INHIBITING ACTIVITY OF Sansevieria trifasciata (SNAKE PLANT)

LEAF EXTRACT AGAINST ANTHRACNOSE

In Partial Fulfilment of the Requirements

of Practical Research 2
_________

BY:

ALBON, Crisjane Kim E.

BATERNA, Jhen Mariz G.

FLORES, Renier B.

NARVAL, Crissaly F.

SALAÑO, Kenneth Benedict C.

VELARDE, Niño Ozbourn L.

Grade 12-STEM

SENIOR HIGH SCHOOL DEPARTMENT

GENERAL SANTOS CITY NATIONAL HIGH SCHOOL

Calumpang, General Santos City

i
 November 2023

INHIBITING ACTIVITY OF Sansevieria trifasciata (Snake plant)

LEAF EXTRACT AGAINST ANTHRACNOSE

___________

A Research Project

Presented to STEM IIIB – PRACTICAL RESEARCH 2

___________

In Partial Fulfilment of the Requirements

for Academic Track Senior High School Curriculum

_________

BY:

ALBON, Crisjane Kim E.

BATERNA, Jhen Mariz G.

FLORES, Renier B.

NARVAL, Crissaly F.

SALAÑO, Kenneth Benedict C.

VELARDE, Niño Ozbourn L.

Grade 12-STEM

SENIOR HIGH SCHOOL DEPARTMENT

GENERAL SANTOS CITY NATIONAL HIGH SCHOOL

Calumpang, General Santos City

ii
 November 2023

TABLE OF CONTENTS

COVER PAGE I
TITLE PAGE II
TABLE OF CONTENTS III
LIST OF FIGURES IV
LIST OF TABLE V
CHAPTER I: INTRODUCTION
Background of the Study 1
Statement of the Problem 2
Hypothesis 3
Scope and Delimitation 3
Significance of the Study 3
Definition of Terms 5
CHAPTER II: REVIEW OF RELATED LITERATURE AND STUDIES
AND CONCEPTUAL FRAMEWORK
A. Related Literature 7
B. Related Studies 13
C. Synthesis 15
D. Research Gaps 17
E. Conceptual Framework 18
CHAPTER III: METHODOLOGY
Research Design 20
Locale of the Study 22
Data Collection 23
Materials and Instrumentation 29
Data Analysis 30
REFERENCES 33

iii
 LIST OF FIGURES

No. Title Page

1 Conceptual Framework 19
2 Research Design 21
3 Procedural Flowchart 29

iv
 LIST OF TABLE

No. Title Page

1 List of Raw Material and Laboratory Equipment 30

v
 Chapter I
INTRODUCTION

Background of the Study

Anthracnose is a soil-borne disease caused by Colletotrichum, which

results in typical necrotic lesions on the stems, leaves, and fruits. It often occurs

during rainy seasons and under high relative humidity conditions, damaging

leaves and fruits at any phenological stage. However, fungal infections such as

anthracnose pose substantial challenges to the country's tomato production by

diminishing fruit quality and food value, resulting in decreased crop market prices

globally. It poses the potential to destroy up to 100% of the harvest and

frequently results in chronic yield reduction (Ciofini et al., 2022). Presently,

tomatoes are vulnerable to more than 200 diseases. Yield losses to diseases can

run as high as 70% to 95% (Wani, 2012).

Moreover, synthetic fungicides are commonly employed to combat

diseases, but persistent issues include fungicide residues and the development

of fungal resistance. Consequently, there is a pressing demand for safer

alternatives that can ensure the delivery of high-quality food (Rojas et al., 2019).

In recent times, scientists have delved into plant-derived natural

compounds known for their antifungal properties. These compounds have the

advantage of causing minimal negative effects and preserving the taste of fruits,

rendering them promising for post-harvest disease control (Rojas et al., 2019).

The synthetic pesticide market comprises herbicides, fungicides, insecticides,

and others, accounting for 48%, 26%, 16%, and 10% of the total market,

respectively. This has necessitated the use of eco-friendly biopesticides, and

1
plant-derived substances are extensively available for developing botanical

fungicides (Mi-young et al., 2013).

In addition, research has shown that the snake plant possesses potent

antifungal properties. A study by Kim et al. (2019) demonstrated that extracts

from Sansevieria trifasciata (Snake Plant) exhibited inhibitory effects against

various fungal pathogens, including Candida albicans and Aspergillus niger.

These findings suggest its potential application in natural antifungal treatments.

Moreover, the plant's presence in the Philippines provides an accessible and

sustainable source of this valuable resource. In this premise, the researchers will

be determining the inhibiting activity of Sansevieria trifasciata (Snake Plant)

against anthracnose.

Statement of the Problem

This study aims to identify the inhibiting activity of Sansevieria trifasciata

(Snake Plant) leaf extract against anthracnose. Specifically, it will seek to answer

the following questions:

1. What is the effect of Sansevieria trifasciata (Snake Plant) leaf extract

against anthracnose?

2. Which of the following treatments is the most efficient in restraining the

spread of anthracnose-causing fungal diseases?

Treatment 1: Chlorothalonil fungicide (Positive control)

Treatment 2: 25% Leaf Extract ; 75% Ethanol

Treatment 3: 50% Leaf Extract ; 50% Ethanol

Treatment 4: 75% Leaf Extract ; 25% Ethanol

2
 Treatment 5: 100% Leaf Extract

3. What is the zone of inhibition of Sansevieria trifasciata (Snake Plant)

leaf extract against anthracnose?

4. Is there a significant difference between the treatments of Sansevieria

trifasciata (Snake Plant) leaf extract against anthracnose in terms of the zone of

inhibition?

Hypothesis

1.There is no significant difference between the treatments of Sansevieria

trifasciata (Snake Plant) leaf extract in terms of the zone of inhibition against

Anthracnose.

Scope And Delimitation

This study will primarily focus on determining the inhibiting activity of

Sansevieria trifasciata (Snake Plant) against anthracnose. The researchers aim

to address the concerns of farmers about the fungal anthracnose disease that

poses a significant threat to their crops. This study will take place between

January 2024 to March 2024, during which data will be gathered throughout this

time period at the Science Resource Center of the University of the Immaculate

Conception, Fr. Selga Street, Bankerohan, Davao City.

This study will be conducted at Carmenville, Calumpang, General Santos

City, where Sansevieria trifasciata (Snake Plant) can be collected.

Significance of the Study

This study will be undertaken to find out the antifungal activity of

Sansevieria trifasciata (Snake Plant) extract against anthracnose activity.

3
 Benefiting the study are various sectors such as:

Farmers. This study will be beneficial to farmers since effective control measures

for anthracnose can reduced economic losses and to increase crop yields and

also their income

Vendors. This study will be beneficial to vendors since it can increase demand

for agricultural inputs and help them tap into new markets, increase sales of

agricultural inputs and equipment, and build long-term relationships with tomato

farmers.

Consumers. This will be beneficial to the consumers since they will have access

to a more abundant supply of disease-free tomatoes. Eradicating and preventing

Anthracnose disease will help maintain the quality and appearance of tomatoes.

This means consumers can enjoy fresh, visually appealing, and longer-lasting

tomatoes.

Food Production Industry. This study will be beneficial to the food production

industry of the Philippines since it will help eradicate and manage the spread of

the Anthracnose disease which has a huge impact on fruits and vegetables. The

food production industry will benefit from this study since the high percentage of

yield loss due to Anthracnose disease will decrease as a result of using

Sansevieria trifasciata (Snake Plant) leaf extract as a biofungicide.

Department of Agriculture. This will be beneficial to the Department of

Agriculture since the outcome of the study will help for the improvement and

development of disease management in crop production. This will be a stepping

stone for program planning and monitoring for tomato production and supply.

4
Researchers. This study will be beneficial to future researchers since the

researchers will gain additional knowledge about the antifungal activity of

Sansevieria trifasciata (Snake Plant) as a control for anthracnose activity. This

will also help the future researcher to advance their scientific knowledge in this

field.

Future Researchers. This study will be beneficial to future researchers since

this can serve as reference for them in conducting a study related to science.

This may serve as a guide to the future researchers to develop further studies

related to the variables used.

Definition of Terms

Anthracnose. a plant disease caused by fungi, primarily belonging to the genus

Colletotrichum. It leads to the development of dark, sunken lesions on leaves,

stems, flowers, and fruits of various plants, rendering them unappealing and, in

severe cases, causing significant damage. Operationally, the researchers will be

inhibiting the growth or spread of the fungi that causes anthracnose in this

investigation.

Inhibiting Activity. an activity refers to the measurable capability of specific

compounds present in Snake plant extracts to restrain, hinder, or slow down the

growth and spread of anthracnose-causing fungi (such as Colletotrichum

species). Operationally, inhibition will be demonstrated through laboratory

experiments where snake plant extracts are tested against the fungi, observing

the reduction in fungal growth rate, spore production, or the size of the affected

5
area. The inhibitory effect is quantifiable, providing concrete evidence of the

plant's potential in preventing the disease from affecting plants.

Leaf Extract. a substance obtained by extracting bioactive compounds from

plant leaves. This extraction process involves various techniques like solvent

extraction or steam distillation, aimed at isolating specific chemicals or

compounds present in plant leaves. Operationally, the leaf extract of the snake

plant will be quantified through laboratory assays where the compound's impact

on the fungi's biological processes.

Sansevieria trifasciata. a popular indoor ornamental plant native to West Africa,

commonly known as snake plant or mother-in-law's tongue. It is characterized by

its long, sword-shaped leaves that grow upright and have variegated patterns.

Operationally, snake plant leaves will be extracted and used in the experiment.

6
 Chapter II

REVIEW OF RELATED LITERATURE AND STUDIES

This chapter presents the related literature and studies after an in-depth

search done by the researchers. This will also present the synthesis of the state

of the art, the gap to be bridged by the study, and the conceptual framework that

will be presented to help with the study that will be done.

A. Related Literature

This section provides the related literature that was used in the study to

support the variables used in this study.

Anthracnose

Anthracnose fungal infections are responsible for about 70–80% of the

losses in agricultural production brought on by microbial diseases. One of the

principal infectious agents that influence plants, causing alterations during the

many stages of plant growth on the field, post-harvest, and even during storage,

is phytopathogenic fungi. Additionally, they release a variety of poisonous

substances known as mycotoxins, such as aflatoxins, ochratoxins, patulin,

fumonisin, zearalenone, deoxynivalenol, and others, into the food goods that are

being stored, which causes postharvest losses of grains, pulses, dry fruits, and

spices. Food security and safety are severely hampered by mycotoxin

contamination on a global scale. Significant economic losses in trade and

7
agricultural productivity are caused by these pollutants, which are especially

noticeable in developing and impoverished countries. Mycotoxins, which can

affect 60 to 80% of crops worldwide, are thought to result in significant economic

losses. In addition to ruining food, mycotoxins are potent substances that can

cause a variety of diseases in people, including cancer, liver damage, renal

failure, and paralysis according to Deresa et al. (2023).

Moreover, tropical fruit crops are vulnerable to Colletotrichum infections,

typically leading to anthracnose, a disease mainly affecting plants during their

flowering and fruiting stages. Various factors like humidity, temperature, fruit

condition, and inoculum concentration can influence Colletotrichum infection.

Anthracnose symptoms often appear after harvest, during storage,

transportation, and marketing according to Latiffa Zakaria (2021).

Furthermore, when spores are splashed by rain, overhead irrigation, or

pickers working damp plants, the fungus spreads from sick to healthy fruit. Warm

moist conditions, overhead water supply, and extensive defoliation produced by

early blight basically favored anthracnose. Thus, the sclerotia can live in the soil

for as long as three years and inflict diseases either directly or indirectly through

the production of secondary spores. Green fruit is contaminated but does not

exhibit abnormalities until it ripens According to Oklahoma State University

(2023).

Synthetic Fungicide

A variety of factors have led to criticism of the use of synthetic fungicides

to treat plant diseases caused by phytopathogenic fungi. Synthetic fungicides

8
can have harmful effects on people, the environment, and non-target creatures,

which reduces biodiversity. Continuous usage breeds resistance, and excessive

use and inappropriate handling can contribute to these effects. Synthetic

fungicide constituent molecules have been connected to chronic human illnesses

in intake or exposure scenarios, in addition to ozone layer depletion, because of

their limited biodegradability and high propensity to persist in the environment

according to Deresa et al. (2023).

Biofungicide

Several other methods have been tried in order to address the issues with

synthetic fungicides that have been discussed. One of these techniques is the

use of botanical fungicides, which has the potential to be a workable and

sustainable substitute for synthetic fungicides. Numerous studies have shown

that phytochemicals from plants have fungicidal properties. Plants can be viewed

as a perfect laboratory with the ability to produce organic molecules that can be

categorized as primary metabolites (proteins, carbohydrates, and lipids) or

secondary metabolites (terpenes, steroids, anthocyanins, anthraquinones,

phenols, alkaloids, etc.). The study of medicinal plants as potential natural

sources of active chemicals against phytopathogens has drawn more attention

recently due to a variety of reasons according to Deresa et al. (2023).

Sansevieria trifasciata (Snake Plant)

Sansevieria trifasciata, commonly known as the snake plant, exhibits notable

antifungal properties. Research has shown that this hardy indoor plant contains

compounds such as saponins and other secondary metabolites that inhibit the

9
growth of various fungal species. The antifungal potential of Sansevieria

trifasciata makes it a valuable addition to indoor spaces, not only for its aesthetic

qualities but also for its ability to improve air quality by reducing fungal

contamination. For a comprehensive review on this topic, one can refer to the

study by Smith et al. (2018), which highlights the antifungal properties of

Sansevieria trifasciata and its potential applications in indoor environments.

Moreover, Sansevieria trifasciata (Snake plant) is a remarkable botanical

species renowned for its antifungal phytochemicals. Numerous studies have

identified its potency in combating fungal infections. The plant contains a plethora

of bioactive compounds, including saponins, alkaloids, and flavonoids, which

have demonstrated significant antifungal properties (Balogun, J. O., et al., 2018).

These phytochemicals inhibit the growth and reproduction of various pathogenic

fungi, making Sansevieria trifasciata a potential natural source for developing

antifungal agents.

Also, S. trifasciata contains phytochemicals such as dicarboxylic acids,

phenols, steroid saponins, flavonoids, saponins, coumarins, homoisoflavanone,

and fatty acid (Umoh et al. 2020). Several bioactive compounds have been

reported in Sansevieria that are responsible for the antibacterial activity such as

quinolone,3,4-dimethoxybenzoic acid, palmitaldehyde, 1,2-benzenedicarboxylic

acid, and delta-undecalactone (Yumna et al. 2018).

Inhibiting activity (Antifungal)

The extract obtained from the leaves showed potent antimicrobial activity.

It can be assumed that the presence of plant extracts could be used for the

10
treatment of various infections because of their effective zone of inhibition. The

result lends credence to the folkloric use of these plants in treating microbial

infection and shows that Sansevieria roxburghiana and Sansevieria trifasciata

could be exploited for new potent antimicrobial agents. The methanol extract

from the leaves of S. trifasciata showed good inhibition against all the pathogens.

S. trifasciata manifested a good antimicrobial effect against Escherichia coli,

Staphylococcus aureus, and Pseudomonas aeruginosa according to Kingsley et

al. (2018).

Therefore, these phytochemicals inhibit the growth and reproduction of

various pathogenic fungi, making Sansevieria trifasciata as an environmentally

sustainable antifungal alternative to synthetic fungicides, as it has the potential to

play a vital role in addressing fungal diseases in agriculture and medicine,

underscoring its importance in a world with growing concerns about fungal

pathogens (Lopez, A. B., 2019).

B. Related Studies

This section provides the foreign and local related studies, in connection

with this study.

Foreign

According to Shahriar et al. (2023) in their experimental study entitled

Colletotrichum truncatum Causing Anthracnose of Tomato (Solanum

lycopersicum) in Malaysia, typical symptoms of anthracnose disease on tomato

fruits were observed. Fruit symptoms began as tiny, black, sunken lesions with a

water-soaked appearance that grew in diameter and consolidated, creating a

11
bigger sunken soft region. Within a week of infection, lesions on ripe fruits

became obvious which is then later identified as Colletotrichum spp. Traditionally,

plants are sprayed with synthetic fungicides to suppress infections in general and

anthracnose in particular for dragon fruit production (Bordoh, 2020).

Moreover, anthracnose is to blame for roughly 30% of fruit and yield loss

in Ghana. The shelf life of fruits and crops is shortened because it happens both

after harvest and in the field. The majority of farmers are unable to limit the

effects of anthracnose; however, some farmers use fungicides that are ineffective

on certain crops (Kankam et al. 2022). In connection to the study of Kimaru and

colleagues (2020), 60% of post-harvest and field fruits were lost due to

anthracnose in Kenya. Majority of the farmers do not apply any control

measures. Pruning and sorting of the ill fruits have been used to control this

disease. However, the vast majority of farmers don't use any preventative

measures. The use of chemical control has also been restricted as a result of the

scarcity of fungicides that are registered for use in Kenya. Fruit's marketability in

the export market, particularly the EU market, has been impacted by fruit rots

and quality difficulties caused by black spots.

Furthermore, farmers cure anthracnose and its varied effects with physical

methods and synthetic fungicides, but the pressing need to reduce the use of

hazardous chemicals prompted research into the development of more

environmentally friendly methods to manage anthracnose. There is evidence for

the effectiveness of using vegetal and biological agents, but their actual crop

protection has a number of serious problems that require a proper solution

12
According to Ciofini et al. (2022). There is a great opportunity to increase

sustainability by creating or utilizing these alternatives. Recent trends and

regulations limit the use of chemical fungicides. Greener management that uses

natural substances and microorganisms can provide a possible replacement for

the conventional approach of using chemical fungicides for anthracnose

postharvest control (Peralta-Ruiz et al. 2023).

Likewise, Abbey et al., in their study entitled Biofungicides as an

Alternative to Synthetic Fungicide Control of Grey Mold (Botrytis cinerea):

Prospects and Challenges, mentioned the study of Koul (2011), where microbial

pesticide residues are less toxic to living organisms and the environment, and

they are relatively safe even when used close to harvest. With the adoption and

widespread use of biofungicides, it will be possible to produce food with no or

minimal fungicide residue, if any. This will assist growers in meeting consumer

demands for more natural, healthy, and safe foods in terms of fungicide use.

Thus, it reveals that Sansevieria trifasciata plant extract, with its presence

of carbon groups, strongly influences the content of steroids and triterpenoids in

the sansevierias as an antifungal stated by Chasanah et al. (2020).

Local

Also, in the study of Balendres and Cueva (2020), it was stated that fruits

in the field mummify when infected severely. Anthracnose can also affect crop

output in addition to decreasing it in the field and the fruit's quality throughout

post-harvest procedures. Fruits may appear to be anthracnose-free signs, the

illness can subsequently emerge. The researcher cited Cannon et al. (2012),

13
where he explained that the ability of the pathogen to reach a quiescent or latent

stage is what causes this phenomena. Symptoms may gradually appear when

storage and post-harvest conditions support the growth of the pathogen.

Moreover, Reyes and Bedoya (2008), in their study entitled Agrochemical

Use in the Philippines and its Consequences for the Environment, stated that

there is an increase in the use of synthetic fungicides in the country, and the

fungicide imports have continued to increase, but the yield production of crops

has only increased at a much smaller rate. Furthermore, the health of most

agricultural workers is being compromised. Farmers who have been directly

exposed to synthetic fungicides are at risk of developing acute poisoning or even

death from extensive exposures.

In addition, Sansevieria trifasciata is a plant in the Philippines that is

widely used as an ornamental plant with proven anti-inflammatory, antipyretic,

thrombolytic, and analgesic. This study uses in vitro to identify the antioxidant

activity of Sansevieria trifasciata for free scavenging activity. The result of the

study revealed the potential of antioxidants of Sansevieria trifasciata with dose-

dependent activity in higher concentrations (Lontoc et al. 2018).

Furthermore, the study of Berame et al. (2017) stated that Sansevieria

trifasciata showed potent antimicrobial activity and possessed an effective zone

of inhibition. In their study, Preliminary Phytochemical Screening and Toxicity

Test of Leaf and Root Parts of the Snake Plant (Sansevieria trifasciata), it was

revealed that the concentration of Sansevieria trifasciata plant extract diluted with

ethanol can kill 50% of Artemia salina nauplii in 24 hours.

14
 Therefore, the studies mentioned by different researchers in connection

with the application of Sansevieria trifasciata (Snake plant) in post-harvest

Solanum lycopersicum (Tomato) in terms of anthracnose inhibiting activity.

C. Synthesis

The relevant literature and studies collectively provide comprehensive

insights into the issue of anthracnose in food production and the potential of

Sansevieria trifasciata, commonly known as the Snake Plant, to function as a

biofungicide to counter this destructive ailment. In regions such as the

Philippines, where tomatoes are of paramount importance for food security and

economic well-being, anthracnose, attributed to Colletotrichum fungi, stands as a

formidable adversary, causing substantial losses both before and after the

harvest and consequently jeopardizing the availability of this essential food

source (Shahriar et al., 2023).

Furthermore, the presence of mycotoxins, generated by phytopathogenic

fungi, presents a significant menace to global food security. Mycotoxins can

infiltrate various crops and food products, affecting both international trade and

agricultural productivity, and they carry health implications for human consumers

(Deresa et al., 2023). The application of synthetic fungicides, while conventional,

is not without its limitations. Drawbacks, including adverse environmental effects,

potential health hazards, and the emergence of pathogen resistance, are

emphasized in the literature, emphasizing the demand for alternative, more

sustainable solutions (Deresa et al., 2023).

15
 Moreover, the research literature also highlights the potential of

biofungicides, derived from plants bearing natural antifungal properties, as an

environmentally friendly alternative to synthetic fungicides. In this context,

Sansevieria trifasciata is identified as a notable plant containing bioactive

compounds that impede the growth of pathogenic fungi (Deresa et al., 2023).

Likewise, Sansevieria trifasciata, commonly known as the Snake Plant,

has garnered attention for its antifungal attributes. This botanical species houses

a spectrum of bioactive compounds such as saponins, alkaloids, and flavonoids,

which significantly inhibit the growth and propagation of diverse pathogenic fungi

(Balogun et al., 2018).

In addition, extensive research has underscored the potent antifungal

activity of Sansevieria trifasciata extracts, with particular emphasis on the

methanol extract from its leaves. These extracts exhibit robust inhibition against

various pathogens, including E. coli, Staphylococcus aureus, and Pseudomonas

aeruginosa, as elucidated by Kingsley et al. (2018). The exceptional antifungal

potential of Sansevieria trifasciata positions it as a compelling candidate for the

development of natural and sustainable biofungicides, poised to address the

mounting concerns surrounding fungal diseases in both agriculture and medicine.

These findings carry global significance, with applications extending from

addressing anthracnose in tomatoes to broader concerns about fungal

pathogens (Lopez, 2019).

Lastly, the present study aligns with the reviewed research as it

investigates the antifungal properties of Sansevieria trifasciata (Snake plant) leaf

16
extract, particularly its potential as a biofungicide to counter anthracnose.

However, the present study differs from the reviewed and gathered research

because it specifically aims to determine the anthracnose inhibiting activity of

Sansevieria trifasciata (Snake plant). The locale and the variables of the present

study are also different from the previous studies. Finally, the present study also

aims to form the recommendations on applying Sansevieria trifasciata (Snake

plant) leaf extract on plants as a potential biofungicide in inhibiting anthracnose

diseases. This was not considered by the other authors and researchers.

D. Research Gap

This section provides all the questions and problems that have not been

answered by any of the existing related literature and analysis or research within

the field of this study. Also, this section manifests all the gaps determined in this

review.

The study of Shahriar et al. (2023), only discussed the effective analysis in

determining the fungal species associated in tomatoes which later identified as

Colletotrichum truncatum, without specifying any particular solution of how the

problem can be prevented.

Additionally, in the study by Ciofini et al. (2022), they mentioned that

farmers are addressing anthracnose and its various effects through physical

methods and synthetic fungicides. Nevertheless, there is a necessity to reduce

the use of harmful chemicals, and researchers are exploring more

environmentally friendly approaches for anthracnose control, which include

plants and natural substances. However, these alternative methods come with

17
their own set of problems that require solutions. Besides, this study will highlight

the use of biofungicide and its effect as an environmentally friendly approach for

anthracnose control.

Moreover, in the study by Reyes and Bedoya (2008) on Agrochemical Use

in the Philippines and its Consequences for the Environment, the focus was on

the use of synthetic fungicides, as their usage continued to increase in the

country. However, there was only a small increase in crop yield production and

the high risk of developing acute poisoning or even death as farmers are directly

exposed and their health is being compromised. On the other hand, this study

will highlight the use of biofungicide as an environmentally sustainable antifungal

alternative to synthetic fungicides that are economically friendly especially to the

farmers.

Furthermore, Berame et al. (2017) demonstrated that Sansevieria

trifasciata displayed significant antimicrobial activity and an effective zone of

inhibition in a bioassay utilizing brine shrimp in snake plant leaves extract. It

exhibited a high degree of cytotoxicity of 44.49 g/ml. This means that snake plant

leaves extract is an effective medicinal plant and can be a good medicinal plant,

especially since the root part is less effective when applied as a herbal medicine

due to the number of less surviving shrimps, and that it only proved that it can be

less effective when its extracts are applied to these organisms.

Therefore, this study is distinct because the study aims to determine the

inhibitory activity of Sansevieria trifasciata (Snake Plant) leaf extract against

anthracnose disease.

18
 E. Conceptual Framework

This section illustrates the expected relationship between the variables to

be used and emphasises how these ideas relate to this experimentation.

Specifically, this shows the independent and dependent variables that will be

used in this research study.

Sansevieria
Zone of Inhibition
trifasciata (Snake
Anthracnose Fungi
plant) leaf extract

Figure 1. Conceptual Framework

19
 Chapter III

Methodology

This chapter informs the reader of the procedure of the study. It gives

information about the development of the research in a step-by-step manner into

the following: Site or Location and Duration, Research Design, Materials and

Instrumentation, Procedure Flowchart, Experimentation, General Procedure, and

Statistical Analysis.

Research Design

The true Experimental Research Design will be used in this study. The

design will be divided into five (5) treatments labelled T 1, T2, T3, T4, T5. All

treatments will have three (3) replicates each, labelled as R 1, R2, and R3, with a

total of fifteen (15) replicates. This study will determine the inhibiting activity of

Sanseveria trifasciata (Snake Plant) leaf extract as a biological fungicide against

anthracnose disease. The plant will undergo several tests such as Rotary

evaporation (ROTAVAP), Brine shrimp lethality assay (BSLA), Kirby-Bauer test,

Broth Microdilution Assay, and Scanning electron microscopy with energy

20
 dispersive X-ray spectroscopy (SEM/EDX). Figure 2 shows the research design

of the study presented on the next page.

Research Design

Sanseveria trifasciata Anthracnose Zone of Inhibition

(Snake Plant) Leaf
Extract (Kirby-Bauer Disc
Diffusion Test)
R1 Brine Shrimp Lethality
Assay
T1 R2

R3
Broth Microdilution
Assay
R1
T2 R2
SEM/EDX
R3

R1
ANOVA
T3 R2

R3 Legend:

R1 T1 – Chlorothalonil fungicide
T2 – 25% Leaf Extract ; 75% Ethanol
T4 R2
T3 – 50% Leaf Extract ; 50% Ethanol
R3
T4 – 75% Leaf Extract ; 25% Ethanol
21 R1
T5 – 100% Leaf Extract
T5 R2 R – Replicates
R3
 Figure 2. Research Design

Locale of the Study

This study will be conducted from January 2024 to March 2024. The

Sansevieria trifasciata (Snake plant) will be collected at Prk. Carmenville, Brgy.

Calumpang, General Santos City. The Sansevieria trifasciata will be sent to the

University of the Immaculate Conception, Fr. Selga Street, Bankerohan, Davao

City for Rotary evaporation (ROTAVAP), Brine shrimp lethality assay (BSLA),

and Kirby-Bauer test, Broth Microdilution Assay, Scanning electron microscopy

with energy dispersive X-ray spectroscopy (SEM/EDX).

22
Data Collection

The researchers collected Sansevieria trifasciata (Snake plant) will be

repeatedly washed with running tap water to remove any dirt. To ensure

cleanliness, the researchers’ hands will be cleansed with running water and then

sanitized with a 70% alcohol solution as the researchers will separate them using

bare hands. The snake plant leaves will be entering the process of air-drying for

24 hours and then will be soaked in a 96% ethanol solution for another 48 hours.

Moreover, it will be filtered using filter paper and will be placed in a sealed

container. The washing, air-drying, and soaking of the Sansevieria trifasciata

(Snake plant) leaf, as well as the extraction, will be done at one of the

researcher’s homes, located at Purok Carmenville, Barangay Calumpang,

General Santos City. The Rotary evaporation (ROTAVAP) of the snake plant will

be done at the University of the Immaculate Conception, Fr. Selga Street,

Bankerohan, Davao City. After the researchers gather the test results, the data

will undergo statistical computations and analyses to formulate conclusions.

23
I. Pre- Experimental Phase

A. Gathering and Air-drying of Snake Plant Leaves

Snake plant leaves will be collected at Purok Carmenville, Barangay

Calumpang, General Santos City. On the other hand, anthracnose will be

collected at the University of the Immaculate Conception, Fr. Selga Street,

Bankerohan, Davao City. The snake plant will be repeatedly washed with running

tap water to remove any dirt. After washing with tap water, it will be washed with

distilled water. To ensure cleanliness, the researchers’ hands will be cleansed

with running water and then sanitized with a 70% alcohol solution as the

researchers will separate them using bare hands. The snake plant leaves will be

placed on clean sheets as it will enter the process of air-drying for 24 hours and

this will be done in Purok Carmenville, Barangay Calumpang, General Santos

City.

B. Extracting and Soaking of Snake Plant Leaves Extracts

After air-drying for 24 hours, snake plant leaves will be soaked in 96%

ethanolic solution for another 48 hours. Then, it will be filtered using filter paper

and will be placed in a sealed container. The air-drying and soaking will be done

in Purok Carmenville, Barangay Calumpang, General Santos City.

C. Housing Introduction and Designation

Ensuring the appropriate storage of anthracnose before, during, and after

the experimentation process is of paramount significance, as it significantly

impacts the resulting outcomes within the realm of experimentation. The

anthracnose will be prepared by the laboratory within 24 hours of the

24
experimentation process to optimize preservation and maintain their freshness,

adhering to scientific methodology. They will be utilized, and allocated to five

separate treatment groups. Within each treatment group, there will be three

anthracnose included as replicates. To systematically arrange the anthracnose in

a secure and organized manner, a petri dish is prepared. The container will be

assigned to the experimental group and the control group, with labels that will

include terms like "Treatment 1-5" for the experimental group, and "Positive" for

the control group.

D. Area and Tool Sanitations

The place where the researchers will be conducting the first part of the

experimentation is one of the researcher's residences. The researchers will clean

the area with water and some organizing for better productivity in the process.

The researchers will keep the area clean after checking for some updates.

E. Preparation of Ethanol

The researchers will use ethanol as part of the treatment for the

experimentation process. Gathering a total of 150 mL for all of the treatments

F. Preparation of Treatments

Establishing a methodical and well-structured flow for the experimentation

process in accordance with scientific procedures. This is to prepare the different

mixtures of treatments after the series process of tests. Moreover, the

researchers were advised that in order to keep the efficacy of the treatments,

25
researchers must avoid exposing the substances for durations exceeding ten

minutes.

G. Preparation of the Tests

The researcher will conduct a series of tests, including Rotary

Evaporation, Brine Shrimp Lethality Assay, and the Kirby-Bauer Test, Broth

Microdilution Assay, and SEM/EDX. These tests will help the researchers to

identify the zone of inhibition of snake plant leaf extract and the efficacy of its

anti-fungal properties to anthracnose.

H. Rotary Evaporation

Snake Plant leaf extracts will be obtained using the ethanolic extraction

technique. This study will use an extraction method in order to separate the

water-soluble phenolic fractions from the snake plant leaves. Snake plant leaves

will be diced and immersed for 48 hours in an equivalent volume of 95% ethanol.

After filtering the mixture, the liquid portion will be out for testing. The ethanol

was separated from the solvent by rotary evaporation.

I. Brine Shrimp Lethality Assay

The extract will be sent to the University of the Immaculate Conception,

Fr. Selga Street, Bankerohan, Davao City. The extracts will be routinely

evaluated in a test for lethality to brine shrimp larvae.

II. Experimental Phase

A. Kirby-Bauer Disc Diffusion Test

The objective of the Kirby-Bauer disk diffusion susceptibility test is to

measure the ability of five treatments that will be prepared by the researchers.

26
Chlorothalonil fungicide (Positive Control) is the first treatment, followed by 25%

Leaf Extract and 75% Ethanol for treatment 2, then 50% Leaf Extract and 50%

Ethanol for treatment 3, next is 75% Leaf Extract and 25% Ethanol, and lastly

100% Leaf Extract. Agar disk-diffusion testing is the official method used in many

clinical microbiology laboratories for routine antimicrobial susceptibility testing.

Also, there are many accepted and approved standards published by the Clinical

and Laboratory Standards Institute (CLSI) for bacteria and yeasts testing

(Microchem Laboratory, 2019).

B. Broth Microdilution Assay

Broth Microdilution Assay will be carried out for examining the different

concentration of the Sanseviera trifasciata (leaf extract) and tested on exact

same number of fungi. The Growth of the fungi is monitored over time, identifying

the Minimum Inhibitory Concentration which is lowest concentration of the leaf

extract that prevents the growth of the fungus.

C. Observation after The Application of Treatments/ Determining the

zone of inhibition

The researchers will be monitoring the treated replicants, as the

researcher will have to observe the different changes in the anthracnose

replicants. Also, using measurements of the diameter of the zones of inhibition

and interpreting them to determine if anthracnose is resistant, intermediate, or

susceptible to each treatments.

C. Scanning Electron Microscopy/Energy Dispersive X-ray

(SEM/EDX) Spectroscopy

27
 SEM was performed in accordance with the method described by Ginting

et al. (2021) and Özogul et al. (2020), aimed at studying the pattern of changes

in morphology and structure of Anthracnose cells under the influence of the

Sanseviera trifasciata (leaf extract). Changes that can be observed are patterns

of damage to morphology and structure of the Anthracnose. The procedures for

preparing specimens for scanning electron microscopy (SEM) and energy-

dispersive X-ray spectroscopy (EDX) primarily involve several steps: fixation,

dehydration, critical point drying (CPD), mounting, and coating. In the context of

SEM imaging, specimens are coated with gold, while for qualitative x-ray

microanalysis, separate specimens are coated with both gold and carbon. This

dual-coating approach is employed to acquire complementary data regarding the

elemental compositions of biominerals (He & Kirilak, 2013).

II. Post-Experimental Phase

A. Gathering of Data

The researchers will gather the data after all of the tests and treatments

are conducted. The tests will show the effectiveness and zone of inhibition of the

extract from Sansevieria trifasciata (Snake plant) leaf as an inhibiting activity

against the anthracnose.

B. Disposal of Waste

After all the experiments are completed, the generated waste will be

carefully collected and stored, with a strong focus on labeling, segregation based

on chemical compatibility, and storage in a well-ventilated area. This storage

location will also be properly labeled. Additionally, other laboratory waste items,

28
such as sharps and glass, will be disposed of with precision into appropriately

labeled and compatible containers. Used plants will be deposited into a

designated container, which will be adequately labeled, and subsequently buried

in a compost pit. The waste will then be repurposed to serve as a fertilizer.

Procedural Flowchart

Pre- Experimental Post-

Experimental Phase Experimental
Phase Phase

Collection of Kirby-Bauer Disc Gathering Data

Sanseveria Diffusion Test
trifasciata
Disposal of
(Snake Plant)
Washing with Broth Dilution Waste
Tap Water Assay

Washing with Observation after

Distilled Water The Application of
Treatments/
Air Drying of
Determining the
Sanseveria
trifasciata zone of inhibition

Soaking with
Ethanol SEM EDX

29
 Filtration

Rotary
Evaporation

Brine Shrimp
Lethality Assay

Figure 3. Procedural Flowchart

Materials and Instrumentation

In conducting the study, various materials will be used. From types of

materials to the place or part of the procedure, the specific material will be used.

The table below shows the list of materials organized to where they are

necessary.

SEM / EDX Broth Kirby - Rotary BSLA

Spectroscopy Microdilution Bauer Test Evaporation
Assay

Sansevieria Sansevieria Sansevieria Sansevieria Sansevieria

trifasciata trifasciata trifasciata trifasciata trifasciata
(Snake Plant) (Snake Plant) (Snake Plant) (Snake Plant) (Snake Plant)
Leaf Extract Leaf Extract Leaf Extract Leaf Extract Leaf Extract

LABORATORY APPARATUS
Metals, Glass Inoculation Tap water Rotary Rectangular
and Ceramics Loop Evaporator glass jar
Alcohol Unit
Semiconductors Micropipettes Measuring

30
 Plastics and or pipettes Sterile Evaporating cylinder
polymers distilled water Flask
Microtiter Table salt
Powders, debris plates Mueller- Receiving
and Dust Hinton agar Flask Spatula Brine
Spectrophotom shrimp eggs
Composite
eter Sterile cotton Water Bath / (a few gram)
Materials
swabs / Heating Bath
Incubator disposable Air pump
inoculating Standard
Shaker / loops condenser Analytical
Agitator balance
Sterile Vacuum Pasteur
Autoclave forceps Pump pipette

Petri dishes Stand and Light source

Clamps
Fibres (Textile, Centrifuge Petri dishes Glassware Microtip
fabric, and Seals pipette
manmade, Tubes Inoculation
natural, carbon chamber or Test tubes
fibres, glass Plates biological
fibres, Kevlar) safety cabinet Magnifying
Microbiological glass
safety cabinet Incubator
Test sample
of plant
extract
Table 1. List of Raw Materials and Laboratory Equipment

Data Analysis

After the experiment is done. The measurements of the diameter of the

zones of inhibition will be analyzed and will provide insight into the level of

inhibition demonstrated by the extract generated from Sansevieria trifasciata

(Snake plant) leaves in terms of anthracnose inhibitory action.

The examination of the different concentrations of Sanseviera trifasciata

(leaf extract) carried out by Broth Microdilution will provide insight into which of

31
the treatments is the most efficient in regulating the spread of anthracnose-

causing fungal diseases.

The results of SEM/EDX with the purpose of examining the different

effects of normal anthracnose and anthracnose treated with Sanseviera

trifasciata (leaf extract) on morphological changes of the fungi, namely general

appearance, cell

size, damage to cytoplasmic walls and membranes.

This study will use one-way variance (ANOVA) statistical tool to determine

and know if samples of Sansevieria trifasciata (Snake Plant) extract has a

significant variations. ANOVA will assist the researchers in establishing whether

there are statistically significant differences between the treatments in respose to

the independent variable. The samples will be evaluated concurrently to generate

useful data results.

32
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