1

CHAPTER I
THE PROBLEM AND SCOPE
Introduction
With the uprising population and commodities in the community, morbidity and
mortality: it is hard to avoid since the people in the society are also experiencing
phenomenal calamities like flood. Floods 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 in Philippines.
In addition, the researchers want to help minimize the expenses in health services
most especially to the medications being bought. 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. This research aims to prove that Carabao Grass (P.conjugatum)
has an antimicrobial agent. 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 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 are not aware about the idea of Carabao Grass as an antimicrobial agent (Ebadi,
2007) and through this research; people in the community are beneficial with regard to
the availability of resources.

Theoretical Framework
The study consists of antimicrobial assay, which then would determine the
attribute of P. conjugatum.
The researcher must endeavor and analyze things to equipped themselves and 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 theories that can support the study of the researchers. Theories
like the Environmental Model. It focuses on the manipulation of the availability of
resources in the environment.
It is in this theory that focusing on changing and manipulating the environment in
order to put the patient in best possible conditions for nature to act. 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

This study aims to pursue the following objectives:
1. To evaluate the antimicrobial activity of Paspalum conjugatum based on the zones
of inhibition against the test organism such as Staphylococcus aureus (Grampositive), Pseudomonas aeruginosa and Escherichia coli (Gram-negative).
2. 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 have been primary source for development of drugs and
some are beneficial in an extensive form to gauge in clinical use (Kala, 2011). This study
will 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. This study would be a tool and be useful as reference in the
study of P. conjugatum other medicinal uses.

Scope and Limitation of the study

This research was 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 antimicrobial diffusion method of antimicrobial assay was
conducted in demonstrating the presence of any possible antimicrobial property. The test
microorganisms were obtained in the microbiology laboratory of biology department.
Gathering of Paspalum conjugatum took place in Iligan City. Then, a Professor of
Botany of the Department of Biological Sciences of Mindanao State UniversityIligan

Institute of Technology (MSU-IIT) checked it. The preparation of the extract was in the
College of Nursing Microbiology Laboratory in Mindanao State University-Iligan
Institute of Technology (MSU-IIT), Iligan City. The stem leaves and roots were extracted
prior to assay.

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
For over a thousand years, human beings primary sources for medicine are the
plants, 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 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
(Bernhoft, 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 are the ones 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 (Hudzicki, 2009).
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 Paspalum conjugatum extracts to determine its biological activity through

putting it to test for its antimicrobial properties.

Developing herbal medicine rather than pure isolation of pure compound is

sought out to this study, a grass known as Paspalum conjugatum.Itis 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 grasses in Jamaica, Hilo in Hawaii and locally known as pad pad
(Burkill, 1985).
Traditionally in some countries, they use this type of grass as a means of
treatment it is known as Paspalum conjugatum. It is used to help these indigenous people
from Amazon area to relieve their headache. These people use 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 Bahamian
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 was found on the grass, the plant has been
traditionally used as a medicine for variety of ethnicity all over the globe (Cambie, et al.
1994).Paspalum conjugatum is one of ethno medicine that is used by variety of people all
over the globe. In Trinidad, people use it as a cure for pleurisy, pneumonia, fever, flu and
fatigue (Austin, 2004). Furthermore, flavonoids have compounds that are known to

synthesize as an antioxidant, antimicrobial, and many more. Other studies found out
those flavonoids are just not antioxidants but also exhibits antiallergenic, antiviral, antiinflammatory and vasodilation actions (Pietta, 2000).
However, to test the antimicrobial activity Paspalum 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 Staphylococcus aureus (Uwaezuoke,
& Aririatu, 2005). Through Kirby-Bauer agar diffusion method,the in vitro study is used
to test the susceptibility of bacteria against the extracts of PaspalumConjugatum, 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 Staphylococcus aureus (Locksley, et al, 1982). As antibacterial becomes to
falter from its efficacy, it also has never ending complaints of the side effects that brought
together upon taken (Uwaezuoke, &Aririatu, 2005). It is also the same 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).
With the majority found on common damp environments, Pseudomonas
aeruginosa is a rod-shaped, strict aerobe. Furthermore, it is regularly found in soilbut
more in waterborne creatures. However, colonization has been found in numerous
different plants and organisms, thus it appeared to bring a colony of more than fifty
percent of the people.
Pseudomonas aeruginosa will be an entrepreneurial pathogen that camwood
actuate infections that regularly bring about hospitalization and are frequently lives

10

undermining. This organism exhibits intrinsic imperviousness to large portions of

antibiotics and could adjust when laid open on antimicrobial operators to create new
safety measures. This permits Pseudomonas aeruginosa to flourish and further develop.
With such survivability, these microscopic organisms may not be best deliberately
monitored alongside hospitals. Its genome will be continually updated under a database,
and it will be comprehensively monitored because of its possibility similarly to as
antibiotic weapon (Joey Silhavy, 2009).
Bacillus subtilis may be a Gram positive, Pole molded bacteria and it is ordinarily
found in soil. It might have been initially named Vibrio subtilis asChristian Gottfried
Ehrenberg might have found it previously during the year 1835. It might have been
renamed Bacillus subtilis clinched alongside 1872 by Ferdinand Cohn. This bacterium
will be also known toward the names feed bacillus, grass bacillus, or Bacillus
globigii.Bacillussubtilis is a endospore framing bacteria, and the endospore types.
Itpermits to withstand an amazing temperature and additionally dry situations.
Bacillussubtilis may be acknowledged and commit aerobicity. The point when over the
vicinity for nitrates or glucoseBacillussubtilis is not recognized pathogenic or harmful.
Furthermore,it will not be an agonizing sickness. Bacillussubtilis needs aflagellum, which
makes motility quicker (Ellen Kirk, 2009).
Escherichia coli (E. coli) microbes ordinarily live in the digestion tracts of
individuals and creatures. Generally, E. coliis innocuous and really is a critical part of the
human intestinal tract. Some E. coli are pathogenic, which means they can bring sickness,
either loose bowels or disease outside of the intestinal tract. The sorts of E. coli that can
bring about loose bowels can be transmitted through tainted water or sustenance, or

11

through contact with creatures or persons (Center for Disease Control and Prevention,
2014).
E. coli comprises of a various gathering of microbes. Pathogenic E. coli strains
are sorted into pathotypes. Six pathotypes are connected with looseness of the bowels and
all things considered are alluded to as diarrhea genicE. coli.

Justification of the Study

The selections of Paspalum conjugatum are as follows. (1) It is commonly used in other
ethnicity and areas; (2) it has been used as a medicinal plant. (3) It is widespread in the
local settings especially in rural areas (4) the extracts of the plant were traditionally used
to treat skin conditions such as wounds and in addition,(5) it can be easily accessible
aside from itswidespread distribution in the country, (6) it can be easily prepared. There
are no journals about the study of Paspalum conjugatum for its antimicrobial activity.
This is the first study to extract the compounds of Paspalum conjugatum, which will then
be used as an effective anti-microbial alternative. With this, researchers are determined to
conduct a novel study regarding its anti-microbial activity.

Synthesis
Treatment of infectious diseases in this millennium shows significant side effects.
Too much intake of this medicine might cause the bacteria to produce resistance to this
drug, thusthis dilemma is inescapable. To help and minimize this problem, an in vitro
study was conducted that would determine the susceptibility of bacteria against the
extract of Paspalum conjugatum.

12

CHAPTER 3
METHODOLOGY

Research Design
The study was carried 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
included two types of bacteria. Two from Gram-positive bacteria (Staphylococcus aureus
and Pseudomonas aeruginosa) and one from Gram negative bacteria (Escherichia coli).
The bacteria were cultured and obtained in the microbiology laboratory of the
Department of Biological Sciences. Kirby-Bauer zone of inhibition test was used in
determining antimicrobial activity of the decoction extract.
In the Kirby-Bauer antimicrobial test, microbial suspension containing bacteria
were spread evenly onto the surface of the solidified medium. The plates were allowed to

13

solidify for 15 minutes before the test discs were placed at equidistance from each other.
The top cover of plates was marked as each filter disc paper was placed to create zones of
inhibition. The petri dish of culture group bacteria consists of four filter paper, two (2) for
the plant extract and two (2) for standard group. The assay was left for 18 to 24 hours.
For the zones of inhibition, Vernier caliper was used in measuring.

Local of the Study

Image source: Google Maps

14

Fresh plant parts were collected near the Andrada heights subdivision, Iligan
City.Fresh plant materials 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)afterdecoction.

Extraction and Preparation of Crude Extracts

Decoction
Prepared the sample of Carabao grass and preheated the pot to 50-60 degree
Celsius. Placed the Carabao grass into the preheated pot for 15 minutes and regulated the
temperature to 50-60 degrees.

Screening of Antimicrobial Activities

The antimicrobial activities of the extracts were carried out in a Kirby-Bauer agar
diffusion method. Sterilized molten agar (20 ml) were dispensed to each sterile
disposable Petri dish (diameter 9 cm) and allowed it to solidify. Nutrient broth with agar
was used in bacterial culture. During the transfer of bacteria, medicine dropper was used
to drop alcohol and inoculation of the string to obtain sample of bacteria in the culture
after which, the string with bacteria were evenly distributed on each corner of the petri
dish. Microbial suspension containing bacteria were spread evenly onto the surface of the
solidified medium. The plates were allowed to dry for 15 minutes before the filter paper
discs was placed at equidistance from each other. Each plate consisted of standard group,

15

the negative and positive group and two other filter paper discs impregnated with various
extracts.
After the assay procedure is completed, the Petri dishes were incubated in an
inverted position at 37C for 18 to 24 hours for bacteria to allow growing. The following
day, observation was made.

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

16

CHAPTER 4
RESULTS AND DISCUSSION

Table 1 displays the results of the antimicrobial assay done with the Carabao
Grass decoction on the four types of organisms namely: Bacillus subtilis, Escherichia
coli, Pseudomonas aeruginosaand Staphylococcus aureus.
The samples given were tested against a positive control, which is
chloramphenicol, a broad-spectrum antibiotic drug, and a negative control, which is
sterile water. The results showed that Carabao Grass decoction has no antimicrobial
activity on all of the test organisms employed in the study. The samples given did not
yield Zones of Inhibition (ZOI).
Table 1 Average Zones of Inhibition (ZOI) in millimeters (mm)

R1

R2

R3

Negative
control
(Distilled
Water)

Bacillus subtilis

22.24

Staphylococcus aureus

17.08

Escherichia coli

12.00

Pseudomonas aeruginosa

4.25

Decoction Extract
Test Organism

Positive control
(Chloramphenic
ol)

*Unit of measurement is in millimeter.

Based on the result, the table showed no antimicrobial activity of the Carabao
decoction extract. It yielded no zones of inhibition (ZOI) from replicates one, two, and
three.

17

The Carabao grass contains chemical compounds such as the flavonoids that are
capable of killing microorganisms. However, the data is in contrary to the study of
Antimicrobial Activity of Betula pendula ,in which the decoction of buds and flowers of
the plant yielded positive inhibition in the Zone of Inhibition with the same bacteria
sample.(Duric et.al., 2013) in which it has antimicrobial effects. The distilled water on
the other hand as negative control yielded an expected zeros result because it does not
contain any antimicrobial properties. The positive control, which is the Chloramphenicol,
yielded antimicrobial activity against the four test organisms such as Bacillus subtilis
(22.24), Staphylococcus aureus (17.08), Escherichia coli (12.00), and Pseudomonas
aeruginosa (4.25 mm).The positive control is a broad-spectrum antibiotic that is
commonly used in the hospital due to its known killing effects on microbes, availability
in the pharmacy, and affordability. In comparison, the decoction extract is no match to the
positive control.

18

CHAPTER 5
SUMMARY OF FINDINGS, CONCLUSIONS AND RECOMMENDATIONS
Summary
The study of the Antimicrobial Activity of Carabao Grass (Paspalumconjugatum)
is a quantitative experimental research. It was done to gather data and facts in the
management of various infections and at the same time a cost-saving intervention to
people in the community that are not financially stable.
The antimicrobial potency of Carabao grass decoction extract was assessed
through the Kirby-Bauer Disc Diffusion Test.
The study was conducted within the period of January to May 2016 in Iligan City,
specifically at MSU- IIT College of Nursing and the Department of Biological Sciences
(DBS) in the College of Science and Mathematics (CSM). As to sample, plants were
gathered in the local area of Iligan City, cleansed from roots to leaves before subject to
decoction. Upon obtaining the sample, extract of Carabao Grass were then forwarded to
laboratory for antimicrobial assay. The samples given were tested against a
chloramphenicol, a broad-spectrum antibiotic as a positive control and distilled water as
negative control. Results of the antimicrobial assay were collected after 24 hours of
determining Zones of Inhibition (ZOI).

19

Conclusion
The results showed that the Carabao grass (Paspalum conjugatum) had no
antimicrobial activity in all three replicates of the four test microorganisms such as the
Bacillus subtilis, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa.
The decoction extract was of no match against the broad-spectrum antibiotic, which is the
Chloramphenicol that yielded antimicrobial activity. In this finding, it could be the said
herbal plant usually used and practiced in the community were not that efficient in
deterring the microbes as used for wound healing. Despite the findings, it was not
conclusive to say that all Carabao grass decoction extract has no effect on killing
microorganisms. Further investigation should be done before we can conclude that the
said sample is not potent for antimicrobial effect.

Recommendation
The following recommendations are drawn on this study based on the results:
1. To conduct a more extensive research and experimentation for Carabao Grass
(Paspalum conjugatum) not only for antimicrobial testing but also for the
antifungal testing and phytochemical analysis.
2. To obtain bigger sample of the Carabo grassfor a better antimicrobial activity . We
also recommend the future researchers to reconduct the antimicrobial testing and
if the result will still be zero they should inform and conduct an awareness
program to the community that this herb or grass is not a potent herb to be used as
an alternative antimicrobial agent to treat their diseases.

20

BIBLIOGRAPHY
BOOKS
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press. 6000 Broken Sound Parkway, Norway.
Cambie, R.C., Ash, J., (1994). Fijian Medicinal Plants. pp. xvi. CSIRO. Australia.
KALA,CP (2006). Medicinal Plants and Sustainable Development. pp. 43-56. Nova
Science Publishers Inc. New York.
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Perry, L.M., (1980) Medicinal plants of East and South East Asia. 620 pp. MIT Press.
Cambridge. Massachusetts.

JOURNAL

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Hollman PCH (2004): Absorption, Bioavailability, and Metabolism of Flavonoids.

Pharmaceutical Biology. 42:7483. Retrieved May 20, 2015 from
http://www.tandfonline.com/doi/pdf/10.3109/13880200490893492
Bernhoft, A. (2013, August). Highlights through the History of Plant Medicine: Journal
of Food Engineering.The Norwegian Academy of Science and Letters,
Oslo,Norway
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aureus:Introduction,transmission and evolution of nosocomial infection.

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Triterpene Products From Different Part of Birch, Bertula pendula, Roth.
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Sheppard Ave E, Toronto, ON M2J 5B5, Canada
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Parr AJ, Bolwell GP (2000). J SciFood Agric: Phenols in the plant and in man.The
potential for possiblenutritional enhancement of the diet by modifying the

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phenols content or profile. 80: 9851012. Institute of Food Research,

Norwich Research Park, Colney, Norwich NR4 7UA, United Kingdom
Pietta PG: (2000). Journal of Natural Products: Flavonoids as antioxidants. 10351042.
American Chemical Society. 1155 Sixteenth Street, NW, Washington, DC
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Ramawat, K.G., Dass, S., & Mathur, (2009). Herbal Drugs: Ethnomedicine to Modern
Medicine., p. 402. Springer. New York City
Rao, V.S. NH, USA. (2000), Principle of Weed Science: Second edition., pp. 437.
Science Publisher, Inc. New York, New York
Sarker SD, Nahar L, Kumarasamy Y (2007): Methods: Microtitre plate-based
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25

APPENDIX
A

26

Mindanao State
University
Iligan Institute of Technology
College of Nursing

November 23, 2015

Prof. Muhmin Michael E. Manting
Faculty
Department of Biological Sciences

Dear Sir,
Good Day!
The students listed below from MSU-IIT College of Nursing will be
conducting an experimental research in partial fulfilment of the requirements
for the degree of Bachelor of Science in Nursing.
In line with this, we would like to request from your department to
authenticate the provided herbal plant to be used of the said experiment.
This will help us fulfil our Nursing Research and hopefully present a research
paper on the same school year.
Hoping for your kind consideration and approval. More power and God
Bless!
Truly yours,

Laeh Mae A. Abugan

Azim M. Baruang
Dieza May O. Teves
Noted By:

27

Prof. Clowe D. Jondonero, MAN, MSciEd

Bernie A. Casera, RN
MSU-IIT, College of Nursing, Dean
Adviser

APPENDIX
B

28

Fig. 1. Determine the location of the inhabited grass

29

Fig. 2. Picking of grass including the roots

Fig. 3. Decoction of the carabao grass

30

Fig. 4. Carabao Grass Escherichia coli 100%

Fig 5. Carabao Grass Bacillus subtilis 100%

31

Fig. 6. Carabao Grass Paseudomonas aeruginosa 100%

Fig. 7. Carabao Grass Staphylococcus aureus 100%