MARIKINA SCIENCE HIGH SCHOOL
Mayor Juan Chanyungco St., Sta. Elena Marikina City
Antimycotic Activity of Ethanolic Extract from Water Hyacinth (Eicchornia
crassipes mart.) Leaves and Stem Against Fusarium sp.
By:
Alkuino, Laurenz T,
Galupo, Angielyn Noelle S.
Malaga, Jamin Pearl G.
Mr. John Paul Arcilla
Research Adviser
.
March 2020
TABLE OF CONTENTS
ABSTRACT ............................................................................................................................................................. 3
INTRODUCTION................................................................................................................................................. 4
MATERIALS AND METHODS .................................................................................................................... 6
Collection of Water Hyacinth...................................................................................................................... 6
Identification and Verification of Plant Species .................................................................................. 6
Ethanolic Extraction of Water Hyacinth ............................................................................................... 6
Desiccation of Leaves and Stem ............................................................................................................... 6
Powdering of Dried Leaves and Stem ..................................................................................................... 6
Filtration of the Immersed Powdered Leaves and Stem .................................................................... 7
Preparation of Water Hyacinth Ethanoic Extract Ratios ............................................................... 8
Preparation of Potato Dextrose Agar (Culture Media) .................................................................... 8
Anti-Fungal Assay ............................................................................................................................................ 8
RESULTS AND DISCUSSION\ ................................................................................................................... 10
CONCLUSION .................................................................................................................................................... 14
RECOMMENDATIONS ................................................................................................................................. 14
ACKNOWLEDGEMENTS ............................................................................................................................ 14
APPENDICES ...................................................................................................................................................... 16
Appendix A: Collection of Water Hyacinth (Eicchornia Crassipes) ......................................... 17
Appendix B: Water Hyacinth Certificate of Authentication ....................................................... 18
Appendix C: Ethanoic Extraction of Water Hyacinth ................................................................... 19
Appendix D: Preparation of Water Hyacinth Ethanoic Extract Ratio ................................... 20
Appendix E: Preparation of Potato Dextrose Agar (Culture Media) ...................................... 21
Appendix F: Microscopic Images of Fusarium sp. ........................................................................... 22
Appendix G: Antifungal Assay ................................................................................................................. 23
Appendix H: Antifungal Assay Day 1 .................................................................................................... 24
Appendix I: Antifungal Assay Day 2...................................................................................................... 25
Appendix J: Results Antifungal Assay .................................................................................................. 26
REFERENCES ..................................................................................................................................................... 27
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ABSTRACT
Water Hyacinth, also known as Eicchornia crassipes, are one of the most abundant
aquatic native plant in Marikina City that causes its excessive overpopulation. There have
been studies proven that these plants possess secondary metabolites that are significant in
potential antimicrobial activity based on its phytochemical analysis. The objective of this
study is to utilize water hyacinth as a possible component for its antifungal activity
amongst plants destructed by Fusarium species. Ethanol extraction from the pulverized
water hyacinth was executed through the process of distillation. The extracted solution
was mixed into four concentrations as the setups. Miconazole was used as the control
variable in this experiment. Through three days, the zones of inhibition were measured in
accordance to the fungi’s growth. Results for each analysis were compared to the
Miconazole, in which none of the concentrations were able to withstand the growth of the
fungi. Nonetheless, the 75% concentration ratio was able to hold the growth of the fungi
temporarily. The results proved that water hyacinth does cannot be a potential antimycotic
agent against Fusarium species but still a possible antimicrobial agent for other pathogens.
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INTRODUCTION
Evidence had been provided that water hyacinth (Eicchornia crassipes mart.)
contains antimicrobial properties primarily for the needs of the vulnerable plant species.
It has shown the presence of secondary metabolites that are valiant in its potential
antimicrobial activity as presented in its phytochemical analysis.1 On the other hand, water
hyacinths are also known as an aquatic weed for its vast reproduction on the water forms.
Here in the Philippines, many local government units (LGUs) were already imposing ecofriendly methods to minimize the volume of these aquatic plants. The Pasig River
Rehabilitation Campaign (PRRC) had responded wisely to the proliferation of water
hyacinth in the Pasig River through utilizing them to provide livelihood opportunities to
the residents.2 Researches and improvisation from this known aquatic weed paved the way
for its hidden benefits to the living life.
Moreover, there are certain pathogenic fungi that continues to inhibit plants
hindering their growth and development. One example of this pathogenic fungi are the
Fusarium species (Fusarium sp.). Currently, this fungus is estimated to comprise at least
300 genealogically exclusive species.3 Some of the species’ well-known groups of this
species are the following: F. fujikoroi, F. graminearum, F. solani, and F. oxysporum.
[1] Jayanthi, P. and P. Thamaraiselvi. “Preliminary studies on phytochemicals and antimicrobial activity
of solvent extracts of Eichhornia crassipes (Mart.) Solms”. Pelagia Research Library. 2012.
https://pdfs.semanticscholar.org/4ff2/282a377f77df9986d906cf3ae94532684f56.pdf
[2] Jonathan Mayuga. “Water Hyacinth: Bane or boon?”. Business Mirror. 2019.
https://businessmirror.com.ph/2019/06/23/water-hyacinth-bane-or-boon/
[3] Melissa Petruzello. “Fusarium Wilt”. The Editors of Encyclopaedia Brittanica.
https://www.britannica.com/science/fusarium-wilt
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In accordance to the purpose of the study, Fusarium sp. will be the primary fungi to be
treated as it attacks agronomically important crops.4 It has characterized on plants through
the sudden paling of leaves, which can eventually decay the roots and die if left unsolved.5
These pathogens, as an overall, has an alter effect to the agricultural sector as Philippines
is a major distributor in the economic world.
The objective of this study is to utilize water hyacinth as a possible component for
its antifungal activity amongst plants destructed by Fusarium wilt. This study also seeks
for the use of organic materials that are considered as a waste to the environment. The
research study determines if the leaves and stems from the water hyacinth can have an
antimycotic activity using its ethanoic extract through an anti-fungal assay against
Fusarium sp.. Specifically, this study will evaluate the zone of inhibitions of each
concentration ratio of the water and ethanoic extract to the fungi. This study aids for the
needs of future researches upon the development of water hyacinth as an antimycotic
agent and for the reduction of wastes from various aquatic forms.
[4] Aoki, Takayuki, Kerry O’Donnell, and David M. Geiser. "Systematics of Key Phytopathogenic
Fusarium Species: Current Status and Future Challenges." Journal of General Plant Pathology 80, no.
3 (2014): 189-201. doi:10.1007/s10327-014-0509-3.
[5] Melissa Petruzello. “”Fusarium Wilt”. The Editors of Encyclopaedia Brittanica.
https://www.britannica.com/science/fusarium-wilt
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MATERIALS AND METHODS
Collection of Water Hyacinth
Water Hyacinth were collected from the Marikina River with the approval and
assistance of River Parks Authority (RPA).
Identification and Verification of Plant Species
Water Hyacinth samples that are subject for ethanolic extraction were
taxonomically verified and identified in the Herbarium in University of the Philippines
Diliman located in Quezon City.
Ethanolic Extraction of Water Hyacinth
Desiccation of Leaves and Stem
Materials: Water Hyacinth, Scissors, Paper bag, Oven
The leaves and stem of the Water Hyacinth (Eicchornia crassipes mart.) were
cut into small pieces measuring approximately 1 inch. These pieces were then
contained in a paper bag and was put inside the oven for the complete removal of
moisture.
Powdering of Dried Leaves and Stem
Materials: Blender, Container, Scissors
The dried leaves and stem of the Water Hyacinth (Eicchornia crassipes mart.)
were blended, cut and grinded until finely powdered.
Immersion of Powdered Leaves and Stem
Materials: Ethanol (1.2 L), Powdered Water Hyacinth Leaves and Stem (800 g),
Beaker, Watch Glass
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The powdered leaves and stems were immersed in ethanol with a ratio of 1 gram
is to 15 mL of ethanol solution for 2 days.
Filtration of the Immersed Powdered Leaves and Stem
Materials: Ethanol-Immersed Powdered Water Hyacinth Leaves and Stem, Funnel,
Erlenmeyer Flasks, Filter Paper, Cotton, Glass Bottle
The Ethanol-Immersed Powdered Water Hyacinth Leaves and Stem were poured
over funnel with cotton inside an Erlenmeyer flask. It was then filtered with a filter
paper inside a funnel twice to remove the remains of the Water Hyacinth. After the
filtration, the aqueous solution was contained in a glass bottle. An amount of 600 ml
of the aqueous solution was yielded.
Distillation of the Water Hyacinth with Ethanol Solution
Materials: Distillation Set-up, Ethanol and Water Hyacinth with Ethanol Solution
A distillation set-up was prepared for the distillation. The solution was
poured inside the distilling flask and was boiled maintaining a temperature of 78˚ C
(boiling point of ethanol).[6] The condenser was constantly filled with cold water and
be replaced as it heats up. This process will continue until the ethanol is separated
from the extract. The extract was lit with a match stick to test if there are still remains
of ethanol.
[6] “Ethanol Molecule.” The Ethanol Molecule -- Chemical and Physical Properties. Accessed February
21, 2020. https://www.worldofmolecules.com/fuels/ethanol.htm.
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Preparation of Water Hyacinth Ethanoic Extract Ratios
Varying ratios of water hyacinth extract with distilled water was prepared was
Table 1. Ratio of Water Hyacinth Ethanoic Extract and Distilled Water
Water Hyacinth Ethanoic Extract
Distilled Water
1
2
3
4
Percentage
100%
75%
50%
25%
Amount
25.00 mL
18.75 mL
12.50 mL
6.25 mL
Percentage
25%
50%
75%
Amount
0 mL
6.25 mL
12.50 mL
18.75 mL
Preparation of Potato Dextrose Agar (Culture Media)
Materials: Potato, Distilled water, Agar powder, Glucose, Autoclave, Petri Dish,
cheese cloth
200 grams of peeled potatoes were boiled for 30 minutes and were sifted through
a cheesecloth. The extract collected was mixed with 4.75 grams of agar and 1.90
grams of table sugar. Distilled water was added to make the solution 1 liter and was
placed inside and autoclave at 121˚ C for 15 – 20 minutes. The solution was then
transferred in petri dishes.
Anti-Fungal Assay
Materials: Culture media, Fungal culture, Metal rod, Filter Paper Discs, Water
Hyacinth Ethanoic Extract, Miconazole (control), Alcohol Lamp
The discs were measured according to the estimated timeframe where the fungi
had spread to reach the extract and the control variable. For the measurements, the
fungus was placed at the center of an 85 mm diameter of petri dish (42.5 mm), while
the extract and control variable were placed 12.5 mm apart from the fungi.
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Beforehand, the researchers already placed the Potato Dextrose Agar in the discs of
3 mm thickness. It is important that the Potato Dextrose Agar that had been swapped
in the disc shall be sterilize first before spreading it to the disc to prevent
contamination.
For the final appearance of each samples, filter paper discs were made out of
a punched hole of filter paper. These filter paper discs were settled in the
concentration extracts (25%, 50%, 75%, and 100%) and the controlled variable
(miconazole). Afterwards, the researchers sterilized the forceps before picking up
the cellulose discs to the assigned designation on the plates. Consistently,
sterilization through aseptic technique was performed all throughout the discs to
avoid outside contamination in the observation. The plates were settled at room
temperature and clean environment for accurate results.
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RESULTS AND DISCUSSION\
Table 1 shows the zone of inhibitions of each plate with their respective ratios and
control. It can be observed that only the concentration with 75% water hyacinth extract
was able to withstand the growth of the fungus within a span of three days measuring 2
mm with each duplicate.
Table 1. Results of Anti-Fungal Assay
Concentration
100%
75%
50%
25%
Duplicate
1
2
1
2
1
2
1
2
Zone of Inhibition
Extract
Control
0 mm
3 mm
-1 mm
2 mm
2 mm
1.5 mm
2 mm
3 mm
0 mm
3 mm
0 mm
3 mm
0 mm
3 mm
-3 mm
3 mm
Table 1 illustrates that the distribution of the data is uniform (16 mm) for all
concentration ratio since the setups had just been done during the day.
Figure 1. Box-and-Plot Graph for the First Day of Observation
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In the second day of observation, it can be seen that the zone of inhibition for each
of the concentration ratios and the control variables started to decrease. The ratios for the
50% and 25% have the same Interquartile Range (IQR). On the other hand, the 75%
concentrations have the highest zone of inhibition amongst all variables.
Figure 2. Box-and-Plot Graph for the Second Day of Observation
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For the third day of observation, it shows that there are various thin boxes on the
graph. It means that the data do not have much variation in terms of the zone of inhibition.
It is also illustrated in the graph that the 75% concentration is the only concentration that
has a positive value for its zone of inhibition compared to the rest.
Figure 3. Box-and-Plot Graph for the Third Day of Observation
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The conducted Kruskal-Wallis Test shows that the test statistic has no significant
difference for all the days of observation. As said in the decision made for each null
hypothesis, the researchers have concluded that the measurements for the zone of
inhibition for most of the concentration ratio are almost the same in a way that there is no
zone of inhibition manifested in the plates. Nevertheless, it can be also seen that the test
statistic decreases each day, which entails that the values across the zone of inhibition of
the concentration ratios varied at a minimal amount. Although, there are still no values
that satisfy the confidence level.
Table 2. Hypothesis Test Summary for the Days of Observation and the Variables
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CONCLUSION
In conclusion, water hyacinth does not have a potential anti-mycotic activity
against Fusarium sp. It does not possess secondary metabolites that have the capacity to
withhold the presence of the fungi. However, it is evident that the 75% concentration has
the ability to hold temporarily the fungi compared to the other concentrations. In addition,
most of the data gathered in the zone of inhibition testing for all concentrations are
identical and does not have variations.
RECOMMENDATIONS
The researchers recommend the following for the furtherance of the results and
implications of the research:
1. Using this study as baseline to other studies in further studying water hyacinth’s
anti-fungal properties using different extraction methods.
2. Other studies should allot 1 week or more before investigating the zones of
inhibition.
3. Future researchers should test the water hyacinth extract with other pathogenic
fungi that destructs high valued crops.
ACKNOWLEDGEMENTS
First of all, we would like to thank God for giving us knowledge, strength, courage
and love which helped us overcome different obstacles and hindrances that came our way
while conducting the research. One of the most important factor of our success is through
Mr. John Paul Arcilla. The person who taught us everything about research, our biggest
supporter and teacher.
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This work would not have been possible without the critical consultation and
suggestions of Dr. Lourdes Alvarez, Ms. Zephaniah Kesh Doctor-Abayon and Mr. Jerald
Bongalos. Thank you for giving us your endless support and opinion despite your busy
schedules. Thank you for permitting us to conduct some parts of our research methods in
the PUP Mycology Laboratory. Without this big of a help, we wouldn’t be able to conduct,
observe and analyze our research data.
Another factor of our success are our classmates and batch mates who gave us
unconditional support and encouragement by giving their opinions, encouragement and
giving us necessary information especially our ever supportive mentor, Mr. Yuan Ureña.
Nobody has been more important to us in the pursuit of our research project than
our families. We would like to thank our parents who supported us financially,
emotionally and physically. Thank you for supporting us throughout the whole process.
We would also like to thank our principal for the support and endorsement, Ms.
Janet S. Amurao, principal, Ms. Felicitas A. Perez, assistant principal and our subject
teachers, Ms. May F. Villanueva, Ms. Hazel M. Castro, and, Mr. Ronald E. Escorpiso in
excusing us in our missed activities, quizzes, and projects.
Our research team is very grateful for all the love, support and encouragement all
of these people have given to us. If not for all of their efforts, the team wouldn’t be able
to go through all the problems and struggles. Your faith in us and in our study are the one
that pushed us to do better every day.
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APPENDICES
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Appendix A: Collection of Water Hyacinth (Eicchornia Crassipes)
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Appendix B: Water Hyacinth Certificate of Authentication
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Appendix C: Ethanoic Extraction of Water Hyacinth
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Appendix D: Preparation of Water Hyacinth Ethanoic Extract Ratio
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Appendix E: Preparation of Potato Dextrose Agar (Culture Media)
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Appendix F: Microscopic Images of Fusarium sp.
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Appendix G: Antifungal Assay
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Appendix H: Antifungal Assay Day 1
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Appendix I: Antifungal Assay Day 2
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Appendix J: Results Antifungal Assay
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REFERENCES
Jayanthi, P. and P. Thamaraiselvi. “Preliminary studies on phytochemicals and
antimicrobial activity of solvent extracts of Eichhornia crassipes (Mart.) Solms”.
Pelagia
Research
Library.
2012.
https://pdfs.semanticscholar.org/4ff2/282a377f77df9986d906cf3ae94532684f56.pdf
Jonathan Mayuga. “Water Hyacinth: Bane or boon?”. Business Mirror. 2019.
https://businessmirror.com.ph/2019/06/23/water-hyacinth-bane-or-boon/
Melissa Petruzello. “Fusarium Wilt”. The Editors of Encyclopaedia Brittanica.
https://www.britannica.com/science/fusarium-wilt
Aoki, Takayuki, Kerry O’Donnell, and David M. Geiser. "Systematics of Key
Phytopathogenic Fusarium Species: Current Status and Future Challenges." Journal of
General Plant Pathology 80, no. 3 (2014): 189-201. doi:10.1007/s10327-014-0509-3.
Melissa Petruzello. “”Fusarium Wilt”. The Editors of Encyclopaedia Brittanica.
https://www.britannica.com/science/fusarium-wilt
“Ethanol Molecule.” The Ethanol Molecule -- Chemical and Physical Properties.
Accessed February 21, 2020. https://www.worldofmolecules.com/fuels/ethanol.htm.
Alkuino; Galupo; Malaga; 3 | 27