September ,2014

DEAR SIR/MA’AM,

Kindly excuse Nychi Airon Q. SItchon of IV-Kelvin for not attending your class today

because she is scheduled to go to the VIRGEN MILAGROSA UNIVERSITY, San Carlos, Pangasinan

to conduct her phytochemical analysis for her science investigatory project/thesis.

Hoping for your kind consideration. Thanks you very much!

Respectfully Yours,

MRS. JOVELYN BLANCO

Research II Teacher
Phytochemical Analysis and Microbial Assay of Guava
(Psidium guavaja) Leaf Extract Against
Staphylococcus aureus

Nychi Airon Q. Sitchon

IV-Kelvin
STATEMENT OF THE PROBLEM

This study aims to determine the antimicrobial potential of guava (Psidium guajava) leaf

extract against Staphylococcus aureus.

Specifically, it seeks to answer the following questions:

1. What are the chemical substances present in the guava leaf extract that makes it potential

in antimicrobial activity against Staphylococcus aureus?

2. What treatment yields the best antimicrobial activity against Staphylococcus aureus?

3. Is there a significant difference between the guava leaf extract and Ciprofloxacin against

Staphylococcus aureus in terms of:

a. Inhibition Zone

b. Antibacterial Activity

HYPOTHESES

H0 :

There is no significant difference between the phytochemical analysis and microbial

assay activity of guava leaf extract Psidium guajava and Ciprofloxacin against Staphylococcus

aureus.

Ha:

There is a significant difference between the phytochemical analysis and microbial assay

activity of guava leaf extract Psidium guajava and Ciprofloxacin against Staphylococcus aureus.
 RESEARCH PARADIGM

Fig 1.0 This research study aims to determine the efficiency of antimicrobial property of

guava leaf extract tested on Staphylococcus aureus.

PROCESSES

 Phytochemical analysis

 Extraction of variables
INPUT OUTPUT
 Formulation of
Guava Leaf Antimicrobial
Extract treatments

 Testing of treatments

against Staphylococcus

aureus

 Microbial Assay

CONCEPTUAL FRAMEWORK

This study utilized the Guava Leaf Extract as a source of antibacterial agent that was

tested with Staphylococcus aureus as compared to Ciprofloxacin.

This underwent the following processes: Collection of Samples, Preparation of Samples,

Extraction of Samples, Formulation of Treatments, Phytochemical Analysis, and Testing of

Treatments to Staphylococcus aureus and Analysis of Data.

 This research study will help the reasercher to know the efficacy of antimicrobial

property of guava Psidium guavaja leaf extract tested on pathogenic bacteria named

Staphylococcus aureus.

SCOPE AND DELIMITATIONS OF THE STUDY

This research study aims to determine the bacterial effect of Guava Psidium guajava leaf

extract against pathogenic bacteria named and Staphylococcus aureus. The researcher gathered

her independent variable in the front yard of the house. This research study was conducted from

at the Virgen Milagrosa University, San Carlos, Pangasinan for the Phytochemical Analysis and

on Bureau of Fisheries and Aquatic Resources for the Microbial Assay. This research study is

limited to June 2014 to March 2015.

DEFINITION OF TERM

Antibiotics - also known as antibacterial, are types of medications that destroy or slow

down the growth of bacteria. It is also used to treat infections caused by bacteria.

Bacteria - are often maligned as the causes of human and animal disease (like this one,

Leptospira, which causes serious disease in livestock).

Ciprofloxacin- is an antibiotic useful for the treatment of a number of bacterial infections. It is a

second-generation fluoroquinolone.

Disinfection – the process of killing pathogenic organisms or rendering them inert.

Extraction - the separation of a substance from a matrix.

Gram-Positive Bacteria - are those that are stained dark blue or violet by Gram staining. Gram-

positive organisms are able to retain the crystal violet stain because of the high amount of

peptidoglycan in the cell wall.

Inhibition Zone - It is used clinically to measure antibiotic resistance and industrially to test the

ability of solids and textiles to inhibit microbial growth.

Microbial Assay - assays is a type of bioassay and are designed to analyse the compounds or

substances which have effect on micro-organisms.

Pathogen - A pathogen or infectious agent is a biological agent that causes disease or illness to

its host.

Phytochemical Analysis - any of various bioactive chemical compounds found in plants, as

antioxidants, considered to be beneficial to human health.

 CHAPTER III

METHODOLOGY

This contains research design, the variables and procedures in data gathering and the
experimental flow chart of the study and the statistical treatments to be used.

The experimental design to be used is the Completely Randomized Design (CRD) in

order to find the effectiveness of Guava Leaf Extract as an antimicrobial agent against
Staphylococcus aureus compared to the commercial antibacterial Ciprofloxacin. Numbers of
treatments were assigned to the experimental subject.

Table 1.1 Treatments according to their concentrations

TREATMENTS INDEPENDENT VARIABLE EXTRANEOUS VARIABLE

T0 25 mL Ciprofloxacin Solution
T1 25 mL Guava Leaf Extract 0 mL water

T2 15 mL Guava Leaf Extract 5 mL water

T3 10 mL Guava Leaf Extract 10 mL water

There were three treatments made in each replication for the control group and

experimental group.

Replication:

(Treatment) x (no. of trials) x (replications)

(4) x (3) x (6) = 72

The control and experimental group in three replications.

PROCEDURES

PHASE I

Phytochemical Analysis

The researcher has undergone the Phytochemical Analysis at the Virgen Milagrosa

University, San Carlos, Pangasinan. Chemical tests for the screening and identification of

bioactive chemical constituents in the guava were carried out with the extracts using the standard

procedure. About 50 mL of Guava Leaf extract was set in an Erlenmeyer Flask covered with

funnel; 100 mL of 80% of ethyl alcohol is added; the flask were then placed over a hot water

bath and were refluxed for an hour, the mixture was poured in another empty flask through a

glass funnel with filter paper, the filtrate was finally tested under controlled conditions and made

ready for the Preliminary Tests with the aid of chemical reagents and scientific processes.

PROCESSES OF PHYTOCHEMICAL SCREENING

A. Preparation of Guava Leaf Extract

The 100 grams of Guava Leaves were gathered and washed and were extracted by

a hot ethanol. The extract of the Guava Leaves were poured in the Erlenmeyer

flask with the glass funnel.

B. Reflux Method

Eighty percent of ethyl alcohol (100 mL) was poured in an Erlenmeyer flask with the leaf

extract and the flask was placed over a water bath and reflux in a hot plate
PHASE II

A. Preparation of Variables

The laboratory apparatus and materials to be used were gathered from the

Microbiology Laboratory of the Bureau of Fisheries and Aquatic Resources at

Binloc, Dagupan City.

Preparation of Plant Extract

The leaf samples were collected from the guava trees growing at the

researcher’s residence. Random leaf samples were collected into plastic zip lock

bags with appropriate labeling and stored in an ice cooler until being transported

to the laboratory for extraction. The control variable Ciprofloxacin was bought

from the Pharmacia Petilla, San Fabian, Pangasinan.

B. Extraction Methods Used on Guava Leaves

The leaf samples were washed in tap water and were placed into an

ethanol solvent. The ethanol solvent has a polarity of (>99.5%), and boiling distilled water were

used for the maceration extraction procedure. The leaf powder was added to each of solvents to

make a 20% concentration.

Formulation of Different Treatments

The percentages of the extracts were put into the test tubes prior to their concentration. A

clean cap was used to cover up the test tubes which were put on the test tube rack and

refrigerated for 24 hours, for the microbial assay.

The researcher formulated three (3) treatments that was made in each replication for the

experimental and control group.

Treatment 0 (T0) - Ciprofloxacin

Treatment 1 (T1) - Contains 25 mL of Guava Leaf Extract

Treatment 2 (T2) - Contains 15 mL of Guava Leaf Extract and 5 mL of water was prepared

Treatment 3 (T3) - Contains 10 mL of Guava Leaf Extract and 10 mL of water was prepared

PHASE III

Evaluation of Parameters

A. Inhibition Zone Measurement

Inhibition Zone Measurement is used clinically to measure antibiotic resistance and

industrially to test the ability of solids and textiles to inhibit microbial growth.

Interpretation of Results

Inhibition zone were measured after 24 hours incubation period with the use of a

millimetre ruler. Inhibition zone were indicated if its measurement is:

 Table 1.2 Rating Scale

Inhibition Zone (mm) Rating Scale

<20 Inactive against test organism

20-25 Partially active against test organism

>25 Very active against test organism

Antibacterial Activity

Agar well diffusion method was used to screen antibacterial activity of Psidium guajava

extract. The dried extracts were dissolved in 1% dimethyl sulfoxide and prepared at

concentration of 200 mL Ciprofloxacin at concentration of 100 mL was used as standard

antibacterial drug. Plates were inoculated with test bacterial strains and extracts were place in

well, incubated at 37˚C for 24 hrs & Zone of inhibition measured. Each experiment was carried

out in triplicate.

PHASE IV

The research study used the experimental method, Complete Randomized Design

(CRD), Analysis of Variance (ANOVA), and the T-Test statistical tools to test if there is a

significant difference between Guava Leaf Extract and the commercial variable (Ciproflolxacin)

in averting the growth of pathogen bacteria names Staphylococcus aureus.

Testing of Treatments against Staphylococcus aureus

Microbial assay by Agar Well Diffusion Method

The microbial assay of Psidium guavaja was evaluated against bacterial strains in

ethanolic and hot water extracts by using Agar well Diffusion method. Nutrient agar plates were

prepared for all extracts, 50 mL of inoculums of selected bacterium was uniformly spreaded on

agar plates with the help of glass spreader. The equal volume (50 mL) of antibiotic

(Ciprofloxacin), distilled water and guava leaf extract were poured into the wells. The plates

were incubated for 24- hours at 37 °C.

Fig. 3 Microbial assay by Agar Well Diffusion Method

10 mL Negative

15 mL 25 mL