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
2
THE PROBLEM Introduction
1
Theoretical Framework
1
Conceptual Framework
3
Objectives of the Study
5
Hypothesis of the Study
5
Significance of the Study
6
Scope and Delimitation
7
Definition of terms
8
REVIEW OF RELATED LITERATURE AND STUDIES
9
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 wasn’t 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 it’s 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.
Mayer’s 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 37°C 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
0
0
0
0
22.24
0
Staphylococcu s aureus
0
0
0
0
17.08
0
0
0
0
0
12
0
0
0
0
0
4.25
0
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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