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INHIBITION OF ESCHERICHIA COLI USING THE ETHANOLIC EXTRACT OF ELEUSINE INDICA IN

TREATING URINARY TRACT INFECTION

Inhibition of Escherichia coli using the Ethanolic Extract of EleusineIndica in Treating

Urinary Tract Infection

Cruz, Jericho G.

Moreno, Cyryll Josh C.

Mangiliman, Fae Nicole D.

Pinto, Elijah Kate C.

Marcelo H. del Pilar National High School

February 2019
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INHIBITION OF ESCHERICHIA COLI USING THE ETHANOLIC EXTRACT OF ELEUSINE INDICA IN
TREATING URINARY TRACT INFECTION

Introduction

Eleusineindica is considered as an agricultural and environment weed mostly

found in at least 42 countries. In the Philippines it is widely known as its local name as

“Paragis”. This plant is also reasoned to be called as “serious weed” because of the

properties that it contains. Many agricultural experts call this as a dominant weed due to

the fact that it grows rapidly and produces abundant seedlings (Randall, 2012). E. indica

invades habitats in natural areas and at the edges of natural forests, grasslands, marshes,

stream banks and coastal areas. These weeds are also visible along roads, pavements, and

power line corridors. Paragis is an annual, erect, tufted, adventitious, glabrous grass, 10

centimeters to 1 meter in height. Leaves are 10 to 30 centimeters long, sometimes

involute when dry, 3 to 7 millimeters wide, distichous, rather flaccid, with flattened

sheaths. As of today, E. indica is listed as invasive in several countries in Europe, Asia,

Central and South America, the Caribbean and on many islands in the Pacific Ocean.

Eleusineindica has been proved to be diuretic, antihelmintic, diaphoretic, febrifuge.

Studies also have shown anti-inflammatory, antiplasmodial, antidiabetic,

phytoremediative, pancreatic lipase inhibitory, antioxidant, antileishmanial, antioxidant,

cytotoxic, anticonvulsant, antibacterial properties that can be tested on this plant.

The use of natural plant extracts as an antibacterial treatment for humans and

livestock has long been practiced especially in the Philippines where some rural areas

still relies heavily on using alternative and unconventional medicine. The popularity of

using Paragislabelled as a “miracle grass” of many arose, due to its properties that seem
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INHIBITION OF ESCHERICHIA COLI USING THE ETHANOLIC EXTRACT OF ELEUSINE INDICA IN
TREATING URINARY TRACT INFECTION

beneficial for the human and animal health including having Antibacterial, Anti-diabetic,

Antiplasmodial, and Anti-inflammatory properties that can be able to treat and serve as a

counter part for commercially produced drugs.

Escherichia coli is a gram-negative bacteria and mostly found in the lower intestine of

warm-blooded organisms. Some serotypes of the E. coli can cause serious food poisoning

in their hosts and are occasionally responsible for product recalls due to food

contamination. In 1999, the estimated people that are infected by E. coli are 73,

000.Escherichia coli can be from cows, meats and mostly in waters, also from a milk that

is not pasteurized. The bacteria can be possibly get from food (69%), water (18%),

animal (8%) and from another person (6%) (Clark, 2014). Bloody diarrhea, stomach

cramps, nauseas and vomiting can be experienced with people who are infected with E.

coli. One severe complication associated with E. coli infection is hemolytic uremic

syndrome (HUS). The infection produces toxic substances that destroy red blood

cells,and initially cause infection in the urinary tract leading to kidney injury. HUS can

require intensive care, kidney dialysis, and transfusions.

Escherichia coli is a type of bacteria that normally live in the intestines of people and

animals. However, some types of Escherichia coli, particularly Shiga toxin-producing E.

coli (STEC), can cause severe kidneyinfection. Symptoms of the infection include

diarrhea, abdominal pain, and fever. More severe cases can lead to bloody diarrhea,

dehydration, or even kidney failure. People with weakened immune systems, pregnant

women, young children, and older adults are at increased risk for developing these
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INHIBITION OF ESCHERICHIA COLI USING THE ETHANOLIC EXTRACT OF ELEUSINE INDICA IN
TREATING URINARY TRACT INFECTION

complications. This bacteria has a record of 73, 000 records in the whole world per year,

it also has a record of almost 10, 000 cases and 50 deaths per year (Rangel et al., 2010).

About 8 percent of those who are infected develop hemolytic uremic syndrome

(HUS), a condition in which the red blood cells are damaged. This can lead to kidney

failure, which can be life-threatening, especially for children and the elderly. HUS

generally begins about five to 10 days after the onset of diarrhea (Hopkins, 2013).

Escherichia coli can be obtain from contaminated water and contaminated foods or the

surface water (creek, stream, or irrigation) may be contaminated with Escherichia coli

and the most common foods that can contain harmful.

Escherichia coli are raw, undercooked, or ground beef, pork, lamb and poultry.

Unwashed fruits and vegetables such as lettuce or fallen apples can become contaminated

with Escherichia coli bacteria through soil infected with manure or contaminated

irrigation water. One of the reasons is poor hygiene, person may not wash their hands

thoroughly after using the toilet (and coming into contact with Escherichia coli

contaminated feces). If someone contacts the transferred bacteria, then touches their

mouth, they may become infected.

In a study entitled “In vitro antimicrobial evaluation of whole-plant extracts of

Eleucineindica” (Bassey&Ettebong, 2017) “The Ibibio people of Southern Nigeria have a

great wealth of knowledge of medicinal plants used in the treatment of wounds, skin

infections and various ailments. Eleucineindica is widely used in various disease states,

especially in feverish conditions. This study was aimed at evaluating the in vitro
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INHIBITION OF ESCHERICHIA COLI USING THE ETHANOLIC EXTRACT OF ELEUSINE INDICA IN
TREATING URINARY TRACT INFECTION

antimicrobial potentials of whole-plant extracts of Eleucineindica. Four typed cultures of

bacteria such as Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus,

Bacillus subtilis and one clinical strain of fungi, Candida albicans, were assessed using

agar well diffusion method. The extracts that exhibited antimicrobial activity were then

tested to determine the Minimum Inhibitory Concentration for each bacterial or fungal

sample. The ethyl acetate extract showed the widest zone of inhibition (25.0 mm),

followed by chloroform when tested against Staphilococcusaureus. The ethyl acetate

extract also exhibited broadspectrum antibacterial activity against Pseudomonas

aeruginosa, E. coli and B. subtilis. None of the extracts showed any inhibitory effect

against the fungal strain of Candida albicans. Both chloroform and ethyl acetate extracts

exhibited similar potencies having MIC of 50 mg/ml against E. coli and P. aeruginosa

respectively. The antibacterial activities of this plant may be ascribed, at least in part, to

the presence of phytochemical constituents such as flavonoids, alkaloids and tannins in

the extracts.”

In a study entitled “Eleucineindica Possesses Antioxidant, Antibacterial and

Cytotoxic Properties” (Abdul, 2011) “The use of evidence-based complementary and

alternative medicine is increasing rapidly.Eleucineindica (EI) is traditionally used in

ailments associated with liver and kidneys. The therapeutic benefit of the medicinal

plants is often attributed to their antioxidant properties. Therefore, the aim of this study

was to screen the hexane, dicholoromethane, ethyl acetate (EA) and methanol extracts

(MeTH) of EI for their antioxidant, antibacterial and anti-cancer effects using total

phenolic contents (TPCs) and DPPH, disc diffusion method and MTT cytotoxicity
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INHIBITION OF ESCHERICHIA COLI USING THE ETHANOLIC EXTRACT OF ELEUSINE INDICA IN
TREATING URINARY TRACT INFECTION

assays, respectively. The MeTH was showed to have the highest TPC and scavenging

activity (77.7%) on DPPH assay, followed by EA (64.5%), hexane (47.19%) and DCM

(40.83%) extracts, whereas the MeTH showed no inhibitory effect on all tested bacteria

strains. However, the EA extract exhibited a broad spectrum antibacterial activity against

all tested bacteria except Bacillus subtilis, in which this bacterium was found to be

resistant to all EI extracts. Meanwhile, hexane extract was demonstrated to have a

remarkable antibacterial activity against methicillin resistant Staphylococcus aureus

(MRSA) and Pseudomonas aeruginosa, while the dicholoromethane extract did not

exhibit significant activity against P. aeruginosa. None of the extracts showed significant

cytotoxic activity towards MCF-7, HT-29 and CEM-SS human cancer cell lines after 72?

h incubation time (IC50 > 30?g/ml). These results demonstrate that the extract prepared

from the EI possesses antioxidant activity in vitro in addition to antibacterial properties.

Further investigations are needed to verify the antioxidant effects in vitro and in vivo.”

In a study entitled "Antimicrobial And Anthelmintic Activity Of EleusineIndica”

(Morah, 2015) “Antimicrobial activity of Elusineindica was carried out using Agar well

diffusion method on selected pathogens usually associated with diarrhea or other stomach

problems such as Shigelladysenteriae Escherichia coli, Salmonella typhi, Staphylococcus

aureus, Streptococcus facealis and Lactobacillus lactis. The aqueous extract was the most

susceptible while the ethanol extract was the least susceptible to these microorganisms.

The observed antimicrobial activity is concentration dependent. The work also shows that

the plant has marked anthelmintic activity against Strongyloidesstercoralis. The work
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INHIBITION OF ESCHERICHIA COLI USING THE ETHANOLIC EXTRACT OF ELEUSINE INDICA IN
TREATING URINARY TRACT INFECTION

therefore gives credence to the use of Eleusineindica against diarrhea and worm

infections.”

In a study entitled "A Review on the Extraction Methods Use in Medicinal Plants,

Principle, Strength and Limitation" (Azwanida, 2015) “Medicinal plants are gaining

much interest recently because their use in ethno medicine treating common disease such

as cold, fever and other medicinal claims are now supported with sound scientific

evidences. The study on medicinal plants started with extraction procedures that play a

critical role to the extraction outcomes (e.g. yield and phytochemicals content) and also

to the consequent assays performed. A wide range of technologies with different methods

of extraction is available nowadays. Hence, this review aim to describe and compare the

most commonly used methods based on their principle, strength and limitation to help

evaluating the suitability and economic feasibility of the methods.”

Statement of the Problem

The general problem of this study is the potential effectiveness of Eleusineindica

(Indian Goose Grass) against Escherichia coliin treating Urinary Tract Infection.

Specifically, this study addresses the following questions:

1. What is the population of Escherichia coli for each set upinterms of colony

forming unit?
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INHIBITION OF ESCHERICHIA COLI USING THE ETHANOLIC EXTRACT OF ELEUSINE INDICA IN
TREATING URINARY TRACT INFECTION

2. How will the aqueous solution (Ethanolic Extract) from E. indica will be utilized

in each treatment by different levels of concentration?

3. How will the level of concentration of Indian Goose Grass extract will fight

against the Escherichia coli?

4. What implications can be drawn from the results of the study?

Conceptual Framework

Independent Variable Dependent Variable

Amount of Ethanolic Extract

from Eleusine indica in Escherichia coli inhibited in
terms of Millilitres terms of colony forming unit
(mL) (cfu)

Ho: There is no significant effect on using the aqueous solution (Ethanolic Extract) of

Eluesineindica as an antibacterial treatment on Escherichia coli for treating Urinary Tract

Infection.

Ha: There is a significant effect on using the aqueous solution (Ethanolic Extract) of

Eluesineindica as an antibacterial treatment on Escherichia coli fortreatingUrinary Tract

Infection.

Significance of the Study

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INHIBITION OF ESCHERICHIA COLI USING THE ETHANOLIC EXTRACT OF ELEUSINE INDICA IN
TREATING URINARY TRACT INFECTION

This study aims to inhibit Escherichia coli with the use of Eleusineindica to stop the

growth of the population of the people that are infected with the bacteria.This study

targets to uncover possible treatments and prevent infected people to lost their lives.The

output of this study is an extract that can be use by microbiologist for people that are

infected. The proposed study can also open development of the related study.The findings

of this study will redound to the benefits it could give to the following people or group of

people in the society.

To the consumers of meat - This study could bring a wide scope collection of information

on how this bacteria affects the whole condition not only of the domestic swine or cattle

but also to the whole livestock animals to which the humans consume, unknown to the

possible effects of the transmission of this bacteria to the human body. The findings of

this study can bring awareness to the whole community to be knowledgeable and having

the basic knowledge on this type of bacteria.

For the field of medicine – This study could bring a leap to the current studies

regarding to the antibacterial potential use of Eleusineindica against E. coli. The findings

of this study could gain new information for the much reliable use of unconventional

medicine such as the use of Eleusineindica and to innovate new approaches on how to

deal with this type of bacteria residing inside an organism.

To the future researchers – The study can be beneficial to the future study of

proving the antibacterial activity of Eleusineindica in other type of bacteria that

commonly affects the condition and the health of the organism to which it can be found.
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INHIBITION OF ESCHERICHIA COLI USING THE ETHANOLIC EXTRACT OF ELEUSINE INDICA IN
TREATING URINARY TRACT INFECTION

The more efficient the information and the result of this study will provide the more

effective result it could give to the future studies to be conducted. Thus, the accuracy and

the precision of the results in this study will give a huge impact not only to the consumers

but also to the field of medicine in which gradually evolving through newly published

research.

Scope and Delimitation

The study focuses on the antibacterialc activity of Eleusineindica against

Escherichia coli. The coverage of the study is to test the antibacterial activity of

Escherichia coli only. The actual study was held in the year 2019-2020. The time frame

for the study is one month, December to January. The study will be beneficial since this

problem hampers cure. The study were done in one residence in Bulacan and the paragis

(Eleusineindica) were collected in Calumpit, Bulacan while the Escherichia coli were

cultured at the laboratory. The researchers examined the effects of different ratio of

Eleusineindica’s extract to E. coli. The researchers assigned 4 experimental set ups and

with a total of 5 replicates for each experimental set up, 5 5 for the 25% concentration, 5

for the 50% concentration, 5 for the 75% concentration and 5 for the 100% concentration.

The experimentation takes place in a laboratory to observe the progress of the study.

This study do not cover the other genus or family of Eleusine, only the specie

ofEleusineindica since the researchers wants to focus on the not that well known grass

that can be found in the Philippines. Moreover, the researchers only tests and observedthe

antibacterial activity of Eleusineindica and not the other properties that it possesses.
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INHIBITION OF ESCHERICHIA COLI USING THE ETHANOLIC EXTRACT OF ELEUSINE INDICA IN
TREATING URINARY TRACT INFECTION

Definition of Terms

Anthelmintic Activity - expelling or destroying parasitic worms and other internal

parasites from the body

Ethanolic Extract- relating to, containing, or derived from ethyl alcohol Indian Goose

Grass- is a species of grass in the family Poaceae. It is a small annual grass distributed

throughout the warmer areas of the world to about 50 degrees latitude. It is an invasive

species in some areas

Escherichia coli- also known as E. coli, is a Gram-negative, facultative anaerobic, rod-

shaped, coliform bacterium of the genus Escherichia that is commonly found in the lower

intestine of warm-blooded organisms

Trypticase soy agar or tryptone soya agar- growth media for the culturing of bacteria.

They are general-purpose, nonselective media providing enough nutrients to allow for a

wide variety of microorganisms to grow.

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INHIBITION OF ESCHERICHIA COLI USING THE ETHANOLIC EXTRACT OF ELEUSINE INDICA IN
TREATING URINARY TRACT INFECTION

Methods

Type of Research

The researchers utilized experimental research design in conducting the study; it is

defined as a systematic and scientific approach to research in which the researcher

manipulates one or more variables, and controls and measures any change in other

variables. (2013, Explorable) It is also defined as how the participants are allocated to the

different levels of conditions (or IV levels) in an experiment. (2017, Mcleod) An

experimental research design determines the relationship between the factors and the

variables affecting the process of the experimentation. Moreover, the researchers thought

that it is necessary to use the experimental research design since the study will focus on

determining if the antibacterial property of the Ethanolic extract from Eleusineindica

shows an effectivity against Escherichia coli.

Sample and Sampling Method

The Escherichia coli were gathered by culturing the said bacteria in the laboratory, the

streaking of the bacteriaand the incubation of the Tryptic Soy Agar with the bacteria. The

researchers prepared a total of 4 experimental set ups having 6 replicates for each set up.

The researchers used four different levels of concentration for the application of

Ethanolic extract, 25, 50, 75 and 100 level concentration for the experimental set up.
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INHIBITION OF ESCHERICHIA COLI USING THE ETHANOLIC EXTRACT OF ELEUSINE INDICA IN
TREATING URINARY TRACT INFECTION

Also, the researchers utilized clustered sampling method in culturing the E. coli to be

utilize in the study; In a clustered sample, subgroups of the population are used as the

sampling unit, rather than individuals. The population is divided into subgroups, known

as clusters, which are randomly selected to be included in the study (2016, Bhat). The

researchers used cluster sampling method in order to show the bacteria that will be

cultured on each replicates.

Data Collection Procedure

The researchers gatheredEleusineindica (Indian Goose Grass) from Malolos,

Bulacan. The collected grass were properly washed and separated from rocks or other dirt

and then dried for fourteen (14) days. Then, the dried grass were cut into small pieces for

it to undergo the maceration process for the extraction. During this process the

researchers used 95% ethanol were the grass soaked for three (3) days. Then the product

of the maceration process are kept in a cold place until the experimentation has begun.

The researchers then proceeded in the streaking process for culturing the

Escherichia coli. The researchers placedTryptic Soy Agar on the petri dishes and then

conducted the streaking process. After finishing the process on all of the replicates, the

researchers incubated it for 24 hours. The researchers then proceeded with the Kirby

Bauer Test by swabbing the bacteria onto the petri dish with Agar. Then, the researchers

place the disks in the middle of the agar where the extracts were placed afterwards. The

dispensing method were continued for the extracts containing different levels of

concentration were dispensed on the disks and this is where the zone of inhibition will
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INHIBITION OF ESCHERICHIA COLI USING THE ETHANOLIC EXTRACT OF ELEUSINE INDICA IN
TREATING URINARY TRACT INFECTION

exhibit. After doing it in all replicates, the replicates were incubated for 24 hours. After

the incubation of the replicates, the researchers checked the zone of inhibition which

showed the antibacterial property of the Ethanolic extract from Eleusineindica shows an

effectivity against Escherichia coli.

Statistical Analysis

The researchers utilized T-test in the statistical analyzation of the data. T-test is defined

as one type of inferential statistics. It is used to determine whether there is a significant

difference between the means of two groups. (2016, UCONN) T-test as the statistical test

in the research study could determine the significant difference between the applied level

of concentration for the samples 25%, 50%, 75% and 100% level of concentration to the

Escherichia coli. Moreover, the researcher thought that it is necessary to use T-test since

the number of replicates of the study is below 30.

In order to present and analyze the data to be collected in this study, the following

statistical tools will be used:

a. Table. To be able to present the progress of the elimination of Escherichia coli (in

terms of the zone of inhibition). Also, to show and compare the results of the

experimental set ups.

c. Weighted Mean. To be able to describe what concentration is more recommended

to use for the elimination Escherichia coli..

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INHIBITION OF ESCHERICHIA COLI USING THE ETHANOLIC EXTRACT OF ELEUSINE INDICA IN
TREATING URINARY TRACT INFECTION

d. T-test. To be able to see the difference between the sample means of the results on

the elimination of Escherichia coli (in terms of the zone of inhibition).