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7/29/2019 SYNERGISTIC ANTIBACTERIAL ACTIVITY OF AMOXICILLIN AND BAKHAW (Rhizophora mucronata) BARK CRUDE METHANOLIC AND AQUEOUS EXTRACTS AGAINST Staphylococcus au
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Genessa A. Buenafe
Celestino Ray G. Monroy
Vir Nigel A. Tagongtong
Janine B. Dalisay
Jasmine Marie A. Jegonia
Demi Marie S. Oto
Angelie A. Virgo
Mrs. Christine Alog-Villanueva
Adviser
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Throughout the history of mankind, infectious diseases have
remained a major cause of death and disability. Recently,
studies indicate the presence of antibiotic resistant microbesthat is the cause of common infections. Infectious diseases
caused by bacteria affect millions of people worldwide.
Thus, the formulation and production of potent antibiotics
against the infectious agents is a major undertaking for
pharmaceutical institutions nowadays. However, according to
Chanda and Rakholiya, over the past few decades these healthbenefits are under threat as many commonly used antibiotics
have become less and less effective against certain illnesses.
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One approach to treat infectious diseases is the use of plant
extracts individually or as an alternative approach is the useof a combination of antibiotics with plant extracts. This latter
approach, combination therapy or synergistic therapy; against
resistant microorganisms may lead to new ways of treating
infectious diseases and probably this represents a potential
area for further future investigations (Chanda, 2011).
One of the plants in the Philippines that show great potential
in its phytochemical activity as an antibacterial agent are
mangroves, specificallyRhizophora spp.
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According to Abeysinghe, most of the plant extracts of
mangroves showed promising antibacterial activity against
bacteria. As such is the Stilt Mangrove or locally known as
Bakhaw (Rizophora mucronata lamk.) which has been
proven by previous studies that possess a potential
antibacterial activity.
Hence, this study looked on the possibility of a synergistic
antibacterial activity of Amoxicillin and Bakhaw (Rhizophora
mucronata) bark crude methanolic and aqueous extracts
against Staphylococcus aureus andEscherichia coli.
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General Objectives
Generally, this research aims to identify the
synergistic antibacterial activity of Amoxicillin
and Bakhaw (Rhizophora mucronata) bark crudemethanolic and aqueous extracts against
Staphylococcus aureus andEscherichia coli.
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Specific Objectives
Specifically, this research aims to:
1. To determine the zone of inhibition against Staphylococcus
aureus andEscherichia coli of the different treatments:
Treatment ACrude Methanolic Plant Extract
Treatment BCrude Aqueous Plant Extract
Treatment CAmoxicillin
Treatment DCrude Methanolic Plant Extract (10 g) +
Amoxicillin (25 g)
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Treatment ECrude Methanolic Plant Extract (100 g)
+ Amoxicillin (25 g)
Treatment FCrude Methanolic Plant Extract (1000 g)
+ Amoxicillin (25 g)
Treatment GCrude Aqueous Plant Extract (10 g) +
Amoxicillin (25 g)
Treatment HCrude Aqueous Plant Extract (100 g) +
Amoxicillin (25 g)
Treatment ICrude Aqueous Plant Extract (1000 g) +
Amoxicillin (25 g)
Positive ControlCo- Amoxiclav
Negative ControlAmoxicillin + distilled water
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Specific Objectives
2. To determine if there is a significant difference
in the mean zone of inhibition among different
treatment concentrations as well as the positiveand the negative control.
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Specific Objectives
3. To determine if there is a significant difference
in the mean zone of inhibition among treatment
concentrations using two methods of extraction(Methanolic and Aqueous extraction).
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Specific Objectives
4. To determine which among the treatments
and mode of extraction shows the highest
synergistic activity against Staphylococcusaureus andEscherichia coli.
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Hypothesis
There is no significant difference between
the different treatment concentrations as well
as the positive and the negative controlsbased on the zone of inhibition.
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Hospitals and Medical Institutions
Pharmaceutical companies
Communities Future researchers
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CONCEPTUAL FRAMEWORK
Independent VariablesDifferent concentrations of thecrude methanolic and aqueous
plant extracts combined withamoxicillin.
Dependent Variables
Diameter of the Zone ofInhibition
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This study is limited only to identifying the
synergistic antibacterial activity of Amoxicillin and
Bakhaw (Rhizophora mucronata)bark crudemethanolic and aqueous extracts against
Staphylococcus aureus andEscherichia coli.
The plant will be taken from Oton, Iloilo and
Bakhaw, Jaro, Iloilo City, only. These mangroves
will be identified by Dr. Junemie Ramos, mangrove
expert, SEAFDEC AQD.
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The study will be conducted at the University of San
Agustin Research laboratory. Likewise, the positive
control (AmoxiClav) as well as the negative control(Amoxicillin- distilled water solution) will be bought
commercially. The Staphylococcus aureus and
Escherichia coli for culture will also be purchased
commercially.
The statistical tool used in analyzing the data will be the
Mean, Two Way Analysis of Variance and will befollowed by the Post HoC analysis with a confidence
level of 0.05.
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Research Design
The research design is an experimental research
design. The independent variables are the
different treatments while the dependentvariables are the zone of inhibition. Quantitative
data will be collected and will be analyzed to
answer the objectives of the study.
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PLANT
COLLECTION
PLANT
IDENTIFICATION
PROCESSING
OF PLANT
SAMPLES
METHANOLIC
EXTRACTION
AQUAEOUS
EXTRACTION
PREPARATION
OF TREATMENT
ANTIBACTERIAL
SUSCEPTIBILITY
TESTING
AGAR WELL
DIFFUSION
METHOD
DISC
DIFFUSION
METHOD
MEASUREMENT
ZONE OF
INHIBITION
PLAN FOR
DATA
ANALYSIS
PROPER WASTE
DISPOSAL
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Research Design
The testing for the presence of the resistant
Enterobacteriaceae will involve the ModifiedHodge Test for its confirmation. Blood
samples from patients with septicemia will be
used for isolating bacterial samples which will
be used for the said test.
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Plant Collection
The Bakhaw (Rhizophora mucronata) plant will be
collected from Oton, Iloilo. The plant bark shall be
collected and shall be transported to laboratory
where the extraction will be done. Regions whereplant barks are taken will be wrapped with moist
paper/membrane to promote healing.
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Plant Identification
The plant identification will be done by Dr.
Junemie Ramos, mangrove expert, SEAFDEC
AQD.
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Processing of plant samples
The bark from the plant samples will be collected
and will be air dried in a dark room for two
weeks. It will weighed and pounded to obtain250g of the Bakhaw (Rhizophora mucronata)
bark.
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Methanolic Extraction
For extraction of crude bioactives, 250g of
powered mangrove material will be extracted
with one liter of methanol for 24 hours. Theextracts will be further concentrated by
recovering excess solvents to thick oily natured
crude in a rotary evaporator at reduced pressure.The extract will then be stored at 4 degrees
Celcius in air- tight plastic vials.
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Aqueous Extraction
For extraction of crude bioactives, 250g of
powered mangrove material will be extracted
with one liter of sterile distilled water for 24
hours. The extracts will be further concentrated
by recovering excess solvents to thick oily
natured crude in a rotary evaporator at reduced
pressure. The extract will then be stored at 4degrees Celcius in air- tight plastic vials.
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Preparation of Treatment
Following the two methods of extraction is the preparation
of the treatments for antibacterial testing. The treatments
will be prepares with the concentration stated in the
following:
Treatment ACrude Methanolic Plant Extract
Treatment BCrude Aqueous Plant Extract
Treatment CAmoxicillin Treatment DCrude Methanolic Plant Extract (10 g) +
Amoxicillin (25 g)
Treatment ECrude Methanolic Plant Extract (100 g) +
Amoxicillin (25 g)
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Treatment FCrude Methanolic Plant Extract
(1000 g) + Amoxicillin (25 g) Treatment GCrude Aqueous Plant Extract (10
g) + Amoxicillin (25 g)
Treatment HCrude Aqueous Plant Extract (100g) + Amoxicillin (25 g)
Treatment ICrude Aqueous Plant Extract(1000 g) + Amoxicillin (25 g)
Positive ControlCo- Amoxiclav
Negative ControlAmoxicillin + distilled water
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Antibacterial Susceptibility
Testing
Parallel antibacterial susceptibility testing will be
done by the three methods namely, Agar well
diffusion method, and Disc diffusion method.
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Agar- Well Diffusion Method In this method, the antimicrobials present in the
plant extract are allowed to diffuse out into the
medium and interact in a plate freshly seeded
with the test organisms. The resulting zones of
inhibition will be uniformly circular as there will
be a confluent lawn of growth. The diameter ofzone of inhibition will be measured in
millimeters.
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REAGENTS:
Muller Hinton Agar Medium (1 L)
The medium will be prepared by dissolving 33.9 g of thecommercially available Muller Hinton Agar Medium (HiMedia)in 1000ml of distilled water. The dissolved medium will beautoclaved at 15 lbs pressure at 121C for 15 minutes. Theautoclaved medium will be mixed well and poured onto 100mmpetriplates (25-30ml/plate) while still molten.
Nutrient broth (1L)
One litre of nutrient broth will be prepared by dissolving 13 g ofcommercially available nutrient medium (HiMedia) in 1000mldistilled water and boiled to dissolve the medium completely.
The medium will be dispensed as desired and sterilized byautoclaving at 15 lbs pressure (121C) for 15 minutes.
Co-Amoxiclav disc (standard antibacterial agent)
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PROCEDURE:
Petriplates containing 20ml Muller Hinton mediumwill be seeded with 24hr culture of bacterial strains:Staphylococcus aureus andEscherichia coli.
Aseptically swab the test organism (Escherichia coliand Staphylococcus aureus) to respective MuellerHinton Agar plate by streaking the swab over theentire surface of the agar plate three times, rotatingthe plate approximately 60 degrees after each
application to ensure an even distribution of theinoculums on the surface of the medium. The platesthen will stand for 5 minutes.
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Wells will be cut using a sterile glass tubing for anequal diameter of the well will be used to stab the
agar to create five wells cleanly cut using aseptictechnique. 20 l of the treatments will be added. Thetreatments will be namely:
Treatment ACrude Methanolic Plant Extract Treatment BCrude Aqueous Plant Extract
Treatment CAmoxicillin
Treatment DCrude Methanolic Plant Extract (10g) + Amoxicillin (25 g)
Treatment ECrude Methanolic Plant Extract (100g) + Amoxicillin (25 g)
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Treatment FCrude Methanolic Plant Extract (1000
g) + Amoxicillin (25 g)
Treatment GCrude Aqueous Plant Extract (10 g) +
Amoxicillin (25 g)
Treatment HCrude Aqueous Plant Extract (100 g)
+ Amoxicillin (25 g) Treatment ICrude Aqueous Plant Extract (1000 g)
+ Amoxicillin (25 g)
Positive ControlCo- Amoxiclav Negative ControlAmoxicillin + distilled water
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The plates will be incubated at 37C for 24
hours. The antibacterial activity will be assayed
by measuring the diameter of the inhibition zoneformed around the well (NCCLS, 1993).
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Disc Diffusion Method
In this method, paper discs impregnated withspecific antibiotics or the test substances areplaced on the surface of the Muller Hinton agar
medium inoculated with the target organisms,which is recommended for the diffusion ofantimicrobial agents as described in NCCLSapproved standard. The plates are incubated and
the zones of inhibition around each disc aremeasured.
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PROCEDURE:
Muller Hinton Agar plates will be prepared and the testmicroorganisms were inoculated by the spread platemethod. Filter paper discs approximately 6mm indiameter will be soaked with 5 l of the treatments:
Treatment ACrude Methanolic Plant Extract Treatment BCrude Aqueous Plant Extract
Treatment CAmoxicillin
Treatment DCrude Methanolic Plant Extract (10 g)
+ Amoxicillin (25 g) Treatment ECrude Methanolic Plant Extract (100
g) + Amoxicillin (25 g)
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Treatment FCrude Methanolic Plant Extract (1000
g) + Amoxicillin (25 g) Treatment GCrude Aqueous Plant Extract (10 g) +
Amoxicillin (25 g)
Treatment HCrude Aqueous Plant Extract (100 g) +
Amoxicillin (25 g) Treatment ICrude Aqueous Plant Extract (1000 g) +
Amoxicillin (25 g)
Positive ControlCo- Amoxiclav
Negative ControlAmoxicillin + distilled water
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Each disc will be pressed down to ensurecomplete contact with the agar surface and
distributed evenly so that they are no closer than
24 mm from each other, center to center. The
agar plates will be then incubated at 37C. After16 to 18 hours of incubation, each plate will be
examined. The diameters of the zones of
complete inhibition will be measured.
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Measurement of Zone of
Inhibition
Every zone of inhibition of each treatment and
control in the petri dish of the three trials for
Escherichia coli and Staphylococcus aureus will
be measured using a ruler in millimeters.
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Plan for Data Analysis
To identify which of the two extraction methods(methanolic and aqueous) of the Bakhaw(Rhizophora mucronata) crude extract plant is moresynergistic with Amoxicillin, the 1-way ANOVA
with p= 0.05 will be used.
Moreover, the POST HOC (Least SignificantDifference) will be used to identify which among
concentrations of the Bakhaw (Rhizophoramucronata) crude extract plant with Amoxicillin isthe most synergistic in both extraction procedures
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Proper Waste Disposal
Apparatus, waste products, as well as test
organisms, shall be disinfected and sterilized
according to the protocols of the laboratory
where the experiment has been done and shouldbe assisted by proper personnel of the laboratory
applying protective measures that are inherent in
the laboratory.
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S l d f i
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Solvent used for active component
extraction
h h i l f kh
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Phytochemical Content of Bakhaw
(Rhizophora mucronata)
Table on the Zone of Inhibition (mm) of R mucronata
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Table on the Zone of Inhibition (mm) ofR. mucronata
extracts based on the study conducted by Nurdiani et al.
(2008)
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Budgetary Requirement
ITEMS AMOUNT TOTAL
Data Collection Expenses
Laboratory Fees and Materials
Meals
Honorarium
5,000 Php
1,000 Php
3,000 Php
9,000 Php
Printing and Binding 3,000 Php
Total 12,000 Php
D T bl
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Dummy Table
Treatment
Zone of inhibition (mm)Mean zone of
Inhibition
Trial 1 Trial 2
R1 R2 R3 R1 R1 R3
A
B
C
D
E
F
G
H
I
J
K
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GANTTS CHART
ACTIVITY JAN. FEB. MAR APRIL MAY JUNE JULY
Writing of
research
proposal
Approval of
research
proposal
Submission of
letter ofpermission to the
University of San
Agustin Research
Laboratory
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ACTIVITY JAN. FEB. MAR. APRIL MAY JUNE JULY
Plant CollectionPlant IdentificationProcessing of plant
samples
Methanolic Extraction
Aqueous Extraction
Antibacterial
Susceptibility Testing
Data collectionWriting of result
analysisFormulation of
conclusion
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ACTIVITY JAN. FEB. MAR. APRIL MAY JUNE JULY
Writing of final
research report
Presentation of
results
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Genessa A. Buenafe
Celestino Ray G. Monroy
Vir Nigel A. Tagongtong
Janine B. Dalisay
Jasmine Marie A. Jegonia
Demi Marie S. OtoAngelie A. Virgo
Mrs. Christine Alog-Villanueva
Adviser