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IN VITRO EVALUATION OF ANTIBACTERIAL ASSAY OF ILLICIUM VERUM (STAR

ANISE) SEED EXTRACT TO WATERS CONTAINING FECAL COLIFORM BACTERIA

A Research Presented to the


Senior High School Department
San Pedro College, Davao City

In Partial Fulfillment
of the Requirements for the Subject
Practical Research II

Michael Angelo A. Garcia


Royce B. Garcia III
Charisa Antonette S. Huelva
Mary Aubrey Rose A. Ledesma
Michael Jan M. Leornas
Mel Christine C. Leynes
Samantha T. Rubia
Blessy Sharinine P. Sumagayan

September 2018
CHAPTER I

INTRODUCTION

Background of the Study

Water pollution is the contamination of water with various waste products,

including untreated sewage and industrial wastes that contain chemicals which are

released in bodies of water. These make the water unsuitable for utilization. This

proliferation of water pollution threatens our health as hazardous water kills more people

each year than war and all other forms of violence combined [CITATION Mel18 \l 1033 ].

As water pollution becomes prevalent in every country, the risk of diseases and

illnesses also becomes higher for people and in the marine life. According to a United

Nations report on August 30, 2016, increasingly polluted rivers in the continents Africa

and Asia, and including Latin America, poses a great disease risk to 300 million people.

This also threatens farming and fisheries in many countries. [ CITATION Mil16 \l 1033 ]

In 2017, the Goletta di Verde, a research ship of Legambiente, which is an Italian

environmental organization analyzed that approximately 40% of coastal seawater of the

260 samples tested contained polluted results with bacterial loads above the legal limits.

This pollution is said to be linked to untreated sewers [ CITATION The17 \l 1033 ].

However, on a recent trip by the Goletta di Verde’s team this year, they analyzed that

pollution is rather increasing. They showed that there was an eight percent increase in

pollution since the previous year. In addition, according to a map by Legambiente that
The Water Purifying…3

outlined the results, it showed that the coastlines in Liguria, Tuscany and Sicily are the

most polluted [ CITATION 48p18 \l 1033 ].

Moreover, Sarno River, a relatively short river also located in Italy, is known as

the most polluted river in Europe. Sarno is a relatively clean upstream where it supports

fish and other small aquatic animals; however, this changes downstream where the river

starts showing chemical foam, which is easily seen in the surface. This chemical waste

was traced to have originated from dumping of industrial and agricultural waste into the

river[CITATION The18 \l 1033 ].

In the Philippines, Pasig River is deemed important as it connects two major

bodies of water: Laguna de Bay and Manila Bay. The river also provides food, livelihood

and transportation to residents. However by the 1990’s, it is considered biologically

dead. Health authorities found that there is high coliform content in the river, thus making

it a site for diseases[ CITATION Mur04 \l 1033 ].

Additionally, in a recent study in 2017, it was found out that Pasig River gained

the 8th spot among the top 20 polluting rivers globally. The report also stated that the

river dumps 63,700 tons of plastic into the ocean every year [ CITATION Riv17 \l 1033 ].

This also made Pasig River, the world’s second worst contributor of plastic waste to the

ocean, in relation to its drainage area [ CITATION Año17 \l 1033 ]. In fact, according to a

report in April 2018, Lt. May Thorssen of the Philippine Coast Guard Auxiliary (PCGA)

stressed on the high number of plastic sachets in the river and said how it has always

been a huge problem for households around the river are not capable of buying their

personal commodities by jug. In the same report, George Oliver dela Rama, public

information officer of the Pasig River Rehabilitation Commission (PRRC) stated how 70

percent of all the waste in the river are domestic, which means these wastes come from
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human feces, garbage and food and compost, while the remaining 30 percent arise from

industrial and chemical wastes from factories [ CITATION Sub18 \l 1033 ].

Despite earning a spot in 2nd place on the 2nd ASEAN Certificates of Clean Air,

Clean Water, and Clean Land in 2014 [ CITATION ASE14 \l 1033 ], Davao City,

unfortunately has their own set of polluted waters. Davao River, which encompasses

Bukidnon and Davao del Norte on either side, has been identified as one of the country’s

most polluted rivers back in 2015. The regional office of the Environment Management

Bureau also elaborated how the river’s water quality deteriorated based on the 5-year

water quality assessment of the said environment department. Moreover, water pollution

has been identified as one of the crucial problems by the Davao River Situational Report

[ CITATION Tac15 \l 1033 ].

Furthermore, in a report published by Sunstar in 2016, it was said that beaches

in Davao City where found to be highly polluted. This is based on a 2015 study

conducted by the Department of Environment and Natural Resources Environmental

Management Bureau (DENR-EMB). It was found that the city’s beaches have high

coliform content. According to Gloria Raut of the Department of Health (DOH), this is

due to the city’s lack of septage treatment facilities [ CITATION Dav16 \l 1033 ]. Coliform

bacteria are commonly associated with feces of animals and humans. Moreover, the

news report specified the most polluted Liberty beach in the mouth of Davao River which

has a total coliform reading that amounted to 160,115 MPN per 100 liters and fecal

coliform reading of 123,433 MPN per 100 liters [ CITATION Dav161 \l 1033 ].

With these findings, it is imperative to conduct a study with an aim of providing

solution to the prevalence of water pollution, not only in Davao City, but also in the whole

world. The objective of this study is to determine the effectivity of Illicium verum as a
The Water Purifying…5

water purifying agent to polluted waters. Likewise, the study aims to provide a low-cost

yet efficient means of purifying water.

Related Literature

Water Pollution

Water pollution is a problem that has existed since humans evolved over the

millennia. According to Denchak (2018), water pollution occurs when pollutants, usually

harmful chemicals and microorganisms, contaminate a body of water making it toxic and

harmful to humans and animals alike. Water pollution is also defined by Woodford (2

017) as a buildup of harmful matter in the bodies of water that could threaten life and

the environment. As a matter of fact, Dybern (1974) stated that it seems to be that no

body of water is totally free from pollution. Even the first-class countries in the world

experience and complain about this dilemma (World Atlas).

According to of Sawe (2017), the most polluted river in the world is found in Italy -

The Sarno River. It is a relatively clean upstream that supports fish and other aquatic

creatures but then became polluted and shows chemical foams upon joining tributaries.

It was traced that the chemical wastes found in the water body is coming from the

dumpings of industrial and agricultural wastes. While in the Philippines, since a rapid rise

in population in the country, that has created poverty, environmental degradation, and

pollution, also affected the bodies of water in the country [ CITATION Mar16 \l 1033 ].

Philippines is known as a country rich in natural resources and natural water

forms; for the reason that it is composed of thousands of islands [ CITATION The181 \l

1033 ]. However, according to the The Borgen Project (2018), out of more than one
The Water Purifying…6

hundred million Filipinos, nine million rely on unsafe water supplies. In fact, water

pollution in the Philippines and a lack of proper sewage kill 55 people every day. This is

the reason why, due to the observed proliferation of pollution, the government decided to

make a law to diminish this problem. The Philippine Clean Water Act of 2004 (Republic

Act 9275) was implemented to protect the country’s water resources from its different

land-based resources. It provided a comprehensive and integrated strategy to prevent

and minimize pollution through a multi-sectoral and participatory approach.

As to that, different people and organizations of this generation proposed the

idea of introducing the process of water purification using different kinds of plants and

methods.

Water Purification Using Different Kinds of Plants

According to Lanfair, Ambulkar, & Scroth (n.d), water purification is a procedure

in which unwanted chemical compounds, organic and inorganic materials, are cleared

out from water. The water purification procedure reduces the concentration of

contaminants like suspended particles, parasites, bacteria, algae, fungi and viruses.

Almost all water being processed is purified for human consumption. It aims to produce

water fit for a variety of purposes, which includes meeting the requirements of medical,

pharmacology, chemical and industrial applications.

In accordance to that, several studies use different plants acting as natural water

purifiers which can also help in the ecosystem of the environment. According to Raine

(n.d.), aquatic plants play a large role in making a body of water healthy and secrete

oxygen for the fish. From the same article, she also mentioned 4 types of plants that can

act as natural water purifiers; these are the flowering shoreline plants, grass-type

shoreline plants, floating plants, and submerged plants.


The Water Purifying…7

Raine (n.d.) stated examples of flowering shoreline plants which are the Golden

cannas (Canna flacida) and the Lanceleaf frogfruit (Phyla lanceolata). Golden cannas

are hardy plants and are in the U.S Department of Agriculture plant hardiness zones 8 to

11. This plant can grow paddle-shaped leaves and can also grow 4 feet tall. There are

many benefits of this plant, one of which is it prevents erosions in bodies of water. It also

is a phytoremediation agent, therefore it helps remove extra nutrients, like nitrogen and

phosphorus from water [ CITATION Wil \l 1033 ].

As to the following plant given, it was not given enough supporting details to

conclude about its usage. For that reason, more experiments and researches are done

to deepen the understanding towards their characteristics with their capacity to be used

as a water purifier.

The Effects and Benefits of Star Anise

Spices come in all shapes and sizes, and star anise (Illicium verum) is a definitive

example. Although this spice is most popularly known for its dark and attractive color

and unique star shape, star anise also contains powerful health benefits that can

contribute to one’s health [ CITATION Mer \l 1033 ]. It is also used as a common flavoring

for medicinal tea, cough mixture and pastilles. In traditional system of medicines, Illicium

verum fruit is used for both culinary and medicinal uses. Its seed oil is used worldwide as

medicine. Due to the potential side effects of synthetic antioxidants, essential oil derived

from natural products can be served as an alternative source for the further improvement

of synthetic antioxidant [ CITATION Ibr16 \l 1033 ]. Also, according to WebMD (2005), star

anise seeds contain ingredients that have activity against bacteria, yeast, and fungi.
The Water Purifying…8

Additionally, according to Mercola (n.d.), star anise is a good source of two

antioxidants: the essential oil linalool and vitamin C that helps protect the body against

cellular damage caused by free radicals and environmental toxins. Also, attached to

Mercola’s article is the study published in the journal Chemico-Biological Interactions

that revealed that cancer development was prevented among animals fed with star anise

[ CITATION Yad07 \l 1033 ].

South Korean researchers proved that star anise extracts, and essential oils

have antifungal abilities, especially against candida albicans, a common yeast infection

(Park, S.H. and Seong I., 2010).

With the given information, it is seen that star anise is known as an effective

herbal medicine towards repairing cells and other possible treatments. It is also

observed that most of the gathered information are inclined with medicinal overview but

are very few when it comes to the usage of becoming a water purifier.

In this proposed study, the researchers envision to know and deepen the

knowledge of using star anise in the process of water filtration.

Related Studies

“Biological Activities and Chemical Constituents of Illicium Verum hook fruits

(Chinese star anise)”, a study conducted by Chouksey, Sharma, and Pawar on 2010

investigated that Chinese star anise (Illicium verum) had been used traditionally as

carminative, digestive, dyspepsia, antispasmodic, and stimulant, antirheumatic and

duretic. Illicium verum has the oil which is therapeutically potential in the treatment of

microbial diseases, specifically as an antifungal and antioxidant. In another study

conducted by Vecchio, M. et al (2016), entitled “Pimpinella Anisum and Illicium Verum:


The Water Purifying…9

The Multifaceted Role of Anise Plants”, showed that star anise (Illicium verum) have

various cultural usage in different countries, both as food and drug. Anise has an

essential oil extracted from its dry ripe that contains anethole, an active chemical

compound that showed functional properties including antimicrobial, antioxidant,

hypoglycemic, hypolipidemic and estrogenic properties.

The previous studies are associated in the present study because both studies

focused on the uses of Illicium verum given that it has a wide range of medicinal

properties and investigates in relatively newer areas of its application. Moreover, the

previous studies and present study will have its focus on the functional properties that

can found in anise.

There were a lot of studies which utilized plants for water purification. One of

which is the study of Jodi, Birnin-Yauri, Yahaya and Sokoto in 2012. The study, entitled

“The Use of Some Plants for Water Purification”, aimed to find the efficacy of powdered

seeds of plants as natural water treatments alternative to the use of artificial chemicals.

Moreover, they tested the effectiveness of each plant mixed with alum. They used

powdered seeds of M. oleifera, H. sabdariffa, and C. tridens. The study found out that M.

oleifera can be used to treat the turbidity in drinking water. Furthermore, they found out

that when each plant was combined with alum, the results were much better. The study

concluded as the researchers obtained values for M. Oleifera and combinations of each

plant with alum, that as observed, were within the World Health Organization (WHO)

maximum acceptable level of turbidity in drinking water. The study of Jodi, Birnin-Yauri,

Yahaya, and Sokoto (2012) can be associated in the present study for the researchers

aimed to use plants as a means of purifying water.

Similar to the previous study, the study of Chauhan, Gupta, and Singh (2015)

noted certain types of plants that can improve the quality of drinking water. During this
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study, they conducted an experiment with plant extracts of Moringa oleifera, Arachis

hypogaea (peanuts), Vigna unguiculata (cowpeas), Vigna mungo (urad) and Zea mays

(corn), to fully analyze the effect of each plant to purifying water. Their methods included

having to examine treated and untreated sample water for heavy metals and microbial

counts. Plant extracts mentioned were applied in water samples for further observation

and results were noted. The results of their study concluded that the plant extracts used

truly decreased the bacteria count in untreated water samples. The study of Chauhan,

Gupta and Singh is related to the present study since both focuses on the utilization of

plants in purifying water. However, the previous study used different plant extracts

whereas the present study will only focus on the efficiency of Illicium verum as a water

purifier.

Moreover, in a different study, entitled Water Filtration using Plant Xylem

[ CITATION Bou \l 1033 ], the researchers tested the potential of plant xylem, from the

sapwood of coniferous trees, to remove bacteria from water through simple pressure-

driven filtration. They found out that approximately 3 cm3 of sapwood can filter several

liters of water each day, which is sufficient to meet a person’s clean water drinking

needs. Additionally, the study aimed to demonstrate a global solution on providing clean

and safe drinking water in order to reduce waterborne diseases in developing countries

and resource-limited areas. The previous study is connected to the present study since

both studies focuses their aim on providing a solution to reduce the great risk of illnesses

on places with dirty waters.


The Water Purifying…11

Conceptual Framework

Independent Variable Process Dependent Variable

1. Gathering of
samples
2. Testing for
EFFECTIVENESS
coliform count
andOF pHTHE IV
level
3. Extracting
FecalIllicium the
verum
coliform
seed
(starand
count anise)
pH seed
level
4. In vitro
of polluted waters
antibacterial
activity
5. Result

Figure 1. A Conceptual Paradigm showing the relationship of the variables in the study

Figure 1 shows the relationship between the independent and dependent

variable where the fecal coliform count and pH levels of each water sample will

determine the effectivity of the antibacterial activity of star anise as a water filter. The

independent variable is Illicium verum seed while the dependent variables are the fecal

coliform count and pH level polluted waters.

Statement of the Problem

The study aims to determine the antibacterial capability of Illicium verum against

polluted waters for filtration. Specifically, it aims to find answers to the following:

1. What is the level of coliform count and pH level of the water sample?

2. Is there a significant difference between the fecal coliform count of water before

and after the treatment?

3. Is there a significant difference between the pH level of the water before and

after the treatment?


The Water Purifying…12

Hypothesis

H0: There is no significant effect from the antibacterial activity of Illicium verum to the

presence of fecal coliform bacteria and pH level of polluted waters.

Ha: There is a significant effect from the antibacterial activity of Illicium verum to the

presence of fecal coliform bacteria and pH level of polluted waters.

Significance of the Study

This study aims to develop awareness that there are other ways to have access

to clean water with the use of water purifying plants. The outcome of the study could

bring contribution to the following:

Students. It will raise awareness that there are alternative ways to acquire clean

water. This study will also discipline the students to realize the importance of having

clean water to drink. Furthermore, it will allow them to push their schools to use this

method of getting clean water.

Community. People in the community may have access to basic drinking water

and sanitation services, but these services do not necessarily provide safe water and

sanitation. This issue puts the health of all people at risk for such diseases. This study

will give the community an opportunity of being able to use a different method in

providing clean water. It will also allow communities in the country and around the world

to obtain clean water for a way to distribute to their respective communities in an

environmentally friendly way.

Future Researchers. The future researchers will benefit from this study since

they will be able to know the specific methods in using plants for purifying water. Also,
The Water Purifying…13

this would give light to further studies in the future in possibly having to deal with world

water crisis wherein people would lack access to clean drinking water. Future

researchers could also possibly find a way of using plants or herbs as a solution for

important worldwide issues.


CHAPTER II

METHODS

Design
The study is quantitative in nature and will use the experimental design.

Quantitative experimental research is a study that is high in casual validity (Mitchell,

2015). According to Mitchell (2015), causal validity is concerned with the accuracy of

establishing cause and effect relationships among the independent variable and the

variations of its effect on the dependent variable. Furthermore, experimental research

can also be defined as to change or to control factors that may alter the result of the

experiment. Experimental researchers attempt to determine the outcome of what may

happen (Key, 1997).

In this study, the experimental research design will help the researchers

determine the antibacterial capacity of star anise seed extract. The researchers find the

need to conduct an experiment and tests to show the effectivity of star anise seed

extract to reduce the fecal coliform count on polluted waters. Furthermore, the design

will help the researchers determine the effect of star anise seed extract to the pH level of

the water.

Setting

The researchers plan to conduct the study in the laboratories within Davao City

namely University of Immaculate Conception (UIC) and/or San Pedro College due to the

apparatus needed in performing the said research.


The Water Purifying…15

Research Subjects

Star anise (Illicium verum) seed ethanolic extract and water containing high fecal

coliform count will be the subjects of the study.

Data Analysis

The researchers will use three kinds of sampling tests to examine and further

observe the study.

In the first statement of the problem, we will determine the fecal coliform count of

each water sample to get the mean. Additionally, in the second and third statement of

the problem, we will be using t-test dependent to know the significant difference before

and after the antibacterial activity of fecal coliform count and Illicium verum seed

extracts.

Lastly, the researchers will use the Analysis of Variance (ANOVA) to see the

different effects of the antibacterial activity in negative and positive variable control.

Research Procedures

In conducting the study, the following steps will be followed for the procedures

and ethical considerations that will be followed by the researchers to get necessary data

in order to determine the antibacterial capacity of star anise seeds.

First, the researchers will ask permission to conduct the study. The researchers

will present a formal letter to the Senior High School, Officer-in-Charge, Prof. Rosario M.

Oconer, MATC, to allow the researchers to conduct the study in the laboratories of San

Pedro College and University of Immaculate Conception.


The Water Purifying…16

Second, the researchers will ask the panelists to review some ethical

considerations. The researchers will write a letter to the panelists namely: Prof. Narlina

A. Espanto, Prof. Rommel C. Gestopa, and Prof. Zarine Cabugsa, MS Bio, allowing us

to check the ethical considerations of the study.

For the subjects

The subjects of the study will be the star anise seeds ethanolic extraction that will

undergo the process of assay bacterial degradation.

For the Valuators

The researchers will find an expert instructor preferably on the field of Biology.

The researchers will give a letter asking to validate the research procedures.

Furthermore, the researchers will orient the valuators as to how the data gathering of the

research will be done and what will be the subjects of the study. Moreover, the

researchers will ask the valuator to write all their recommendations for the improvement

of the study.

Third, the researchers will ask for permission to use the SPC laboratory wherein

the researchers will perform the extraction of seeds. The researchers will make a letter

to the Laboratory Head, Mrs. Helen Ancla, to allow, help and request laboratory

equipment and chemicals needed in the conduct of the experiment.

Fourth, the researchers will ask permission from the University of Immaculate

Conception to allow the researchers to conduct the experiment. The experiment that will

be performed will be to evaluate the antibacterial activity of Illicium verum and its effect

on the presence of fecal coliform in waters. Aforesaid to the administration of the

experiments, the researchers will send a letter to the faculty to allow the researchers to
The Water Purifying…17

conduct an experiment on assay bacterial degradation for the capacity of Illicium verum

seed ethanolic extraction.

Fifth, will be the analysis of the results. The researchers will request a certificate

of analysis from the University of the Immaculate Conception to analyze the process of

the experiment for the study.

Sixth step will be the retrieving and gathering of the data. The data that will be

collected by the researcher will be handed to Prof. Zarine Cabugsa. The data will be

examined and checked by the expert and the results will be systematized by the

researchers.

Lastly will be the analyzation and interpreting of the data gathered. After the

systematization and the calculation of the data, the researchers will discuss, analyze,

and interpret the results profusely to come up with the conclusions and

recommendations.

Scope and Limitations

The study will have its focus only on the effect of the star anise seed extract to

the fecal coliform count and pH level of the waters. The researchers will not include the

effect of the seed extract to the turbidity and the changes in color of the water. Moreover,

the water samples will be taken and studied within Davao City. The study will

immediately take place after the panel has approved the study and will be conducted in

the laboratories of San Pedro College and the University of Immaculate Conception.
References