Research Title - i

TITLE PAGE
ANTI-FUNGAL EFFECT OF ELEUSINE INDICA (GOOSE GRASS) EXTRACT
AGAINST TRICOPHYTON MENTAGROPHYTES

A Research Paper Presented to the Faculty

Of Science, Technology, Engineering and Mathematics

LAGRO HIGH SCHOOL

Quezon City, Metro Manila

In Partial Fulfillment of the Requirements for

Inquiries, Investigation and Immersion

By:

CEREZO, VICTORIANO JR.

RAFANAN, ANGELA

SANTOS, MAIRY RHOSE

MARCH 2020

MA. KRISTINE E. TAMAYO

ADVISER
 Research Title - ii

APPROVAL SHEET

This Research Paper entitled “ANTI-FUNGAL EFFECT OF ELEUSINE INDICA

(GOOSE GRASS) EXTRACT AGAINST TRICOPHYTON
MENTAGROPHYTES”, prepared and submitted by CEREZO, VICTORIANO JR.,
RAFANAN, ANGELA, SANTOS, MAIRY RHOSE in partial fulfilment of the
requirements for Inquiries, Investigations and Immersion, has been examined and
recommended for Research Presentation.

_________________________

Adviser

Approved in partial fulfilment of the requirements for Inquiries, Investigations

and immersion by the Research Committee.

_____________________________
Panel Chairman

_________________________ _________________________
Panel Member Panel Member

_____________________________
Panel Member

Accepted in partial fulfilment of the requirements for Science, Technology,

Engineering, and Mathematics Strand in Inquiries, Investigations and Immersion.

DENNIS M. ANGELES, Ed.D.

Research Director
 Research Title - iii

ABSTRACT

Athlete’s Foot an infection where 15% of the World’s Population suffered from,

Athlete’s foot caused itch and rash to the affected areas and this is due the long exposure

of the feet to the shoes., that caused the fungi to inhibit the said area. It is usually occured

between the toes and this has cause uncomfortable feelings such as itch and rash. This can

be treated by anti-fungal medicines and also traditional remedy. Traditionn al Remedies

explored in the study is the Eleusine indica. This type of grass can be found everywhere has

prove itself to contain numerous phytochemicals that can actually treat various diseases

namely urinary bladder infections and also it showcased anti-microbial property. The clean

and grounded Eleusine indica (30 grams) were boiled on a clean pot with 250 ml of distilled

water in order to get the aqueous substance. And another 30 grams of the said variable

were soaked in 250 grams of Ethyl acetate for 24 hours in order to get the ethyl acetate

substance. The aquaeous substance undergo the phytochemical analysis and the ethyl

acetate substance undergo the Kirby-Bauer Diffusion Method. The Phytochemical Analysis

showed a positive result for the flavonnoid that was a property of an anti-fungal and the

Kirby-Bauer Diffusion Method indicated that the Ethyl Acetate extract showcase an AI of

>4.5 and 1% Clotrimazole.

Key Concepts: Eleusine indica, Anti-fungal, Kirby-Bauer Diffusion Disc, Ethyl

Acetate, Dilution
 Research Title - iv

ACKNOWLEDGEMENT

We greatly thank and appreciate the efforts of the people who gave their time

and attention in making this research possible.

To Mrs. Ma. Kristine Tamayo, our research teacher adviser for the supervision

of the research project.

To all Lagro High School (LHS) teachers, parents, classmates, relatives, our

sincerest and deepest gratitude for your considerations, understanding and support.

Most of all, to our Almighty God, who never failed in guiding us through our

path and journey in making this research succeeded, for looking after us and this

research, we offer Him our effort in doing this project.

 Research Title - v

DEDICATION

The researchers dedicated this study;

To the future researchers who will conduct related study about Eleusine
indica.

To the Laboratories who wanted to conduct a study relating Eleusine indica’s

anti-fungal effect toward Tricophyton mentagrophytes.

To the Pharmaceutical and Medicinal Industry who wanted to use this study
for the production of an anti-fungal product using Eleusine indica, as an alterative
course.

Lastly, to the researcher’s Alma Matter, its staff, and teachers who worked
hard and helped for the possibilities of this research to came with the best results.
 Research Title - vi

RESEARCH PLAN

Researcher/s : CEREZO, VICTORIANO JR., RAFANAN, ANGELA,

SANTOS MAIRY RHOSE

Title of Research : ANTI-FUNGAL EFFECT OF ELEUSINE INDICA

(GOOSE GRASS) EXTRACT AGAINST
TRICOPHYTON MENTAGROPHYTES

Strand : SCIENCE & TECHNOLOGY, ENGINEERING AND

MATHEMATICS

Institution : LAGRO HIGH SCHOOL

Adviser : MA. KRISTINE E. TAMAYO

RATIONALE: Infections such as Athlete’s foot can be somehow so irritating that

it doesn’t have a specific treatment but our industry only possess prevention to it.

However, this study would prove the feasibility of the Eleusine indica as an

alternative source of Anti-Fungal in order to contribute to the production of the Anti-

Fungal Products in the Pharmaceutical and Medicinal Industry.

STATEMENT OF THE PROBLEM:

The study aims to determine the following;

 The significant effect of Eleusine indica extract towards the Trichophyton

mentagrophytes.

 Determining the essential antifungal properties of Eleusine indica that can

mitigate the production of the Trichophyton mentagrophytes

 Research Title - vii

 The efficiency of Eleusine indica extract as an alternative source of anti-

fungal.

RESEARCH HYPHOTHESIS:

H 0: Eleusine indica has no Anti-Fungal Property and doesn’t have any effect towards

Trichophyton Mentagrophytes

H 1: Eleusine indica has high Anti-Fungal Property and does have any effect towards

Tricophyton Mentagrophytes

METHODS/PROCEDURES:

The Eleusine indica that was gathered by the researchers were subjected into

numerous tests. The aqueous extract of Eleusine indica undergo Phytochemical

Analysis to identify the chemical compounds that it possess. The plant that are soaked

in the Ethyl Acetate were tested using Kirby Bauer Disc Diffusion Method to identify

its Antifungal Index and its effectiveness towards the representative fungi.

SUMMARY OF FINDINGS:

Summary

The study investigated the chemical constituents of the Eleusine indica (Goose

grass) and its antifungal potency against the dermatophytes, Trichophyton

mentagrophytes.

The Eleusine indica were collected from the near empty and grassy lot behind

the SM City Fairview at Novaliches, Quezon City and are washed under running
 Research Title - viii

water until mud and soil are washed out. Goose grass were cut in the size of 30mm or

3cm and are baked inside the oven at 40˚in 3 hours. The plant are then granulated into

a fine powder, thereafter the 30 g of the powder sample are soaked in the 250 ml ethyl

acetate for 24 hours. A mixture that was accumulated was filtered through filter paper

and concentrated to a final volume of 50 ml.

The Eleusine indica extract was delivered by the researchers to Natural

Sciences Research Institute at UP Diliman, Quezon City and the crude plant was

delivered to UP Manila. Phytochemical Analysis and In Vitro Antifungal Assay were

performed. It was then found out that the E. indica have chemical constituent that are

needed in performing antifungal assay which are the Flavonoids for a high antifungal

activity. The researchers also found out that Eleusine indica (Goose grass) extract

showed the determination of zone inhibition represented by the E, indica extract

against T. mentagrophytes of >55mm with an AI of >4.5 and contains 1%

Clotrimazole.

Findings

After conducting the study, the researchers found out that:

1. The Eleusine indica has the presence of Flavonoids that are required to

produce antifungal, anti-allergic, anti-inflammatory, antioxidant and

antimicrobial activities.

2. The Eleusine indica extract has a high antifungal effect and reactivity towards

the dermatophyte, Trichophyton mentagrophytes.

3. The Eleusine indica can be an alternative source of fungicide.

 Research Title - ix

RECOMMENDATIONS:

The researchers recommend searching for more information or more related

literatures about this study. They also recommend performing more assays to know

the other things this plant extract can perform or research for another plant that

contains the same constituents like the Goose grass and perform the same procedure.

The researchers lastly recommend future researchers to use other types of

dermatophytes to have another discovery.

 Research Title - x

TABLE OF CONTENTS

Table of Contents
TITLE PAGE.............................................................................................................................i
APPROVAL SHEET................................................................................................................ii
ABSTRACT............................................................................................................................iii
ACKNOWLEDGEMENT.......................................................................................................iv
DEDICATION..........................................................................................................................v
RESEARCH PLAN.................................................................................................................vi
TABLE OF CONTENTS.......................................................................................................viii
CHAPTER I.............................................................................................................................1
THE PROBLEM AND ITS BACKGROUND.........................................................................1
1.1 INTRODUCTION.........................................................................................................1
1.2 BACKGROUND OF THE STUDY...................................................................................1
1.3 REVIEW OF RELATED LITERATURE/STUDIES..............................................................1
1.4 CONCEPTUAL/THEORETICAL FRAMEWORK..............................................................1
1.5 OBJECTIVES...............................................................................................................1
1.6 STATEMENT OF THE PROBLEM.................................................................................1
1.7 SIGNIFICANCE OF THE STUDY....................................................................................1
1.8 SCOPE AND LIMITATION...........................................................................................1
1.9 DEFINITION OF TERMS..............................................................................................1
CHAPTER II............................................................................................................................2
METHODOLOGY...................................................................................................................2
2.1 RESEARCH DESIGN....................................................................................................2
2.2 RESEARCH VARIABLES...............................................................................................2
2.3 RESEARCH INSTRUMENTS.........................................................................................2
2.4 DATA GATHERING PROCEDURE................................................................................2
2.5 DATA ANALYSIS.........................................................................................................2
CHAPTER III...........................................................................................................................3
RESULTS AND DISCUSSION...............................................................................................3
CHAPTER IV...........................................................................................................................5
SUMMARY OF FINDINGS, CONCLUSIONS AND RECOMMENDATIONS....................5
 Research Title - xi

4.1 SUMMARY OF FINDINGS.................................................................................................5

4.2 CONCLUSIONS.................................................................................................................5
4.3 RECOMMENDATIONS.....................................................................................................5
BIBLIOGRAPHY....................................................................................................................6
APPENDICES..........................................................................................................................7
CURRICULUM VITAE.........................................................................................................33
 Research Title - 1

CHAPTER I

THE PROBLEM AND ITS BACKGROUND

This chapter presents the background of the study, review of related literature

and related studies, framework of the study, statement of the problems, scope and

limitation of the study, significance of the study, and definitions of terms.

1.1 BACKGROUND OF THE STUDY

Skin infections in one of the common problems around the world especially

for those people who encounters direct contact to many things in their everyday lives.

This direct contact may cause fungi passed through one individual to another. These

fungi are commonly called Dermatophytes. Dermatophytes are fungi that inhabits in

human and animals also can be directly spread through skin to skin contact from other

people. This kind of fungi require keratin for growth and causes various skin

conditions such as athlete’s foot, ring worm, nail infections, scalp infection, and beard

infection. (Bainer,2003)

Infections commonly caused by fungi are treated using bio-active

phytochemicals. Based on a research by Rajesh Verma (2003), “Plant products

traditionally used as biocides in indigenous culture and are being re-evaluate for

safer means of fungi control over synthetic” According to the results, one of the

medicinal plants that can control fungi is Eleusine indica.

Eleusine indica (goose grass) also known as Paragis grass can be found almost

everywhere and have proven itself as traditional treatment and remedy for various
 Research Title - 2

diseases relating to urinary bladder such as cystitis, gallstone, bladder and kidney

problems and minor cuts and wounds on the skin. (Dyer, 2018) Goose grass’ roots

serves as depurative or purifying and detoxifying effects and diuretic used in order

to increase the water and sodium that was eliminated from the body in the form of

urine, laxative that helps in stimulating the evacuation of bowels, febrifuge that

reduce fever, also for the treatment of influenza, high blood pressure, and urinary

problems. (Al-Zubairi et.al.,2011) E. indica is one of the primary listed as an

agricultural and environmental weed (Randall, 2012) and 42 countries were

considered that E. Indica as a “serious weed”. According to (Verma, 2003), Eleusine

indica showcase a 7’mm antifungal potency after being dropped on a disk consisting

of fungi. The use of Eleusine indica as an alternative source of anti-fungal can be one

of the best ways to mitigate the growing concern of humanity through the spreading

of Dermatophytes that leads to common skin problems. This study aims to provide a

cheaper and effective anti-fungal product through the usage of common grass located

from everywhere.

1.2 REVIEW OF RELATED LITERATURE/STUDIES

Eleusine indica

One of the best sources of potent drugs are plants and as human population

face diseases and health problems, discovery of new drugs from plants are increasing.

These living things often give people their essential needs and this is one of the

creator’s best works. Plants have many uses, they give people clean oxygen for

breathing, give them foods when they are hungry, give them nutrients that their body

needs in order to proceed to their daily activities in life. However, it’s just the basic
 Research Title - 3

materials that plants give to people. It also provides people traditional medicines that

are considered a natural and organic way of treatment in the past and even in the

present. Plants give people components for production of new drugs and vaccines.

However, some researchers are focused on a specific plant that is considered one of

the most troublesome weeds found worldwide as stated by (Mueller et al., 2011;

Takano et al., 2016). And this plant is Eleusine indica commonly known as

Goosegrass or Paragis.

Eleusine indica is a common monocotyledonous plant commonly referred to

as "Goosegrass" and may also be known as "Yard grass". This plant is closely related

to the millets, that include Sorghum bicolor (sorghum or great millet), Zea mays

(maize), Pennisetum americanum (pearl millet), Eleusine coracana (finger millet),

Setaria italica (foxtail millet), Paspalum Scrobicu latum (kodo millet), Echinochloa

frumentacea (barnyhard millet) and Eragrostis tef (teff).

According to Al-zubairic, A. et al. (2011), the Eleusine indica is a traditional

medicine plant that can be used for treating liver, kidney and other disease because of

their antioxidant property. Many countries especially in the Southeast Asia and Africa

consider this as one of their medicinal plants until today since modern treatment can’t

reach places such as isolated and rural areas that are far from the civilization. Studies

shows that this plant also contains certain secondary metabolites with antibacterial

activity (Balangcod et al. 2012). The metabolites include the flavonoids, phenols and

phenolic glycoside, saponins, cyanogenic glycoside, unsaturated lactones, and

glucosinolates. These metabolites are considered a help for those people who have

digestion problems due to its property that helps in metabolism and digestion of
 Research Title - 4

foods. Many of the scientists around the world are discovering the possible

antimicrobial compounds of many plants’ secondary compounds that are known to

have diverse biological activity, these may include the antibacterial, antifungal, and

anticancer.

Antibacterial and Antifungal are some of the properties of E. indica. The

whole plant especially the root is depurative, diuretic, febrifuge, and laxative, which is

used for treatment for influenza, hypertension, oliguria, and urine retention. The

ability of E. indica root extract has anti-urolithiathic property to reduce the number of

calcium oxalate crystals which can decrease kidney retention among treatment

groups.

Phytochemicals

One of the compounds of the plants that are naturally occurring is the

phytochemicals that serves as a defense system. Phytochemicals has protective or

disease preventive properties. Leaves of the plants that belong to the Poaceae Family

such as Eleusine indica were collected and were used for the phytochemical analysis.

The results obtained after the phytochemical screening suggested that Eleusine indica

has a high content of Flavonoids, Glycosides, Lignin, Phenols, Saponins and Steroids

and possess antioxidant, anti-inflammatory and antimicrobial activities (Iberahim, R.,

Yaacob, W. A., & Ibrahim, N., 2015). These Phytochemicals; flavonoids has many

different uses such as anti-allergic, anti-inflammatory, anti-oxidant, anti-cancer, anti-

diarrheal; Glycosides as said to give mitigation to heart diseases; Lignin can be an

alternative bio-based petroleum also has an anti-microbial property; Saponins can be

used for cosmetics purposes and its foaming and antibacterial properties that leads it
 Research Title - 5

to one of the components in the formulation of new vaccines; steroids that are used

for anti-inflammatory that reduces soreness, redness and swelling; Phenols that helps

in disinfecting the skin that reduces and give relief to itching; and terpenoids that has

an anti-fungal properties.

Dermatophytes

Skin is the largest organ of a human body and it also protects the internal

organs along with the bones however it cannot be avoided that this organ can also be

damage by foreign and physical matters and may cause some problems on the skin.

Skin infections such as allergies, rashes, warts, cellulitis, boils, and impetigo are

caused by bacteria that sticks to the skin, while infections such as athlete’s foot, ring

worms, nail infections, scalp infections, and other infections that are related to keratin

is a caused by Fungi invading people’s skin. And these fungi are called

Dermatophytes.

Dermatophytosis is a major challenge in public health in many parts of the

world. Trichophyton is one of the several causes of athlete's foot, itch, ringworms and

other infections that inhabit soil, human body, and animals. (Rogers, K. 2008).

One of the kind of Dermatophytes is Trichophyton mentagrophytes. This kind

of fungi cannot identify the morphological features because it has a complex features.

In line with that T. mentagrophyes can infect the keratinized tissues of the human and

the animal’s origin such as hair, skin and nails. Based on the recent study of Beguin et

al. (2012) using the multilocus phylogenetic analysis they conclude that this fungi is

very complex than the other kind of dermatophyes such as the taxonomic changes that

includes the clinical features, cultural characteristics and conidial morphology. The
 Research Title - 6

clinical features of T. mentagrophytes can cause clinical diseases. The cultural

characteristics of t. mentagrophyptes usually have a deep-yellow submerged fringe

and reverse which later becomes dark red in the center. The conidial morphology of t.

mentagrophytes is a rapid growth and it will become matures within 6 to 10 days. The

t. mentagrophytes can be described as an anthropophilic isolates because of having a

downy, powdery or even fluffy texture, however, this funji also described as a

zoophilic isolated because of more granular in appearance.

This skin infection caused a lot of troubles for those people who will be

affected and are currently affected. These problems can affect on how affected people

live on their daily lives, somehow getting bullied, or colleagues, classmates and

friends might avoid him/her as they realized that theses infections are contagious

through a skin to skin contact. People who possess these skin bacteria often have a

hard time especially when itching, redness, swelling, the nail infections, and also the

odor that people hates the most.

Eleusine indica Anti-fungal Activities against the Trichophyton mentagrophytes

Related study of Eleusine Indica stated that it showcases an anti- microbial

property and this study is closely related that this plant hasan anti-fungal property and

has an effect towards the Trichophyton (Balangcod et al., 2012) due to its great

number of phytochemical components particularly flavonoids that carries anti-

microbial, terpenoids that deals with fungi and phenols in the disinfecting skin

properties.

Skin infection often lead into itching, redness, swelling, and bad odor, and the

component phenols of the Goosegrass can be an addition to the mitigation of the

 Research Title - 7

Dermatophytes invading the skin. The Dermatophyte mentioned above can be

controlled by these components of the Eleusine indica and helps in the elimination of

the itching, redness, swelling and reducing the bad odor coming out of the skin. Also

due to the fact that Dermatophytes are fungi and Eleusine indica was proven to have

terpenoids and terpenoids deals with the fungi that eventually are an anti-fungal

property (Mueller et al., 2011; Takano et al., 2016). Studies of (Lum et.al, 2019)

manifest that Paragis is one of the plants that is most effective against the pathogenic

fungi.

According to a study by (Fatima et al., 2018), the ethyl acetate extract of

Eleusine indica exhibit alkaloids, flavonoids, glycosides, phenols, steroids,

terpenoids, saponins, tannins, anthocyanin and coumarin. The flavonoids and phenols

have inhibitory effects against viruses and bacteria while coumarin and terpenoids are

used against viruses, bacteria, and fungi. Ethyl acetate extract combined with

Eleusine indica proves to be an effective antimicrobial solution (Al-Zubairi et al.,

2011)

Synthesis

Skin infections specifically those infections that are caused by the

dermatophytes had caused the reduction of self-confidence and produce social

discomfort for the people who are affected these infections includes the ring worms,

athlete’s foot, jock itch, nail infections and many more. Previous studies show that

Bio-active phytochemicals such as flavonoids possessed an anti-microbial, anti-

fungal, and anti-bacterial property.(Manharan S. & Kaur, J., 2013) And these Bio-

active phytochemicals can be accumulated and acquired from plants.

 Research Title - 8

Plants such as Eleusine indica which is easy to find and people in need can

easily acquire this and they will never undergo a complicated process when acquiring

an efficient and effective treatment for fungal infection. This plant showcases anti-

fungal properties based on numerous experiments and a study that was published a

year ago (Lum et.al, 2019) that this plant has one of the high numbers of effective

phytochemicals against the fungi. Phytochemicals present in Paragis have different

usage and gives light to the new source of anti-fungal.

Eleusine indica’s related researches brings another light to the possibility of

the production of a new alternative anti-fungal product against common skin

problems such as tinea corporis (ringworm), tinea cruris (jock itch), tinea pedis

(athlete's foot), and tinea unguium (fungal infection of the nail bed).

1.3 CONCEPTUAL/THEORETICAL FRAMEWORK

\ INPUT PROCESS OUTPUT

 Collection of Eleusine  Concentration of  Alternative source of

indica Eleusine indica in antifungal for

 Pathogenic fungi such Ethyl Acetate fungicides

as Trichophyton  Ethyl Acetate  New industry of

mentagrophytes extraction method in fungicide made from a

extracting Eleusine medicinal plant

indica

 Testing of the extract

to the thriving

pathogenic fungi such

as Trichophyton

mentagrophytes
 Research Title - 9

Figure 1: Representation of the Conceptual Framework of the Anti-Fungal Effect of Eleusine indica

(Goosegrass) against Trichophyton mentagrophytes

The researchers gathered Eleusine indica and used it to extract concentration

of it using the Ethyl Acetate as indicated in process of the conceptual framework.

When the extract is made, it was brought to University of the Philippines – Natural

Science Research Institute to test the antifungal activity of the Eleusine indica extract

against the dermatophyte Trichopyton mentagrophyte. The researchers were able to

identify that the concentration exhibits antifungal activity against the said

dermatophytes and can be used as an alternative source of fungicide.

1.4 OBJECTIVES

This study aimed to;

 To Determine the essential antifungal properties of Eleusine indica that

can mitigate the production of the Trichophyton mentagrophytes

 To Determine the significant effect of Eleusine indica extract to the

trzichophyton mentagrophytes in terms of size.

 To Determine the efficiency of Eleusine indica extract as an alternative

source of anti-fungal.
 Research Title - 10

1.5 STATEMENT OF THE PROBLEM

The study aimed to determine the following;

 The significant effect of Eleusine indica extract towards the Trichophyton

mentagrophytes.

 Determining the essential antifungal properties of Eleusine indica that can

mitigate the production of the Trichophyton mentagrophytes

 The efficiency of Eleusine indica extract as an alternative source of anti-

fungal.

1.6 SIGNIFICANCE OF THE STUDY

This study of antifungal activity of Eleusine indica is conducted to benefit the

educators and the students as this study can provide topics for classroom discussion

and serves as reference for the subjects related to the study. It can also provide basis

and guide for future researchers undertaking related studies. The researchers, being

the main participants gained knowledge and experience to improve their skills. The

results showed the effectiveness of the plant against Trichophyton mentagrophytes

and this provided proofs that Eleusine indica can also be recognized as an alternative

source of fungicide.

1.7 SCOPE AND LIMITATION

This study focused on the Anti-Fungal Effect of the Eleusine indica

(Goosegrass) against the Dermatophytes such as Trichophyton, Microsporum and

Epidermophyton. These dermatophytes have cause various skin infections to human.

This study aimed to determine if Eleusine indica can be a new source of anti-fungal.
 Research Title - 11

Researchers may be able to gain information and knowledge about the capability of

Eleusine indica to go against these fungi. Eleusine indica will undergo 2 process and

2 tests before verifying it as another source of Anti-fungal product. Researchers e

unveil their insights on the capability of the Eleusine indica as a new alternative

source of fungicide.

1.8 DEFINITION OF TERMS

Eleusine indica  Eleusine indica is a common monocotyledonous plant commonly

referred to as "Goosegrass" and may also be known as "Yard grass". It can be found

almost everywhere and have proven itself as traditional treatment and remedy for

various diseases relating to urinary bladder such as cystitis, gallstone, bladder and

kidney problems and minor cuts and wounds on the skin.

Anti-Fungal  used to prevent fungal growth; active against fungi.

Fungicide a chemical that destroys fungus

Kirby-Bauer Disk Diffusion The disk diffusion test, or agar diffusion test, or

Kirby–Bauer test, is a test of the antibiotic/antifungal sensitivity of bacteria/fungi. It

uses antibiotic/antifungal discs to test the extent to which bacteria/fungi are affected

by those antibiotics/fungicide.

Dilution A simple dilution is one in which a unit volume of a liquid material of

interest is combined with an appropriate volume of a solvent liquid to achieve the

desired concentration
Research Title - 12
 Research Title - 13

CHAPTER II

METHODOLOGY

This chapter presents the different methods approaches and strategies

employed in gathering the data, as well as the processes done in analyzing and

interpreting them. The procedures are discussed in detail and the previous research

works that inspired the chosen methodological framework were also cited.

2.1 RESEARCH DESIGN

This research study is categorized as experimental research. This study is

focused on the characteristic of Eleusine indica as an alternative source of antifungal.

The researchers gathered information to know the potential of E. indica as a source of

controlling the dermatophytes. Since the topic has no other scientific investigation

reported in the evaluation of E. indica's antifungal potential. The researchers aimed to

develop a product using Eleusine indica as an alternative source for controlling the

reproduction against Trichophyton mentagrophytes.

 Research Title - 14

2.2 RESEARCH VARIABLES

Independent
Independent
Variable: Eleusine
Indica
Indica
ANTI-FUNGAL
EFFECT OF
ELEUSINE INDICA
(GOOSE GRASS)
EXTRACT AGAINST
TRICOPHYTON
MENTAGROPHYTE
Dependent S
Variable:
Variable:
Trichophyton
Mentagrophytes

Figure 2: This Figure represents the Independent Variable of the study which is the Eleusine

indica and the Dependent variable of the Study namely, Trihchophyton Mentagorphytes.

2.3 RESEARCH INSTRUMENTS

The aim of this study is to showcase the ability of the Eleusine indica extract

to mitigate and control the production of Trichophyton mentagrophytes. The

researchers utilized the following materials;

 Eleusine indica/Paragis/ Goosegrass

 Ethyl Acetate

 Oven

 Spray Drying Machine

 Trichophyton mentagrophytes

 Kirby-Bauer Disk Diffusion Method

 Research Title - 15

 Distilled Water

2.4 DATA GATHERING PROCEDURE

The experiment is the main tool to gather data. The researchers collected the

data through the test and observation. The materials needed are organic and visible in

their surroundings. It was conducted between the independent and dependent

variables.

Collection and Preparation of the Materials

The Eleusine indica were collected from the near empty and grassy lot behind

the SM City Fairview at Novaliches, Quezon City and are washed under running

water until mud and soil are washed out. Goose grass were cut in the size of 30mm or

3cm and are baked inside the oven at 40˚in 3 hours. The plant are then granulated into

a fine powder, thereafter the 30 g of the powder sample are soaked in the 250 ml ethyl

acetate for 24 hours. A mixture that was accumulated was filtered through filter paper

and concentrated to a final volume of 50 ml.

Kirby Bauer Disc Diffusion Method

Anti-fungal suspensions were prepared from 24-hour old culture of the test

organisms. The suspending medium was 0.1% peptone water.

Pre-poured Nutrient Agar (NA) plates about 3 mm thick were inoculated with

the respective anti-fungal suspension by swabbing the agar surface. The cotton swab

on an applicator stick was dipped into the anti-fungal suspension, rotated several

times and pressed firmly on the inside wall of the tube above the fluid level to remove

excess inoculum from the swab. The swab was streaked over the entire agar surface.
 Research Title - 16

This procedure was repeated two more times, rotating the plate 60°each time to ensure

even distribution of the inoculum. Three (3) equidistant wells were made on the agar

plate using a cork borer (10 mm diameter). Two hundred (200) µl portion of the

sample was placed in each of the wells.

The NA plates were incubated at 35°C and observed after 24 hours.

2.5 DATA ANALYSIS

AI= Diameter of clearing zone – Diameter of well

Diameter of well

The Antifungal Index are used to determine the effectivity of the powder by

measuring the diameter of the clearing zone where the fungi were exposed to it. The

Antifungal Index is utilized as reference tool designed to be used and enhance the

work of professionals in the scientific community concerning microbiology. The

clearing zone are measured in millimeters and the average diameter of the clearing

zone are calculated and if there is no clearing zone then there is no inhibition of

growth of the test organisms. The researchers determined that the Eleusine indica

extract has an antifungal activity against pathogenic fungi such as Tricophyton by the

use of the Antifungal Index.

Phytochemical Analysis

The freshly prepared extract of active plant was chemically tested qualitatively

for the presence of chemical components specifically flavonoids, phenols, terpenoids,

and saponins that may affect the anti-fungal activity on the skin infection causing

fungi.
 Research Title - 17

CHAPTER III

RESULTS AND DISCUSSION

This chapter includes the presentations of the data recorded from the student-

participants, the analyses of these data, interpretations and discussions based on the

problems that were stated in the first chapter.

3.1 RESULTS

Phytochemical Analysis

Table 1. The result of Phytochemical Analysis of Goose grass Aqueous

Extract

Phytochemical Analysis of Goose grass Aqueous Extract

Tests Positive Results Actual Results Indication

For CARBOHYDRATES
Molisch Test Violet ring at the Green ring at Negative
junction junction
For REDUCING SUGARS
Fehling’s Test Formation of Blue colored Negative
brick red solution
precipitate
For FLAVONOIDS
Alkaline Reagent Test Yellow coloration Yellow coloration Positive
which disappears which disappears
upon the addition upon the addition
of dilute acid of dilute acid
Lead Acetate Test Presence of Gel-like Positive
yellow turbidity or precipitate in
precipitate yellow solution
For ALKALOIDS
Hager’s Test Yellow precipitate Clear yellow Negative
or turbid solution solution
Mayer’s Test White precipitate Clear light-yellow Negative
or turbid solution solution
 Research Title - 18

Wagner’s Test Reddish brown or Clear reddish- Negative

turbid solution brown solution
For TANNINS
Ferric Chloride Test Blue solution- Yellow solution Negative
presence of gallic
tannins
Green to black
solution- presence
of catecholic
tannins
For GLYCOSIDES
Keller killani Test Reddish No ring at junction Negative
brown/purple ring
at the junction
For SAPONINS
Froth Test Froth greater than Not persistent Negative
2 cm even after 30 foam formation
seconds
For RESINS
Test for Resins Turbid solution Clear yellow Negative

solution
For PHYTOSTEROLS
Liebermann-Burchard Test Brown ring at No ring at junction Negative
junction, Green
upper layer-
presence of
sterols/
Deep red-
presence of
triterpenoids
For ANTHRAQUINONE
Test for Anthraquinone Red color Clear yellow Negative
solution
For Proteins (Peptide
Bonds)
Biuret Test Purple/ Violet Clear blue solution Negative
color
Antifungal Assay

Table 2. The result of the Antifungal Assay

Clearing zone, mm
Test organism Sample AI
1 2 3
 Research Title - 19

Eleusine indica >55a >55 >55 >4.5

T. mentagrophytes Canesten solutionc , 10 µl 70 6.0
a
No growth of the test organism on the entire plate
b
Contains 1% clotrimazole

The Eleusine indica extract showed antifungal activity against the T.

mentagrophytes with an AI of >4.5. The results are shown in the Table 2.

3.2

Collection of Phytochemical
Materials Analysis

Antifungal
Preparation of
Activity
Extracts

Figure 1. Flowchart representing the process of the study

The summary of the process is shown in Figure 1. The first stage in the

process is the collection of the materials that are needed. The aqueous extract of

Eleusine indica were then subjected in the Phytochemical Analysis to identify the

chemical compounds that it possess. The plant that are soaked in the Ethyl Acetate
 Research Title - 20

undergo Antifungal Assay to identify its effectiveness towards the representative

fungi.

3.3 DISCUSSION

After obtaining the results from the antifungal assay conducted, it was proven

that Eleusine indica has the potential to become an antifungal agent against

Trichophyton mentagrophytes. This is because of the presence of Flavonoids that are

required to produce antifungal, anti-allergic, anti-inflammatory, antioxidant and

antimicrobial activities. The phytochemical analysis prove that this chemical

constituent is present in the plant through its aqueous extract. The result of the

antifungal assay showed the determination of zone inhibition represented by the

Eleusine indica extract against T. mentagrophytes in clearing zone (mm) trial 1 of

>55mm, clearing zone trial 2 of >55mm, and clearing zone trial 3 of >55mm with

Antifungal Index of >4.5 and containing 1% of Clotrimazole. The phytochemical

analysis results showed that the chemical Flavonoids are present in the aqueous

extract while other chemical constituents like carbohydrates, reducing sugars,

alkaloids, tannins, glycosides, saponins, resins phytosterols, anthraquinone and

proteins are not present in the plant aqueous extract. The result of the Phytochemical

Analysis supported the whole study and prove that the property that the Eleusine

indica has can mitigate the production of the dermatophyte, Trichophyton

mentagrophytes.
 Research Title - 21

CHAPTER IV

SUMMARY OF FINDINGS, CONCLUSIONS AND RECOMMENDATIONS

This chapter summarizes the findings of this study and concludes all the

implications based from the findings. It also presents the recommendations for future

action and research following this research.

4.1 SUMMARY OF FINDINGS

The study investigated the chemical constituents of the Eleusine indica (Goose

grass) and its antifungal potency against the dermatophytes, Trichophyton

mentagrophytes.

The Eleusine indica were collected from the near empty and grassy lot behind

the SM City Fairview at Novaliches, Quezon City and are washed under running

water until mud and soil are washed out. Goose grass were cut in the size of 30mm or

3cm and are baked inside the oven at 40˚in 3 hours. The plant were then granulated

into a fine powder, thereafter the 30 g of the powder sample are soaked in the 250 ml

ethyl acetate for 24 hours. A mixture that was accumulated was filtered through filter

paper and concentrated to a final volume of 50 ml.

The Eleusine indica extract was delivered by the researchers to Natural

Sciences Research Institute at UP Diliman, Quezon City and the crude plant was

delivered to UP Manila. Phytochemical Analysis and In Vitro Antifungal Assay were

performed. It was then found out that the E. indica have chemical constituent that are

needed in performing antifungal assay which are the Flavonoids for a high antifungal

activity. The researchers also found out that Eleusine indica (Goose grass) extract
 Research Title - 22

showed the determination of zone inhibition represented by the E, indica extract

against T. mentagrophytes of >55mm with an AI of >4.5 and contains 1%

Clotrimazole.

Findings

After conducting the study, the researchers found out that:

4. The Eleusine indica has the presence of Flavonoids that are required to

produce antifungal, anti-allergic, anti-inflammatory, antioxidant and

antimicrobial activities.

5. The Eleusine indica extract has a high antifungal effect and reactivity towards

the dermatophyte, Trichophyton mentagrophytes.

6. The Eleusine indica can be an alternative source of fungicide.

4.2 CONCLUSIONS

Based from the result of this study and the results of the related studies, the

researchers therefore conclude that the Eleusine indica (Goose grass) extract can be

an antifungal agent on the selected athlete’s foot-causing fungi, the Trichophyton

mentagrophytes due to the presence of chemical constituent Flavonoids that are

required to produce antifungal, anti-allergic, anti-inflammatory, antioxidant and

antimicrobial activities.

4.3 RECOMMENDATIONS

The researchers recommend searching for more information or more related

literatures about this study. They also recommend performing more assays to know

the other things this plant extract can perform or research for another plant that

contains the same constituents like the Goose grass and perform the same procedure.
 Research Title - 23

The researchers lastly recommend future researchers to use other types of

dermatophytes to have another discovery.

 Research Title - 24

BIBLIOGRAPHY

Amoah, D.O., Joson, M.B., & Pajera, M. C. (2007). Antiurolithiatic Potential of

Eleusine indica Linn. (GOOSE GRASS) Root Extract on Ethylene Glycol

Induced Nephrolithiasis in Rattus norvegicus (ALBINO RATS). Biochemical

Science 3(5), 99-108.

Altemimi, A. et al. (2017). Phytochemicals: Extraction, Isolation, and Identification of

Bioactive Compounds from Plant Extracts. Plants 6, 42.

Babu, R. H., & Savithramma, N. (2013). Phytochemical screening of underutilized

species of Poaceae. An Int J, 1(10), 947-51.

Balangcod, T. D., Vallejo, V. L., Patacsil, M., Apostol, O., Laruan, L. M. V. A.,

Manuel, J., & Gutierrez, R. M. (2012). Phytochemical screening and

antibacterial activity of selected medicinal plants of Bayabas, Sablan, Benguet

Province, Cordillera administrative region, Luzon, Philippines.

Beguin H , Pyck N , Hendrickx M , Planard C , Stubbe D , Detandt M . The

taxonomic status of Trichophyton quinckeanum and T. interdigitalerevisited: a

multigene phylogenetic approach . Med Mycol 2012 ; 50 : 871 – 882.

Fatima, I., Kanwal, S., & Mahmood, T. (2018). Evaluation of biological potential of

selected species of family Poaceae from Bahawalpur, Pakistan. BMC

complementary and alternative medicine, 18(1), 27.

 Research Title - 25

Iberahim, R., Yaacob, W. A., & Ibrahim, N. (2015, September). Phytochemistry,

cytotoxicity and antiviral activity of Eleusine indica (sambau). In AIP

Conference Proceedings 1678(1), 030013.

Lum, A. F., Ndifon, E. M. J., Mbong, G. A., Jovita, S., & Chi, G. N. N (2019). Anti-

fungal activity of plant extracts for the management of Fusarium oxysporum f.

sp. elaiedis in vitro. International Journal of Biosciences 14(6), 1-9.

Manoharan, S., & Kaur, J. (2013). Anticancer, Antiviral, Antidiabetic, Antifungal and

Phytochemical Constituents of Medicinal Plants. Am. J. PharmaTech

Research 3(4), 2249-3387.

Rekha, D. N. et al. (2014). Diversity, antimicrobial and antioxidant activities of

endophytic fungi in Chloris barbata and Eleusine indica of Poaceae l

subfamily Chloridoideae. Department of Crop Physiology University of

Agricultural Sciences, 113-124.

Saraswati Jayanthi, R., Nagamani, R., Lavanya, M., & Rao, R. (2016). Incidence and

Culture Characteristics of Dermatophytes Isolated at a Tertiary Care Hospital

in the Outskirts of Hyderabad, India. International Journal of current

Microbiology and Applied Sciences 5(8), 372- 380.

Valentino, M. G., Dulay, R.R., & Lindain, J. G. (2018). Phytochemicals, anti-oxidant

potential and total phenolics of Eleusine indica leaf extracts. International

Journal of Biosciences 13(5), 317-321.

Verma, R., Chaurasia, L., & Katiyar, S. (2008). Potential antifungal plants for

controlling building fungi. Natural Product Radiance 7(4), 374-387.

 Research Title - 26

Wiart, C., Mogana, S., Khalifah, S., Mahan, M., Ismail, S., Buckle, M., & Sulaiman,

M. (2004). Antimicrobial screening of plants used for traditional medicine in

the state of Perak, Peninsular Malaysia. Fitoterapia, 75(1), 68-73.

Research Title - 27
 Research Title - 28

APPENDICES
Research Title - 29
Research Title - 30
Research Title - 31
Research Title - 32
Research Title - 33
Research Title - 34
Research Title - 35
Research Title - 36
Research Title - 37
Research Title - 38
Research Title - 39
Research Title - 40
Research Title - 41
Research Title - 42
Research Title - 43
Research Title - 44
Research Title - 45
Research Title - 46
Research Title - 47
Research Title - 48
Research Title - 49
Research Title - 50
Research Title - 51
Research Title - 52
Research Title - 53
 Research Title - 54

CURRICULUM VITAE