IN VITRO STUDY OF PASPALUM CONJUGATUM FOR

ANTIMICROBIAL ACTIVITY OF CARABAO GRASS (Paspalum conjugatum)

AND DETERMINATION OF PHYTOCHEMICAL
COMPOUNDS

A Research Proposal
Presented to the Faculty of College of Nursing
Mindanao State University- Iligan Institute of Technology

Researchers:
Abugan, Laeh Mae A.
Baruang, Azim M.
Teves, Dieza May O.

May 28, 2015

TABLE OF CONTENTS
Title

Page

Chapter
1

THE PROBLEM
Introduction

Theoretical Framework

Conceptual Framework

Objectives of the Study

Hypothesis of the Study

Significance of the Study

Scope and Delimitation

Definition of terms

REVIEW OF RELATED LITERATURE AND STUDIES

Justification of the Study

Synthesis
3

RESEARCH METHODOLOGY
Research Design

11

Collection and Identification of Plant Material

12

Extraction and Preparation of Crude Extracts

12

Preliminary Phytochemical Analysis

12

Screening of Antimicrobial Activities

14

Zone of Inhibition

14

Data and statistical Analysis

15

References

16

CHAPTER I

THE PROBLEM AND SCOPE

Introduction
With the uprising population and commodities in the community, morbidity and
mortality is hard to avoid since the people in the society is also experiencing phenomenal
calamities. Calamities like flood may carry microorganisms which can contribute to poor
wound healing, the researchers conducted this study since the Carabao Grass (Paspalum
conjugatum) is known to have an antimicrobial property and is widely spread in local
areas and in most communities here in our country. In addition to this, the researchers
want to minimize the expenses in health services most especially to the medications
being bought. Furthermore, there are number of people living in far flung areas that are
unfamiliar to this kind of grass which can be a possible alternative antimicrobial regimen.
Thus, this research aims to prove that Carabao Grass (P.conjugatum) has an antimicrobial
agent and through this, the researchers will be of help in disseminating information to the
people not only in remote areas but also to those people living in the community.
The medicinal benefits of this herb are dated in all the early records of the human
existence. It started during the early Chinese era, which is dated more than 4000 years
ago up to our generation today, the extracts of these herbs and plants are still being used
to treat diseases today. Medicinal herbs are being used extensively in our generation and
are being expanded into much noncomplex forms. This study is then focused on the
application of P.conjugatum extracts to determine its biological activity through putting it
to test for its antimicrobial properties.

Antimicrobial is defined to be an agent that interferes with the growth and

reproduction of microorganism. In this field of study, the researchers aim to know more
about the phytochemical analysis that are found in Carabao Grass (P.conjugatum) and its
potential action as an antimicrobial agent. People wasnt much aware about the idea of
Carabao Grass as an antimicrobial agent and through this research, people in the
community are beneficial with regards to the availability of resources.

Theoretical Framework
The study consists of antimicrobial assay and phytochemical analysis which then
would determine the attribute of P. conjugatum. In defining this, the researcher must have
endeavor and analyze things to equip themselves in such a manner that would be able to
discover the significance of the study. The Kirby-Bauer agar diffusion method of
antimicrobial assay is a convenient and efficient procedure to conduct the study. It is
placing of filter paper disk with a concentration of treatment and control extract. The
diffusion will depend on the agar properties and the molecular weight of the filter paper
disk. Antimicrobial activity may be observed based on the presence of the zones of
inhibition within the span of 24 hours. The slower the diffusion the larger the molecular
weight present in the filter disk. The size of the inhibition zone will depend on the depth
of the agar. There are some varied results due to timing and temperature but it is
economical, and shows good accuracy in results (Davis & Stout, 1971,Sarker, et al, 2007,
& Hudzicki, 2009).
There are some theories that can support the study of the researchers, theories
such as the Environmental Model.

In this theory, it discussed about focusing on changing and manipulating the

environment in order to put the patient in best possible conditions for nature to act. It is
explained that the said study has relation to this theory for it benefits the community to
lessen their expenses in health services and can be an alternative antimicrobial regimen
for people living far away from health centers and pharmacies.

Objectives of the Study

It was only during 1994 that there was no thorough analysis of phytochemicals of
the said sample (Cambie, 1994) and antimicrobial agent of it, with it, this study aims to
pursue the following objectives:
1.

To determine the presence of P. conjugatum, other than flavonoid compound that has

been found in the plants to assert antimicrobial agent of the plant.

2.

To develop two (2) different preparations that can be obtained from P. conjugatum.

Thru fresh juice extract of plant and maceration.

3.

To evaluate the antimicrobial activity of P.conjugatum based on the zones of

inhibition against the test organism such as Staphylococcus aureus (Gram-positive),

Pseudomonas aeruginosa and Escherichia coli (Gram-negative).
4.

To determine the significant relationship of antimicrobial activity of Carabao Grass

Decoction against the Chloramphenicol as positive control.

Significance of the Study
Over the years, plants has been a primarily source for development of drugs and
some are beneficial in an extensive form to gauge in clinical use (Kala, 2011). This study

is to be conducted to provide information about antimicrobial activity of the Carabao

Grass (P. conjugatum) through Kirby-Bauer disc diffusion test against the bacteria
namely, Staphylococcus aureus, Pseudomonas auruginosa and Escherichia coli. The
gathered data for antimicrobial activity of P. conjugatum will be useful to gain
alternatives in combating microbial infection. This study would be beneficial in the
following groups and institutions:
Community. The study is primarily for the beneficial of the community that would be
able to have easily access of antimicrobial agent and that is easily prepared and will much
cost lesser than other antimicrobial drugs. The outcome of the study would be greatly
helpful as it is will be payless to access too. Complication of microbial agent would be
easily tended.
Pharmaceutical Institutions. Delivering result for antimicrobial agent of P. conjugatum
will pull the curiosity of institutions and that would lead to advance study and
development of the plant and pursue an affordable antimicrobial drug.
Government and non-government Health Services Institutions. Of the new discovery
of the antimicrobial agent in P. conjugatum institution like DOH and NGO organization
would be able to enhance and developed the ability of P. conjugatum as antimicrobial and
informed the public about the effectiveness of the drug which then useful in health
services.
Future Researchers. Upcoming researchers may improve, innovate or enhance a more
comprehensive study. They would be able to create a more potent antimicrobial agent
with the aid of this study. Moreover, this study would be useful as reference in the study
of P. conjugatum other medicinal uses.

Scope and Limitation of the study

This research is to be conducted to determine if P. conjugatum as a promising
antimicrobial agent. It is an in vitro study in the settings of College of Nursing laboratory
in Mindanao State University-Iligan Institute of Technology (MSU-IIT), Iligan City.
The Kirby-Bauer agar diffusion method of antimicrobial assay will be conducted
to demonstrate the presence of any possible antimicrobial property. The test
microorganisms were obtained in the microbiology laboratory of biology department.
Gathering of P. conjugatum will take place in Iligan City. Then it would be
checked by a designated professor of botany of the Department of Biological Sciences of
Mindanao State University Iligan Institute of Technology (MSU-IIT). The preparation
of the extract will be in College of Nursing Microbiology Laboratory in Mindanao State
University-IliganInstitute of Technology (MSU-IIT), Iligan City. The stem, leaves and
roots are extracted prior to assay, while maceration happen a day before the procedure.

Definition of Terms
Antimicrobial is an agent that kills microorganism or inhibits their growth.
Antimicrobial Assay- a method for quantitatively determining the concentration of an
antibiotic by its effect in inhibiting the growth of a susceptible microorganism.
Bacteria are microscopic living organism, usually one-celled, that can be found
everywhere.

Kirby-Bauer agar diffusion method - is a test which uses antibiotic-impregnated wafers

to test whether bacteria are affected by antibiotics
Minimum inhibition concentration is the lowest concentration of an antimicrobial
that will inhibit the visible growth of a microorganism after overnight incubation.
Mueller Hinton Agar is a microbial growth medium that is commonly used for
antibiotic susceptibility testing.
Zone of inhibition is an area of growth inhibition around a point source, within a
cultured organism on a solid medium, due to the action of growth-inhibitory substance
such as antimicrobial present at the source.

CHAPTER II
Review of Related Literature
Over a thousand years human beings primarily source for medicine is plants and
still, until now people are developing newer medicines that are basically derived from
plants. Then and now has no apparently significant changes that are worthy to be say that
people no longer use plants as a one of sources for medicines (Kala, 2011). On the 20 th
century plant become the emerging medicinal products that have been discovered using
extract and some modern medicines comes from pure compounds isolated from plants.
Ethno medicine helps researcher to conduct new study with basis of this traditional plant
(Paulsen, 2008).
The medicinal effect of the plants that is used as ethno medicines or for the
development of new medicine is the reason that plant produces secondary metabolites
that are no longer needed but these metabolites is the one that give effect for medicinal
use (Kala, 2000& Parr, et al, 2000). Developed countries are using this secondary by
isolating the compound to create a more potent and effective medicine however,
developing countries like our country opted for herbal medicines, due to the lack of
facilities and fund to able isolate pure compounds (Shah, 2009).
Developing herbal medicine rather than pure isolation of pure compound is
sought out to this study, is it about a grass known as Paspalum conjugatum its a type of
grass that grows about 20-40 cm and some might reach 60 cm that grows in an open area
and spread quickly, due to its feature as a creeping grass (Rao, 2000). The grass is known
as buffalo grass; sour grass in Jamaica, Hilo in Hawaii and locally known as pad
pad (Burkill, 1985).

Traditionally in some countries, some countries use a type of grass as a means of

treatment it is known P. conjugatum it is used to help these indigenous people from
Amazon area to relieve their headache, these shuar people uses the grass by infusion
(Russo, 1990). Also, in other country like Cuba, they apply this by mixing the grass in
their bath to relieve malaria; others create a medicine by grinding the grass to formulate
an alternative medicine for pleurisy, pneumonia, flu, fever, and fatigue. The Bahamians
people prepared it with prickly pear (Opuntia) and wood ashes as a medicine for
tuberculosis (Austin, 2004). In the local settings, indigenous people use the grass in
relieving diarrhea problems, fever and wound, the fact that this kind of grass can be
found elsewhere in the local areas (Carabao Grass for Kidney Disease, 2014).
Aside from the phytochemicals that is found in the grass is also the fact that the
plant has been traditionally used as a medicine for variety of ethnicity all over the globe
(Cambie, et al. 1994).P. conjugatum is one of ethno medicine that is used by variety of
people all over the globe. In Trinidad it has many uses that people there use it as a cure
for pleurisy, pneumonia, fever, flu and fatigue (Austin, 2004). It also used as a medicine
in the local community as a reliever for diarrhea, fever and wound (Carabao Grass for
Kidney Disease, 2014). Furthermore, flavonoids have compounds that known to
synthesize as antioxidant, antimicrobial, and many more. Other studies found out that
flavonoids is just not antioxidants but also exhibits antiallergenic, antiviral, antiinflammatory and vasodilation actions (Pietta, 2000).
However, to test the antimicrobial activity P. conjugatum and is to conduct
an in vitro study by which testing it through susceptibility of common bacteria that are
commonly the causes of infections such as S. aureus (Uwaezuoke, & Aririatu, 2005).

Through Kirby-Bauer agar diffusion method the in vitro to test the susceptibility of
bacteria against the extracts of P. Conjugatum which is efficient and reliable in screening
compound of natural extract of the plant (Sarker, et al, 2007).
It has been alarming that more and more of bacteria are resistant to drugs
including S.aureus (Locksley, et al, 1982). As antibacterial becomes to falter from its
efficacy is also the never ending complain of side effect of it that brought together when
taken (Uwaezuoke, & Aririatu, 2005). It is the same also in the locality but rather the
inevitable growing population and poverty, that drags health services to implacably
unavailable to those who needed it most (Chan, 2010 & WHO, 2011).
Justification of the Study
Behind the selection of P. conjugatum in this study of it are as follows. (1) it is
commonly used in other ethnicity and areas (2) it have long been used as medicinal
plant. (3) it is widespread in the local settings especially in rural areas (4) the extracts of
plant were traditionally used to treat skin conditions such as wounds and in addition it
can be easily accessible apart from its to widespread distribution in the country it would
be easily prepared. There are no journals about the study of P. conjugatum for
antimicrobial, and it is the first. With this, researchers are determined to conduct a novel
study regarding the antimicrobial activity and phytochemicals determination.
Synthesis
Treatment of infectious diseases in this millennium shown significant side effect
of either side the physical side effect after taking medicine and the later side effect that
bacteria become resistant to drug. The dilemma is inescapable. Too add more is the
growing population of the country which then parallel by poverty and then health. To

help and minimize this an in vitro study will be conducted that would determine the
susceptibility of bacteria against the extract of P. conjugatum

CHAPTER 3
Methodology of the Study

Research Design
The study will carry out through in vitro study where a strict and proper
observation is required in conducting the antimicrobial assay and keeping it from
contamination wherein samples are cultured bacteria and isolated in a prepared agar. It
will include bacteria of in two different stains types. Two from the For Gram positive the
bacteria is (S. aureus and P. aeruginosa), and one from the Gram-Negative bacteria (E.
coli) for gram-negative stain. The bacteria were are cultured from the microbiology
laboratory of the Department of Biological Sciences. in different group in a Nutrient
broth with agar and Sabouraud dextrose agar. Using Kirby-Bauer zone of inhibition test
was use in determining antimicrobial activity of the decoction extract. agar diffusion
method for screening of an antimicrobial activity of P. conjugatum extract. The
preparation of extract is easy squeezing the plant for fresh juice and the other is
maceration for 24 hours in a room temperature. The control group for this study is the
ethanol and penicillin as negative and positive group respectively.

In the antimicrobial activity will be determined through Kirby-Bauer

antimicrobial test, agar diffusion method by microbial suspension containing bacteria that
was spread evenly onto the surface of the solidified medium. The plates were allowed to
dry solidify for 15 minutes before the test discs were placed at equidistance from each
other. The top cover of plates where were marked as each filter disc paper would be was
placed to create enable have baseline for the zones of inhibition. The petri dish off culture
group bacteria would consists of four filter paper, two for the plant extract and (2) for
standard group. The assay would be were left for 18 to 24 hours. For the zones of
inhibition, overnight and on the next day will be the examination of zone inhibition using
Vernier caliper is used in measuring as a means of measurement. The control and
treatment group would be determined the significance using statistical method with
Statistical Package for the Social Science (SPSS).

Collection and Identification of Plant Material

Fresh plant parts were collected randomly on a widespread distribution in Iligan
City, such as the hill top, backyards and fields. The plants would then be checked by a
designated professor of botany of the Department of Biological Sciences of Mindanao
State University Iligan Institute of Technology (MSU-IIT). Fresh plant material will
then be were washed and cleaned thoroughly under running tap water, and then brought
to the College of Nursing (CON) of Mindanao State University Iligan Institute of
Technology (MSU-IIT) after for preparation of decoction.

Extraction and Preparation of Crude Extracts

Decoction

Prepare the sample of Carabao grass and preheat the pot to 50-60 degree
celsius. Place the carabao grass into the preheated pot for 15 minutes and regulate the
temperature to 50-60 degrees.

Mayers reagent. Solution I: 1.36 g HgCl2 was dissolved in 60mL water. Solution II: 5 g
KI will be dissolved in 10mL water. Both solutions will be combined and diluted with
water to 100 mL. If alkaloids are present a white to yellowish precipitate will appear.
Wagner reagent. 1.27 g I2 (sublimed) and 2 g KI will be dissolved in 20mL water and
filled with water up to 100 mL. A brown precipitate indicates the presence of alkaloids.
Saponins.
About 2.5 g of the plant material will be extracted with boiling water. After
cooling, the extract will be shaken vigorously to froth and will then be allowed to stand
for 15 20 min and classify for saponin content as follows: (no froth = negative; froth
less than 1cm = weakly positive; froth 1.2 cm high = positive; and froth greater than 2cm
high = strongly positive).

Screening of Antimicrobial Activities

The antimicrobial activities of the extracts would be carried out in a Kirby-Bauer
agar diffusion method. Sterilized molten agar (20 ml) will be dispensed to each sterile
disposable Petri dish (diameter 9 cm) and allow it to solidify. Nutrient broth with agar is
used in bacterial culture. The transfer of bacteria is by dripping of alcohol and inoculation
of the string to obtain sample of bacteria in the culture after which, the string with
bacteria will be evenly distributed on each corner of the petri dish. Microbial suspension
containing bacteria will spread evenly onto the surface of the solidified medium. The

plates will be allowed to dry for 15 minutes before the filter paper discs will be placed at
equidistance from each other. Each plate consisted of standard group the negative and
positive group, and two other filter paper discs impregnated with various extracts.
After the assay procedure is completed the Petri dishes will be incubated in an
inverted position at 37C for 18 to 24 hours for bacteria to allow growing. The following
day, observation would be made.

Zones of Inhibition
The diameters of the zone of inhibition (ZOI), defined by the clear area devoid of
growth, will be measured twice. The anti-microbial activities will be determined by the
ratio of the ZOI diameters of the extracts to that of the standard antibiotic that is in the
same petri dish. This is measured through caliper of the distance on the inhibition from
filter paper disc.

Data and statistical analysis

Data will be expressed by mean standard deviation (SD) of triplicates. Two-way
ANOVA will be used to analyze the effect of the plant extraction methods on the
extraction yields. The tests employed Bonferroni post hoc analysis. T- test will be used to
compare antimicrobial activity of the extracts against the standard antibiotic. All
statistical analyses will be ran through using Statistical Package for the Social Science
(SPSS) software (v.12, SPSS, USA)

CHAPTER 4
Results and Discussion

Results for the antimicrobial assay of Carabao Grass leaf extract

ZONE OF INHIBITION (average, in millimeters)
STOCK
SOLUTION

75%
OF
STOCK
SOLUTION

50%
OF
STOCK
SOLUTION

25%
OF
STOCK
SOLUTION

Positive
(Chlorampeni
col)

Negative

Bacillus
subtilis

22.24

Staphylococcu
s aureus

17.08

12

4.25

Escherichia
coli
Pseudomonas
aeruginosa

Carabao Grass (P.conjugatum) does not have an antimicrobial agent in the plant.
The table above shows the zone of inhibition is measured in millimeters. As shown in the
result, the carabao grass decoction extract has no antimicrobial activity against Bacillus

subtilis, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa in 100%,

75%, 50%, and 25% concentration.

CHAPTER 5
Summary of Findings, Conclusions and Recommendations

Summary
The purpose of this study is to determine the presence of antimicrobial property in
the plant called Carabao Grass (P.conjugatum). To develop two (2) different preparations
that can be obtained from P. conjugatum thru fresh juice extract of plant and maceration.
The sample extract has been obtained through the process of decoction. The research
instrument being used in this experiment is Kirby-Bauer agar diffusion method to
determine if the sample has an antimicrobial activity when interacted with the bacterias
being prepared.
Conclusion
The results of the study have allowed the researchers to come up with their
conclusions:
There is no antimicrobial agent can be found in Carabao Grass (P.conjugatum)
The zone of inhibition shows that there is no antimicrobial activity against Bacillus
subtilis, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa
No significant relationship of antimicrobial activity of Carabao Grass decoction against
positive control

Recommendation
The following recommendations are drawn on this study based on the
conclusions.
Pharmaceutical Institution. Inform the pharmaceutical institution about the need to
further do a study regarding Carabao Grass
Government and non-government Health Services Institution. Allocate
sufficient funds to the local researchers in order to conduct different testing for this kind
of plant.
Future Researchers. Conduct a more extensive research and experimentation for
Carabao Grass (P.conjugatum) not only for antimicrobial testing but also for the
antifungal testing and phytochemical analysis.

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