 by Braz. Genetic plasticity promotes evolution and a wide

range of diversity in Escherichia coli strains ranging from avirulent to highly pathogenic,

including the emergence of virulent hybrid microorganisms. This ability also aids in the

development of antimicrobial resistance. These pathogenic hybrid E. coli (HyPEC),

aggregative adherent bacteria with the potent Shiga-toxin, are emerging threats, such as

O104:H4 from the 2011 European outbreak. We briefly went over the details of these E.

coli classic and hybrid pathogens, the rise in antimicrobial resistance in the context of a

genetically empowered multifaceted and versatile bug, and the growing need for alternative

therapies to combat these infections.

As indicated in the study entitled "Escherichia coli as a Multifaceted Pathogenic

and Versatile Bacterium" conducted in 2020 by Braz. Escherichia coli, also known as E.

coli, is a Gram-negative, adaptable bacterium that is simple to find and susceptible to a

random and natural genetic mutation. Sequenced E is available in large quantities. coli

genomes that show a wide variety within the same bacterial species, including varied sizes

and genetic diversity among commensals and pathogens. They are made up of non-

pathogenic bacteria that can function as commensals and are a normal component of the

gut microbiota of many animals and humans. The facultative bacteria can behave as

opportunistic pathogens and cause various extraintestinal diseases when they are present

outside of their native habitat in the intestinal system. On the other hand, intestine-

obligatory pathogenic variations can result in deadly infections under a variety of

circumstances, from mild diarrhea to more serious instances (Kaper et al., 2004; Köhler

and Dobrindt, 2011).

Synthesis

In the study of Hilario (2015), street food vendors lacked training in handling and

serving food and were unaware of hygienic practices. And in the study of Aruna and Rajan

(2017) vendors who prepare street foods need to enroll in classes on hygienic preparation

techniques right away.

The researchers believe that bad hygienic practices of vendors is a factor that

contribute to the presence of E. coli and other bacteria of street foods.

According to Baliga and Kurian (2012), ixora coccinea Lynn. (Syn. I. grandiflora),

sometimes called the flame of wood, jungle geranium, and Vethi, is a stunning shrub with

a number of therapeutic benefits. It has a wide range of biological and therapeutic effects,

including hepatoprotective. From the study of Muhammad (2020), all I have antimicrobial

properties samples of coccinea roots were tested against 4 bacterial strains as well.
Additionally, assessed by HPLC were the composition of specific phenolic compounds and

the content of polyphenolic classes. And in another study of Annapurna et al. (2003);

Agashikar et al. (2010); and Ratnasooriya et al. (2005), pharmacological studies, the leaves

have antibacterial, antidiarrheal, and antinociceptive properties. Lastly, in the study of

Benerjee (2020), ixora coccinea's leaves and flowers were extracted using ethanol and

methanol, and both showed greater antibacterial activity against Staphylococcus aureus

than they did against Pseudomonasaeruginosa. Therefore, according to the results of the

current investigation, these plant parts exhibit more antistaphylococcal than

antipseudomonal activity. The study concludes that this plant may be utilized to treat pus

infections and wounds brought on by hardy bacteria. Further research into these plants'

potential is also necessary in order to create an alternative therapy for diseases brought on

by bacteria resistant to antibiotics (Benerjee, 2022).

The researchers conclude that santan has many therapeutic benefits; the leaves and

flowers showed great antibacterial activity that can be utilized to treat infections bought by

bacteria and an alternative therapy for diseases bought by bacteria.

 CHAPTER 3

METHODOLOGY

Research Design

This study will use a quantitative form of research. Quasi-experimental research

will be used by the researcher as their research design in this research project. Quasi-

experimental research goal is to demonstrate the cause-and-effect between an independent

and dependent variable, and does not rely on random assignment.

Research Locale

The extraction and conduction of the research study will be conducted in the school

of Luis Palad Integrated High School located in the City of Tayabas in Quezon Province.

It is reachable and convenient for the researchers to conduct in Tayabas City, since this is

where the researchers reside. It is also advantageous for the researchers to conduct in

Tayabas City because it will not be difficult for them to conduct the research since the

researchers are familiar with the places in Tayabas City.

The detection of Escherichia coli bacteria and the determination of the antibacterial

content of red santan (Ixora coccinea) leaves extract will be brought in the laboratory of

UP Los Baños located in the towns of Los Baños and Bay in the Province of Laguna.
Data Gathering Procedure

Phase 1: Gathering Equipment and Materials

The researcher will gather the 1000 grams of dried red santan (Ixora coccinea)

leaves, raw or uncooked 4 sticks per street food samples, and 650 ml of 95% ethyl alcohol.

The equipment is 4 beakers, knife/blender, and 4 containers, the equipment will be clean

with warm water to ensure the cleanliness and safety.

Phase 2: Safety Procedures in Extraction and Conduction

The researchers will follow the safety protocols in using the laboratory and in

extracting the red santan (Ixora coccinea) leaves. The researchers will also ensure that no

accident will occur during the leaf’s extraction and in the conduction of the study. The

researchers will wear Personal Protective Equipment (PPE) such as laboratory gown, face

mask, and surgical gloves in extracting and conducting the study. After that, the researchers

will clean the laboratory and all of the equipment that is used will also be cleaned.

Phase 3: Preparation of Red Santan (Ixora coccinea) Leaves Extract

The collected red santan (Ixora coccinea) must be dried and it will be chopped

using a knife or grounded using a blender into smaller pieces. In extracting the red santan

(Ixora coccinea) leaves, the 1000 grams of dried red santan leaves will be held in a beaker

together with the 650 ml of 95% ethanol alcohol and let it soak for 24 hours.

The Whatman filter paper 1 will be use to filter all of the mixtures. By using a rotary

evaporator and temperatures below 50°C, it will concentrate to a volume of about 10 ml of

the result filtrates. The researchers will record the amount of dried plant material in grams
per milliliter of the produced extracts as their concentration. The researchers will prepare

the plant extracts at 25%, 50%, 75%, and 100% concentrations.

After that, the researchers will label the 4 containers with their plant name, plant

extract concentration, and extract date. The extract of the red santan leaves will be store in

a cold temperature between 0˚ to -5˚ C

Phase 4: Detection of the Escherichia Coli and Antibacterial Content of Red Santan (Ixora

coccinea) Leaves Extract

The researchers will bring the independent variables which are: 25%, 50%, 75%,

and 100% plant extract concentration of red santan (Ixora coccinea) leaves extract to the

laboratory of UP Los Baños to determine the antibacterial contents. For the detection of

the Escherichia coli, it will also bring the samples of raw or uncooked 4 sticks per street

food samples to determine the Escherichia coli bacteria.

Phase 5: Application of the Different Plant Concentration to the Street Food Samples

The different plant extract concentration of red santan (Ixora coccinea) will be

mixed and soaked the 4 sticks per street food in a closed container for 24 hours and labelled

it depending on what street food samples and what plant extract concentration was used.

After 24 hours, it will be put in a styrofoam with ice.

Phase 6: Detection of Escherichia coli After the Conduction

The researchers will bring the 4 labelled containers to the laboratory of UP Los

Baños for the detection of Escherichia coli of the street food samples.
Cost-Efficiency

While creating the antibacterial solution for the antibacterial activity the vendors

will come across an expense which is relatively cheaper than a product made by machines.

The cost efficiency of the solution revolves around less than 200 pesos depends on the size

of usage. Estimated cost of santan (Ixora coccinea) is free as santan plants are mostly found

on a public garden, the researchers conclude it as a non-costing ingredient. The next

component is the food grade alcohol, the researchers conducted many researches to

estimate the range of a food grade alcohol, estimated price of the alcohol is 150 to 200 PHP

which comes on a large size which can occupy a large use.

Test

This research study will use 4 different tests in order for the researchers to get the

specific data that is needed in the study and know the significant differences between our

santan (Ixora coccinea) leaves extract and the following street food samples. The

researcher will use the following tests:

Chemical test. The researcher will use chemical test as it will determine the antibacterial

content of red santan (Ixora coccinea) leaves extract.

PCR-based detection of Escherichia coli. The researcher will use PCR-based detection

of Escherichia coli (per sample) on various food samples of UP Los Baños' BIOTECH

Laboratory in order to efficiently detect the presence of the E. coli bacteria before and after

the appliance of the various concentrations of santan leaf extract on the street food samples.
Kirby-Bauer test. The researchers will also make use of the Kirby-Bauer test in order to

test the resistance of pathogenic bacteria to the different concentrations of the santan leaf

extract. Specifically, it will determine the effectivity of the red santan (Ixora coccinea)

leaves extract to the Escherichia coli of the street food samples

ANOVA test. The researcher will also use ANOVA test to analyze the difference between

the means of more than two groups, Specifically, this will determine the significance

difference of the different plant extract concentration and the significance difference of

before and after applying the red santan (Ixora coccinea) leaves extract to the Escherichia

coli bacteria of the street food samples.

Statistical Treatment of Data

ANOVA or Analysis of Variance will be used to determine significant differences

before and after applying the different plant extract concentration of santan (Ixora

Coccinea) leaves extract to the Escherichia coli activity of street food samples in terms of:

isaw-isaw, betamax, and chicken barbeque.

Where:

F = ANOVA coefficient

MST = Mean sum of squares due to treatment

MSE = Mean sum of squares due to treatment

 In addition, the researchers would also likely to determined the best plant extract

concentration as an antibacterial activity against Escherichia coli of street food samples.

Hence, ANOVA or Analysis of Variance will be used to determine significant differences

of the different santan (Ixora coccinea) plant extract concentration in terms of its

effectivity.

Where:

F = ANOVA coefficient

MST = Mean sum of squares due to treatment

MSE = Mean sum of squares due to error

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 CHAPTER 4

PRESENTATION, ANALYSIS AND INTERPRETATION OF DATA

 CHAPTER 5

SUMMARY, CONCLUSION AND RECOMMENDATIONS

Summary of Findings

Conclusions

Recommendations