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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

Throughout the history of mankind, infectious diseases have remained a major cause of death and disability. Recently, studies indicate the presence of antibiotic resistant microbes that 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 health benefits are under threat as many commonly used antibiotics have become less and less effective against certain illnesses.

One approach to treat infectious diseases is the use of plant extracts individually or as an alternative approach is the use of 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, specifically Rhizophora spp.

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 and Escherichia coli.

General Objectives
Generally, this research aims to identify the synergistic antibacterial activity of Amoxicillin and Bakhaw (Rhizophora mucronata) bark crude methanolic and aqueous extracts against Staphylococcus aureus and Escherichia coli.

Specific Objectives
Specifically, this research aims to:
1. To determine the zone of inhibition against Staphylococcus aureus and Escherichia coli of the different treatments:

Treatment A Crude Methanolic Plant Extract Treatment B Crude Aqueous Plant Extract Treatment C Amoxicillin Treatment D Crude Methanolic Plant Extract (10 g) + Amoxicillin (25 g)

Treatment E Crude Methanolic Plant Extract (100 g) + Amoxicillin (25 g) Treatment F Crude Methanolic Plant Extract (1000 g) + Amoxicillin (25 g) Treatment G Crude Aqueous Plant Extract (10 g) + Amoxicillin (25 g) Treatment H Crude Aqueous Plant Extract (100 g) + Amoxicillin (25 g) Treatment I Crude Aqueous Plant Extract (1000 g) + Amoxicillin (25 g) Positive Control Co- Amoxiclav Negative Control Amoxicillin + distilled water

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 positive and the negative control.

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).

Specific Objectives
4. To determine which among the treatments and mode of extraction shows the highest synergistic activity against Staphylococcus aureus and Escherichia coli.

Hypothesis

There is no significant difference between the different treatment concentrations as well as the positive and the negative controls based on the zone of inhibition.

Hospitals and Medical Institutions Pharmaceutical companies Communities Future researchers

CONCEPTUAL FRAMEWORK

Independent Variables Different concentrations of the crude methanolic and aqueous plant extracts combined with amoxicillin.

Dependent Variables Diameter of the Zone of Inhibition

This study is limited only to identifying the synergistic antibacterial activity of Amoxicillin and Bakhaw (Rhizophora mucronata) bark crude methanolic and aqueous extracts against Staphylococcus aureus and Escherichia 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.

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 be followed by the Post HoC analysis with a confidence level of 0.05.

Research Design

The research design is an experimental research design. The independent variables are the different treatments while the dependent variables are the zone of inhibition. Quantitative data will be collected and will be analyzed to answer the objectives of the study.

PLANT COLLECTION

PLANT IDENTIFICATION

PROCESSING OF PLANT SAMPLES

PREPARATION OF TREATMENT

AQUAEOUS EXTRACTION

METHANOLIC EXTRACTION

ANTIBACTERIAL SUSCEPTIBILITY TESTING

AGAR WELL DIFFUSION METHOD

DISC DIFFUSION METHOD

PROPER WASTE DISPOSAL

PLAN FOR DATA ANALYSIS

MEASUREMENT ZONE OF INHIBITION

Research Design
The testing for the presence of the resistant Enterobacteriaceae will involve the Modified Hodge 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.

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 where plant barks are taken will be wrapped with moist paper/membrane to promote healing.

Plant Identification

The plant identification will be done by Dr. Junemie Ramos, mangrove expert, SEAFDEC AQD.

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 obtain 250g of the Bakhaw (Rhizophora mucronata) bark.

Methanolic Extraction

For extraction of crude bioactives, 250g of powered mangrove material will be extracted with one liter of methanol 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 4 degrees Celcius in air- tight plastic vials.

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 4 degrees Celcius in air- tight plastic vials.

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 A Crude Methanolic Plant Extract Treatment B Crude Aqueous Plant Extract Treatment C Amoxicillin Treatment D Crude Methanolic Plant Extract (10 g) + Amoxicillin (25 g) Treatment E Crude Methanolic Plant Extract (100 g) + Amoxicillin (25 g)

Treatment F Crude Methanolic Plant Extract (1000 g) + Amoxicillin (25 g) Treatment G Crude Aqueous Plant Extract (10 g) + Amoxicillin (25 g) Treatment H Crude Aqueous Plant Extract (100 g) + Amoxicillin (25 g) Treatment I Crude Aqueous Plant Extract (1000 g) + Amoxicillin (25 g) Positive Control Co- Amoxiclav Negative Control Amoxicillin + distilled water

Antibacterial Susceptibility Testing

Parallel antibacterial susceptibility testing will be done by the three methods namely, Agar well diffusion method, and Disc diffusion method.

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 of zone of inhibition will be measured in millimeters.

REAGENTS:

Muller Hinton Agar Medium (1 L) The medium will be prepared by dissolving 33.9 g of the commercially available Muller Hinton Agar Medium (HiMedia) in 1000ml of distilled water. The dissolved medium will be autoclaved at 15 lbs pressure at 121C for 15 minutes. The autoclaved medium will be mixed well and poured onto 100mm petriplates (25-30ml/plate) while still molten.

Nutrient broth (1L) One litre of nutrient broth will be prepared by dissolving 13 g of commercially available nutrient medium (HiMedia) in 1000ml distilled water and boiled to dissolve the medium completely. The medium will be dispensed as desired and sterilized by autoclaving at 15 lbs pressure (121C) for 15 minutes.
Co-Amoxiclav disc (standard antibacterial agent)

PROCEDURE:

Petriplates containing 20ml Muller Hinton medium will be seeded with 24hr culture of bacterial strains: Staphylococcus aureus and Escherichia coli. Aseptically swab the test organism (Escherichia coli and Staphylococcus aureus) to respective Mueller Hinton Agar plate by streaking the swab over the entire surface of the agar plate three times, rotating the plate approximately 60 degrees after each application to ensure an even distribution of the inoculums on the surface of the medium. The plates then will stand for 5 minutes.

Wells will be cut using a sterile glass tubing for an equal diameter of the well will be used to stab the agar to create five wells cleanly cut using aseptic technique. 20 l of the treatments will be added. The treatments will be namely:
Treatment A Crude Methanolic Plant Extract Treatment B Crude Aqueous Plant Extract Treatment C Amoxicillin Treatment D Crude Methanolic Plant Extract (10 g) + Amoxicillin (25 g) Treatment E Crude Methanolic Plant Extract (100 g) + Amoxicillin (25 g)

Treatment F Crude Methanolic Plant Extract (1000 g) + Amoxicillin (25 g) Treatment G Crude Aqueous Plant Extract (10 g) + Amoxicillin (25 g) Treatment H Crude Aqueous Plant Extract (100 g) + Amoxicillin (25 g) Treatment I Crude Aqueous Plant Extract (1000 g) + Amoxicillin (25 g) Positive Control Co- Amoxiclav Negative Control Amoxicillin + distilled water

The plates will be incubated at 37C for 24 hours. The antibacterial activity will be assayed by measuring the diameter of the inhibition zone formed around the well (NCCLS, 1993).

Disc Diffusion Method

In this method, paper discs impregnated with specific antibiotics or the test substances are placed on the surface of the Muller Hinton agar medium inoculated with the target organisms, which is recommended for the diffusion of antimicrobial agents as described in NCCLS approved standard. The plates are incubated and the zones of inhibition around each disc are measured.

PROCEDURE:
Muller Hinton Agar plates will be prepared and the test microorganisms were inoculated by the spread plate method. Filter paper discs approximately 6mm in diameter will be soaked with 5 l of the treatments: Treatment A Crude Methanolic Plant Extract Treatment B Crude Aqueous Plant Extract Treatment C Amoxicillin Treatment D Crude Methanolic Plant Extract (10 g) + Amoxicillin (25 g) Treatment E Crude Methanolic Plant Extract (100 g) + Amoxicillin (25 g)

Treatment F Crude Methanolic Plant Extract (1000 g) + Amoxicillin (25 g) Treatment G Crude Aqueous Plant Extract (10 g) + Amoxicillin (25 g) Treatment H Crude Aqueous Plant Extract (100 g) + Amoxicillin (25 g) Treatment I Crude Aqueous Plant Extract (1000 g) + Amoxicillin (25 g) Positive Control Co- Amoxiclav Negative Control Amoxicillin + distilled water

Each disc will be pressed down to ensure complete 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. After 16 to 18 hours of incubation, each plate will be examined. The diameters of the zones of complete inhibition will be measured.

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.

Plan for Data Analysis

To identify which of the two extraction methods (methanolic and aqueous) of the Bakhaw (Rhizophora mucronata) crude extract plant is more synergistic with Amoxicillin, the 1-way ANOVA with p= 0.05 will be used. Moreover, the POST HOC (Least Significant Difference) will be used to identify which among concentrations of the Bakhaw (Rhizophora mucronata) crude extract plant with Amoxicillin is the most synergistic in both extraction procedures

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 should be assisted by proper personnel of the laboratory applying protective measures that are inherent in the laboratory.

Solvent used for active component extraction

Phytochemical Content of Bakhaw (Rhizophora mucronata)

Table on the Zone of Inhibition (mm) of R. mucronata extracts based on the study conducted by Nurdiani et al. (2008)

Budgetary Requirement
ITEMS Data Collection Expenses AMOUNT TOTAL

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

Dummy Table
Zone of inhibition (mm) Treatment Trial 1 R1 A B C D E F G H I J K R2 R3 R1 Trial 2 R1 R3

Mean zone of Inhibition

GANTTS CHART
ACTIVITY JAN. FEB. MAR APRIL MAY JUNE JULY

Writing of research proposal Approval of research proposal Submission of letter of permission to the University of San Agustin Research Laboratory

ACTIVITY
Plant Collection Plant Identification Processing of plant samples

JAN.

FEB.

MAR.

APRIL

MAY

JUNE

JULY

Methanolic Extraction

Aqueous Extraction Antibacterial Susceptibility Testing

Data collection Writing of result analysis Formulation of conclusion

ACTIVITY Writing of final research report Presentation of results

JAN.

FEB.

MAR.

APRIL

MAY

JUNE

JULY

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