Green Synthesis of Silver Nanoparticles (AgNPs) Using Brassica oleracea var.

italica and
the Evaluation of Its Bactericidal Properties

Chapter 1

Background of the Study

- Antibiotic resistance occurs when bacteria develop the ability to defeat the drugs designed
to kill them.
- A nanoparticle is a microscopic particle with one dimension ranging from 1 nanometer to
100 nanometers (0.0000000001m).
- Most conventional AgNPs production methods require many chemicals, which are not only
expensive but can also produce hazardous residues (Charan et al., 2005).
- Plants are utilized for the NPs synthesis as their extract is rich in phytochemicals which
act as both reducing and stabilization agent (Ramesh et al., 2015)
- This study aims to synthesize AgNPs using B. oleracea var. italica and determine its
bactericidal activity against Staphylococcus aureus and Klebsiella pneumoniae.
- Pricing:
o 25mL silver nanoparticles in colloidal solution – Php 10 000 (Sigma-Aldrich)
o Biosynthesized silver nanoparticles – Php 300

Statement of the Problem

This study aims to synthesize AgNPs using the aqueous extract of B. oleracea var. italica.
Subsequently, the biosynthesized AgNPs will be evaluated for its bactericidal activity against
common drug-resistant bacteria.

Specifically, this study aims to seek answers to the following questions:

1. How does increasing the concentration of the plant extract affect the biosynthesized
AgNPs?

2. What functional groups or phytochemicals present in the B. oleracea var. italica extract
capped and stabilized the AgNPs?

3. What sizes and shapes were observed from the microscopic analysis of the
biosynthesized AgNPs?

4. What is the inhibitory activity of the biosynthesized AgNPs against Staphylococcus

aureus?

5. What is the inhibitory activity of the biosynthesized AgNPs against Klebsiella

pneumoniae?
Significance of the Study

The findings of this study will be beneficial to the following:

- Field of Medicine
- Field of Chemistry
- Field of Engineering
- Future Researchers

Scope and Delimitation

- Researchers will synthesize the AgNPs

- Only broccoli florets will be used in the study
- Only inhibitory activity will be discussed and correlated with various literatures
- Bacteria subjects will only be based from the World Health Organization – list of common
drug-resistant bacteria

Chapter 2

Antibiotic Resistance

- occurs when bacteria or fungi develop the ability to defeat the drugs designed to kill them.
- 2 million people are being infected with antibiotic-resistant bacteria each year, and at
least 23,000 people die as a result (CDC, 2018)
- bacteria can neutralize an antibiotic by changing it in a way that makes it harmless.

Pharmacological Applications of Silver Nanoparticles

- Anti-microbial
- Anti-fungal
- Anti-oxidant
- Anti-cancer
- Anti-viral

Conventional Methods of Synthesizing Silver Nanoparticles

A. Chemical Reduction Method

B. Polysaccharide Method
C. Tollens Method

Phytochemicals Responsible for In Capping Nanoparticles

- phytochemicals are non-nutritive plant chemicals that work together with nutrients and
dietary fiber to protect against diseases.
- they can act as reducing agent in the synthesis of nanoparticles (Bhavesh et al., 2011)
- phenolics and alkaloids were responsible for capping and stabilizing of nanoparticles
(Amin et al., 2013)

Hypotheses
 H0 – The biosynthesized AgNPs have an average size of greater than 100 nanometers;

and possesses no inhibitory activity against the bacteria subjects.

Ha– The biosynthesized AgNPs have an average size of less than 100 nanometers; and

possesses strong inhibitory activity against the bacteria subjects.

Paradigm of the Study

 Collection of Plant  Characterization of  Surface Morphology

Materials Biosynthesized of AgNPs is
 Extract of Plant AgNPs (FTIR, UV- determined
 Preparation of Vis, SEM)  Phytochemicals
Reagents  Phytochemical present in Plant are
 Synthesis of Silver Screening of Plant determined
Nanoparticles  Antimicrobial  Inhibitory Activities of
Inhibition Assay of AgNPs are evaluated
AgNPs

Figure 2.1: IPO model of the Paradigm of the Study

Chapter 3

Research Design

- Descriptive
- Experimental

Research Instruments

- The following instruments will be used to gather data in this study:

FTIR Spectrometer, UV-Vis Spectrophotometer, Scanning Electron Microscope, Vernier
Calipers.
Research Procedure

Phase 1: Collection of Plant Materials

- Broccoli was bought from a local market at San Jose del Monte, Bulacan.

Phase 2: Preparation of Aqueous Plant Extract

- 50 g of powdered broccoli was dissolved in 200 mL of deionized water.

- Solution was subjected under water bath for 15 mins at a constant 65oC temp.

Phase 3: Qualitative Phytochemical Screening

- Tannins
 - Flavonoids
- Alkaloids
- Terpenoids
- Cardiac Glycosides
- Phenolic Compounds

Phase 4: Synthesis of Silver Nanoparticles

- 0.001 M AgNO3 was prepared via dilution of 0.1 M stock concentration.

- Plant Extract was added dropwise to 50 mL AgNO3 at increasing ratio (1:4, 1:3, 2:5, 1:2)

- Color change from colorless to reddish brown indicates formation of silver nanoparticles

- To ensure homogeneity the colloidal solution was placed under sonication for 20 mins.
 - The solution was then centrifuged at 10,000 rpm for 20 mins.

Phase 5: Characterization of Synthesized Silver Nanoparticles

- UV-Vis Spectroscopy
- FTIR Spectroscopy
- SEM Imaging

Phase 6: Evaluation of Antimicrobial Activity

- Kirby-Bauer disk diffusion Assay.

- Test Subjects: Staphylococcus aureus, and Klebsiella pneumoniae