SYNERGISTIC ANTIBACTERIAL ACTIVITY OF AMOXICILLIN AND BAKHAW (Rhizophora mucronata) BARK CRUDE METHANOLIC AND AQUEOUS EXTRACTS AGAINST Staphylococcus aureus and Escherichia coli

7/29/2019 SYNERGISTIC ANTIBACTERIAL ACTIVITY OF AMOXICILLIN AND BAKHAW (Rhizophora mucronata) BARK CRUDE METHANOLIC AND AQUEOUS EXTRACTS AGAINST Staphylococcus au

1/55

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


2/55


3/55

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.


4/55

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.


5/55

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.


6/55


7/55

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.


8/55

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)


9/55

Treatment ECrude Methanolic Plant Extract (100 g)

+ Amoxicillin (25 g)

Treatment FCrude Methanolic Plant Extract (1000 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


10/55

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.


11/55

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).


12/55

Specific Objectives

4. To determine which among the treatments

and mode of extraction shows the highest

synergistic activity against Staphylococcusaureus andEscherichia coli.


13/55

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.


14/55


15/55

Hospitals and Medical Institutions

Pharmaceutical companies

Communities Future researchers


16/55

CONCEPTUAL FRAMEWORK

Independent VariablesDifferent concentrations of thecrude methanolic and aqueous

plant extracts combined withamoxicillin.

Dependent Variables

Diameter of the Zone ofInhibition


17/55


18/55

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.


19/55

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.


20/55


21/55

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.


22/55

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


23/55

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.


24/55

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.


25/55

Plant Identification

The plant identification will be done by Dr.

Junemie Ramos, mangrove expert, SEAFDEC

AQD.


26/55

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.


27/55

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.


28/55

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.


29/55

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)


30/55

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)




31/55

Antibacterial Susceptibility

Testing

Parallel antibacterial susceptibility testing will be

done by the three methods namely, Agar well

diffusion method, and Disc diffusion method.


32/55

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.


33/55

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)


34/55

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.


35/55

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 DCrude Methanolic Plant Extract (10g) + Amoxicillin (25 g)

Treatment ECrude Methanolic Plant Extract (100g) + Amoxicillin (25 g)


36/55

Treatment FCrude Methanolic Plant Extract (1000


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)


Positive ControlCo- Amoxiclav Negative ControlAmoxicillin + distilled water


37/55

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).


38/55

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.


39/55

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 DCrude Methanolic Plant Extract (10 g)

+ Amoxicillin (25 g) Treatment ECrude Methanolic Plant Extract (100



40/55

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)




41/55

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.


42/55

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.


43/55

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


44/55

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.


45/55

S l d f i


46/55

Solvent used for active component

extraction

h h i l f kh


47/55

Phytochemical Content of Bakhaw

(Rhizophora mucronata)

Table on the Zone of Inhibition (mm) of R mucronata


48/55

Table on the Zone of Inhibition (mm) ofR. mucronata

extracts based on the study conducted by Nurdiani et al.

(2008)


49/55

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


50/55

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


51/55

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


52/55

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


53/55

ACTIVITY JAN. FEB. MAR. APRIL MAY JUNE JULY

Writing of final

research report

Presentation of

results


54/55


55/55

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

Documents

SYNERGISTIC ANTIBACTERIAL ACTIVITY OF AMOXICILLIN AND BAKHAW (Rhizophora mucronata) BARK CRUDE METHANOLIC AND AQUEOUS EXTRACTS AGAINST Staphylococcus aureus and Escherichia coli