1
CHAPTER I
THE PROBLEM AND SCOPE
Introduction
With the uprising population and commodities in the community, morbidity and mortal mor tality ity:: it is har hard d to avoi avoid d si since nce the people in the society society are also exp experi erienci encing ng phenomenal calamities like flood. Floods may carry microorganisms which can contribute to poor wound healing. Thee re Th rese sear arch cher erss co condu nduct cted ed th this is st study udy si since nce th thee Ca Cara rabao bao r ras asss ! Paspalum Paspalum conjugatum) is conjugatum) is known to have an antimicrobial property and is widely spread in local areas and in most communities in "hilippines. #n addition, the researchers want to help minimi$e the expenses in health services most especially to the medications being bought. There are number of people living in far flung areas that are unfamiliar to this kind of grass which can be a possible alternative antimicrobi antim icrobial al regimen. This research research aims to prove that Carabao rass (P.conjugatum) has an antimicrobial agent. Through this, the researchers will be of help in disseminating information to the people not only in remote areas but also to those people living in the community. The researchers aim to know more about the phytochemical analysis that are found in Carabao rass (P.conjugatum) (P.conjugatum) and and its potential action as an antimicrobial agent. "eople are not aware about the idea of Carabao rass as an antimicrobial agent !%badi, &''() and through this research* people in the community are beneficial with regard to the availability of resources.
2
Theoretical Framewor
The stu study dy con consis sists ts of ant antim imicr icrobi obial al ass assay ay,, whi which ch the then n wou would ld det determ ermine ine the attribute of P. of P. conjugatum conjugatum.. The researcher must endeavor and analy$e things to e+uipped themselves and be able to discover the significance of the study. The irby-auer agar diffusion method of antimicrobial assay is a convenient and effi ef fici cient ent pr proc ocedu edure re to co condu nduct ct th thee st study udy.. #t is pl placi acing ng of fi filt lter er pa pape perr di disk sk wi with th a concentration of treatment and control extract. The diffusion will depend on the agar properties and the molecular weight of the filter paper disk. /ntimicrobial activity may be observed based on the presence of the $ones of inhibition within the span of &0 hours. The slower the diffusion, the larger the molecular weight present in the filter disk. The si$e of the inhibition $one will depend on the depth of the agar. There are some theories that can support the study of the researchers. Theories like the 1%nvironmental 2odel3. #t focuses on the manipulation of the availability of resources in the environment. #t is in this theory that focusing on changing and manipulating the environment in order to put the patient in best possible possible conditions conditions for for nature to act. The said study has relation to this theory for it benefits the community to lessen their expenses in health services and can be an alternative antimicrobial regimen for people living far away from health centers and pharmacies.
3
O!"ecti#e$ o% the Stud&
This study aims to pursue the following ob4ectives: 5. To eva evaluat luatee the antimi antimicro crobia biall activity activity of of Paspalum conjugatum based conjugatum based on the $ones of inh inhibi ibitio tion n agai against nst the tes testt org organi anism sm suc such h as Staphy Staphylococcu lococcuss aureus !ram positive), Pseudomonas positive), Pseudomonas aeruginosa and and Escherichia Escherichia coli !ram-negative). determine the signi significant ficant relationship relationship of antim antimicrobi icrobial al acti activity vity of Carabao 2. To determine rass 6ecoction against the Chloramphenicol as positive control.
Si'ni%icance o% the Stud&
7ver the years, plants have been primary source for development of drugs and some are beneficial in an extensive form to gauge in clinical use !ala, &'55). This study will wi ll pr prov ovid idee in info form rmat atio ion n ab about out an anti timi micr crobi obial al ac acti tivi vity ty of th thee Ca Cara rabao bao r ras asss ! P. P. conjugatum)) thr conjugatum through ough ir irbyby-au auer er dis discc dif diffus fusion ion tes testt aga agains instt the bact bacteri eriaa nam namely ely,, Staphylococcus aureus, aureus, Pseudomonas auruginosa and and Escherichia Escherichia coli. The gathered data for antimicr antimicrobi obial al act activi ivity ty of P P.. conjugatum will be useful to gain alternatives in combating microbial infection. This study would be beneficial in the following groups and institutions: Communit& : The study is primari primarily ly for the beneficial beneficial of the community community that would would be
able to have easily access acc ess of antimicrobial agent and that is easily prepared an d will much cost lesser than other antimicrobial drugs. The outcome of the study would be greatly
4
helpful as it is will be payless to access too. Complication of microbial agent would be easily tended. Pharmaceutical In$titution$ : 6elivering 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. (o#ernment and non)'o#ernment Health Ser#ice$ In$titution$ : 7f the new discovery
of the antimicrobial agent in P. conjugatum institution like 678 and 97 organi$ation would be able to enhance and developed the ability of P. conjugatum as antimicrobial and informed the public about the effectiveness of the drug which then useful in health services. Future Re$earcher$ : pcoming 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.
Sco*e and Limitation o% the $tud&
This research was conducted to determine if P. conjugatum as a promising antimicrobial agent. #t is an in vitro study in the settings of College of 9ursing laboratory in 2indanao ;tate niversity-#ligan #nstitute of Technology !2;-##T), #ligan City. The irby-auer antimicrobial diffusion method of antimicrobial assay was conducted in demonstrating the presence of any possible antimicrobial property. The test microorganisms were obtained in the microbiology laboratory of biology department. athering of Paspalum conjugatum took place in #ligan City. Then, a "rofessor of otany of the 6epartment of iological ;ciences of 2indanao ;tate niversity<#ligan
5
#nstitute of Technology !2;-##T) checked it. The preparation of the extract was in the College of 9ursing 2icrobiology =aboratory in 2indanao ;tate niversity-#ligan #nstitute of Technology !2;-##T), #ligan City. The stem leaves and roots were extracted prior to assay.
De%inition o% Term$ Antimicro!ial + is an agent that kills microorganism or inhibits their growth. Antimicro!ial A$$a&) a method for +uantitatively 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. ,ir!&)Bauer a'ar di%%u$ion method ) is a test which uses antibiotic-impregnated wafers
to test whether bacteria are affected by antibiotics Minimum inhi!ition concentration + is the lowest concentration of an antimicrobial
that will inhibit the visible growth of a microorganism after overnight incubation. Mueller Hinton A'ar + is a microbial growth medium that is commonly used for
antibiotic susceptibility testing. -one o% inhi!ition + is an area of growth inhibition around a point source, within a
cultured organism on a solid medium, due to the action of growth-inhibitory substance such as antimicrobial present at the source.
6
7
CHAPTER II
RE.IE/ OF RELATED LITERAT0RE
For over a thousand years, human beings3 primary sources for medicine are the plants, until now people are developing newer medicines that are basically derived from plants. Then and now has no apparently significant changes that are worthy to say that people no longer use plants as a one of sources for medicines !ala, &'55). 7n the &'th century plant become the emerging medicinal products that have been discovered using extract and some modern medicines comes from pure compounds isolated from plants. %thno medicine helps researcher to conduct new study with basis of this traditional plant !ernhoft, &''>). The medicinal effect of the plants that is used as ethno medicines or for the development of new medicine is the reason that plant produces secondary metabolites that are no longer needed but these metabolites are the ones that give effect for medicinal use !ala, &'''? "arr, et al, &'''). 6eveloped countries are using this secondary by isolating the compound to create a more potent and effective medicine however, developing countries like our country opted for herbal medicines, due to the lack of facilities and fund to able isolate pure compounds !8ud$icki, &''@). The medicinal benefits of this herb are dated in all the early records of the human existence. #t started during the early Chinese era, which is dated more than 0''' years ago up to our generation today, the extracts of these herbs and plants are still being used to treat diseases today. 2edicinal herbs are being used extensively in our generation and are being expanded into much noncomplex forms. This study is then focused on the
8
application of Paspalum conjugatum extracts to determine its biological activity through putting it to test for its antimicrobial properties.
6eveloping herbal medicine rather than pure isolation of pure compound is sought out to this study, a grass known as Paspalum conjugatum.#tis a type of grass that grows about &'-0' cm and some might reach A' cm that grows in an open area and spread +uickly, due to its feature as a creeping grass !Bao, &'''). The grass is known as 1buffalo grass3*3 sour grasses in amaica, 18ilo3 in 8awaii and locally known as 1pad pad3 !urkill, 5@>D). Traditionally in some countries, they use this type of grass as a means of treatment it is known as Paspalum conjugatum. #t is used to help these indigenous people from /ma$on area to relieve their headache. These people use the grass by infusion !Busso, 5@@'). /lso, in other country like Cuba, they apply this by mixing the grass in their bath to relieve malaria* others create a medicine by grinding the grass to formulate an alternative medicine for pleurisy, pneumonia, flu, fever, and fatigue. The ahamian people prepared it with prickly pear !Opuntia) and wood ashes as a medicine for tuberculosis !/ustin, &''0). #n the local settings, indigenous people use the grass in relieving diarrhea problems, fever and wound, the fact that this kind of grass can be found elsewhere in the local areas !Carabao rass for idney 6isease, &'50). /side from the phytochemicals that was found on the grass, the plant has been traditionally used as a medicine for variety of ethnicity all over the globe !Cambie, et al. 5@@0). Paspalum conjugatum is one of ethno medicine that is used by variety of people all over the globe. #n Trinidad, people use it as a cure for pleurisy, pneumonia, fever, flu and fatigue !/ustin, &''0). Furthermore, flavonoids have compounds that are known to
9
synthesi$e as an antioxidant, antimicrobial, and many more. 7ther studies found out those flavonoids are 4ust not antioxidants but also exhibits antiallergenic, antiviral, antiinflammatory and vasodilation actions !"ietta, &'''). 8owever, 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 !wae$uoke, ? /ririatu, &''D). Through irby-auer agar diffusion method,the in vitro study is used to test the susceptibility of bacteria against the extracts of PaspalumConjugatum, which is efficient and reliable in screening compound of natural extract of the plant !;arker, et al , &''(). #t has been alarming that more and more of bacteria are resistant to drugs including Staphylococcus aureus !=ocksley, et al, 5@>&). /s antibacterial becomes to falter from its efficacy, it also has never ending complaints of the side effects that brought together upon taken !wae$uoke, ?/ririatu, &''D). #t 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, &'5' ? W87, &'55). With the ma4ority found on common damp environments, Pseudomonas aeruginosa is a rod-shaped, strict aerobe. Furthermore, it is regularly found in soilbut more in waterborne creatures. 8owever, coloni$ation has been found in numerous different plants and organisms, thus it appeared to bring a colony of more than fifty percent of the people. Pseudomonas aeruginosa will be an entrepreneurial pathogen that camwood actuate infections that regularly bring about hospitali$ation and are fre+uently lives
10
undermining. This organism exhibits intrinsic imperviousness to large portions of antibiotics and could ad4ust 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. #ts genome will be continually updated under a database, and it will be comprehensively monitored because of its possibility similarly to as antibiotic weapon !oey ;ilhavy, &''@). Bacillus subtilis may be a ram positive, "ole molded bacteria and it is ordinarily found in soil. #t might have been initially named Eibrio subtilisG asChristian ottfried %hrenberg might have found it previously during the year 5>HD. #t might have been renamed Eacillus subtilisG clinched alongside 5>(& 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. #tpermits to withstand an ama$ing temperature and additionally dry situations. Bacillussubtilis may be acknowledged and commit aerobicity. The point when over the vicinity for nitrates or glucose Bacillussubtilis is not recogni$ed pathogenic or harmful. Furthermore,it will not be an agoni$ing sickness. Bacillussubtilis needs aflagellum, which makes motility +uicker !%llen irk, &''@). Escherichia coli (E. coli) microbes ordinarily live in the digestion tracts of individuals and creatures. enerally, E. coliis innocuous and really is a critical part of the human intestinal tract. ;ome E. coli are pathogenic, which means they can bring sickness, either loose bowels or disease outside of the intestinal tract. The sorts of E. coli that can bring about loose bowels can be transmitted through tainted water or sustenance, or
11
through contact with creatures or persons !Center for 6isease Control and "revention, &'50). E. coli comprises of a various gathering of microbes. "athogenic E. coli strains are sorted into pathotypes. ;ix pathotypes are connected with looseness of the bowels and all things considered are alluded to as diarrhea genic E. coli.
1u$ti%ication o% the Stud&
The selections of Paspalum conjugatum are as follows. !5) #t is commonly used in other ethnicity and areas* !&) it has been used as a medicinal plant. !H) #t is widespread in the local settings especially in rural areas !0) the extracts of the plant were traditionally used to treat skin conditions such as wounds and in addition,!D) it can be easily accessible aside from itswidespread distribution in the country, !A) it can be easily prepared. There are no 4ournals about the study of Paspalum conjugatum for its antimicrobial activity. This is the first study to extract the compounds of Paspalum conjugatum, hich 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.
S&nthe$i$
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 minimi$e this problem, an in vitro study was conducted that would determine the susceptibility of bacteria against the extract of Paspalum conjugatum.
12
CHAPTER 2
METHODOLO(3
Re$earch De$i'n
The study was carried out through in vitro study where a strict and proper observation is re+uired 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 ram-positive bacteria !Staphylococcus aureus and Pseudomonas aeruginosa) and one from ram negative bacteria ! Escherichia coli). The bacteria were cultured and obtained in the microbiology laboratory of the 6epartment of iological ;ciences. irby-auer $one of inhibition test was used in determining antimicrobial activity of the decoction extract. #n the irby-auer antimicrobial test, microbial suspension containing bacteria were spread evenly onto the surface of the solidified medium. The plates were allowed to
13
solidify for 5D minutes before the test discs were placed at e+uidistance from each other. The top cover of plates was marked as each filter disc paper was placed to create $ones of inhibition. The petri dish of culture group bacteria consists of four filter paper, two !&) for the plant extract and two !&) for standard group. The assay was left for 5> to &0 hours. For the $ones of inhibition, ernier caliper was used in measuring.
Local o% the Stud&
#mage source: oogle 2aps
14
Fresh plant parts were collected near the /ndrada heights subdivision, #ligan City.Fresh plant materials were washed and cleaned thoroughly under running tap water, and then brought to the College of 9ursing !C79) of 2indanao ;tate niversity < #ligan #nstitute of Technology !2;-##T)afterdecoction.
E4traction and Pre*aration o% Crude E4tract$ Decoction
"repared the sample of Carabao grass and preheated the pot to D'-A' degree Celsius. "laced the Carabao grass into the preheated pot for 5D minutes and regulated the temperature to D'-A' degrees.
Screenin' o% Antimicro!ial Acti#itie$
The antimicrobial activities of the extracts were carried out in a irby-auer agar diffusion method. ;terili$ed molten agar !&' ml) were dispensed to each sterile disposable "etri dish !diameter @ cm) and allowed it to solidify. 9utrient broth with agar was used in bacterial culture. 6uring the transfer of bacteria, medicine dropper was used to drop alcohol and inoculation of the string to obtain sample of bacteria in the culture after which, the string with bacteria were evenly distributed on each corner of the petri dish. 2icrobial suspension containing bacteria were spread evenly onto the surface of the solidified medium. The plates were allowed to dry for 5D minutes before the filter paper discs was placed at e+uidistance from each other. %ach plate consisted of standard group,
15
the negative and positive group and two other filter paper discs impregnated with various extracts. /fter the assay procedure is completed, the "etri dishes were incubated in an inverted position at H(IC for 5> to &0 hours for bacteria to allow growing. The following day, observation was made.
-one$ o% Inhi!ition
The diameters of the $one of inhibition !J7#), defined by the clear area devoid of growth, were measured twice. The anti-microbial activities were determined by the ratio of the J7# diameters of the extracts to that standard antibiotic is in the same petri dish. This was measured through ernier caliper of the distance on the inhibition from filter paper disc.
16
CHAPTER 5
RES0LTS AND DISC0SSION
Ta!le 6 displays the results of the antimicrobial assay done with the Carabao
rass decoction on the four types of organisms namely: Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosaand Staphylococcus aureus.
The
samples given
were tested against a positive control,
which is
chloramphenicol, a broad-spectrum antibiotic drug, and a negative control, which is sterile water. The results showed that Carabao rass decoction has no antimicrobial activity on all of the test organisms employed in the study. The samples given did not yield Jones of #nhibition !J7#).
Ta!le 6 /verage Jones of #nhibition !J7#) in millimeters !mm)
)1
)2
)3
Negative contro !Distie" #ater$
Bacillus subtilis
0
0
0
0
22.24
Staphylococcus aureus
0
0
0
0
17.08
Escherichia coli
0
0
0
0
12.00
Pseudomonas aeruginosa
0
0
0
0
4.25
Decoction Extract Test Organism
%ositive contro !&'oram('enic o$
Knit of measurement is in millimeter.
ased on the result, the table showed no antimicrobial activity of the Carabao decoction extract. #t yielded no $ones of inhibition !J7#) from replicates one, two, and three.
17
The Carabao grass contains chemical compounds such as the flavonoids that are capable of killing microorganisms. 8owever, the data is in contrary to the study of /ntimicrobial /ctivity of Betula pendula ,in which the decoction of buds and flowers of the plant yielded positive inhibition in the Jone of #nhibition with the same bacteria sample.!6uric et.al., &'5H) in which it has antimicrobial effects. The distilled water on the other hand as negative control yielded an expected $eros result because it does not contain any antimicrobial properties. The positive control, which is the Chloramphenicol, yielded antimicrobial activity against the four test organisms such as Bacillus subtilis !&&.&0), Staphylococcus aureus !5(.'>), Escherichia coli !5&.''), and Pseudomonas aeruginosa !0.&D mm).The positive control is a broad-spectrum antibiotic that is commonly used in the hospital due to its known killing effects on microbes, availability in the pharmacy, and affordability. #n comparison, the decoction extract is no match to the positive control.
18
CHAPTER 7
S0MMAR3 OF FINDIN(S8 CONCL0SIONS AND RECOMMENDATIONS
Summar&
The study of the /ntimicrobial /ctivity of Carabao rass ! Paspalumconjugatum) is a +uantitative experimental research. #t was done to gather data and facts in the management of various infections and at the same time a cost-saving intervention to people in the community that are not financially stable.
The antimicrobial potency of Carabao grass decoction extract was assessed through the irby-auer 6isc 6iffusion Test.
The study was conducted within the period of anuary to 2ay &'5A in #ligan City, specifically at 2;- ##T College of 9ursing and the 6epartment of iological ;ciences !6;) in the College of ;cience and 2athematics !C;2). /s to sample, plants were gathered in the local area of #ligan City, cleansed from roots to leaves before sub4ect to decoction. pon obtaining the sample, extract of Carabao rass were then forwarded to laboratory for antimicrobial assay. The samples given were tested against a chloramphenicol, a broad-spectrum antibiotic as a positive control and distilled water as negative control. Besults of the antimicrobial assay were collected after &0 hours of determining Jones of #nhibition !J7#).
19
Conclu$ion
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. #n 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. 6espite 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 stud y based on the results: 5. To conduct a more extensive research and experimentation for Carabao rass ! Paspalum conjugatum) not only for antimicrobial testing but also for the antifungal testing and phytochemical analysis. &. 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 $ero 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
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&'5A Escherichia
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from
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24
25
APPENDI9
A
26
Mindanao State University Iligan Institute of Technology College of Nursing
Novem*er 23, 2015 %ro+. -'min ic'ae E. anting ac-t/ De(artment o+ ioogica ciences
Dear ir, oo" Da/ T'e st-"ents iste" *eo +rom T &oege o+ N-rsing i *e con"-cting an ex(erimenta researc' in (artia +-ment o+ t'e re-irements +or t'e "egree o+ ac'eor o+ cience in N-rsing.
n ine it' t'is, e o-" i:e to re-est +rom /o-r "e(artment to a-t'enticate t'e (rovi"e" 'er*a (ant to *e -se" o+ t'e sai" ex(eriment. T'is i 'e( -s +- o-r N-rsing )esearc' an" 'o(e+-/ (resent a researc' (a(er on t'e same sc'oo /ear. ;o(ing +or /o-r :in" consi"eration an" a((rova. ore (oer an" o" ess Tr-/ /o-rs,
im . ar-ang Die>a a/ O. Teves Note" /?
27
Prof. Clowe D. Jondonero, MN, MSci!d "ernie . Casera, #N T, &oege o+ N-rsing, Dean ="viser
APPENDI9
B
28
Fig. 5. 6etermine the location of the inhabited grass
29
Fig. &. "icking of grass including the roots
Fig. H. 6ecoction of the carabao grass
30
ig. 4. &ara*ao rass Escherichia coli 100@
ig 5. &ara*ao rass Bacillus subtilis 100@
