DE LA SALLE ARANETA UNIVERSITY

Pioneer in Agriculture and Veterinary Medicine

Don Salvador Araneta Campus, Victoneta Avenue, Malabon City
BASIC EDUCATION DEPARTMENT
JUNIOR HIGH SCHOOL DIVISION

An Investigatory Project Proposal entitled

Green Synthesis of Copper Nanoparticles using

Makabuhay (Tinospora rumphii boerl) leaf extract as
Biocompatible Novel Antioxidant Agent
In Partial Fulfillment on the Subject Requirements of
Agriculture 10, English 10 and Science 10

(GROUP 1)
(GRADE 10 – BR. ARKADIUS)

Submitted by:
Alcomendas, April Ronald T.
Barrameda, Benedict G.
Dineros, Shaina M.
Liwanag, Kyla Camille A.
Tamesis, Maureen Anne S.

Submitted to:

Mr. Danilo H. Dogillo Jr.

Agricultural Arts Teacher

Ms. Ma. Theresa DC. Libnao

English Teacher

Mr. Jann Andre H. Padua

Science Teacher

1
 Table of Contents

Abstract…………………………………………………………………………….…….3
Acknowledgement………………………………………………………………….…...4
Chapter 1: The Problem and its Background

- Background of the Study…….……………………………………………………5-6

- Statement of the Problem….………………………………………………………..7
- Hypothesis….…………………………………………………………………………7
- Significance of the Study.....………………………………………………………8-9
- Scope and Limitations………..………………………………………………………9
- Definition of Terms………………………….………………………………………10
Chapter 2: Review of Related Literature……………………………………..11-18
Chapter 3: Methodology

- Materials………………………………………………………………………….….19
- Procedures…………….…………………………………………………………19-22
- Tests to be conducted…………………………….…………………………….21-22
Chapter 4: Results and Discussion……………..…………………...…….…23-28
Chapter 5: Summary of Findings, Conclusion, and Recommendations

- Summary of Findings…...…………………………………………..………………29
- Conclusions.…………………………………………………………………………29
- Recommendations……………………………...…………………….…………….29
Appendices…………………………………………………………………..……..30-32
Bibliography……………………………………………………………………...…36-39

2
 Abstract

The aim of our study is to make a green synthesis copper nanoparticle that

will serve as an effective safer antioxidant and antibacterial agents that will perform

in different strains of bacteria. It will also help in lessening the bacteria in the

environment for people to prevent diseases. At last, it will avoid harm to the

environment since it is completely made from plants.

The study was divided into three processes namely: (1) UV-Vis

spectroscopy, (2) FTIR (Fourier Transform Infrared Spectroscopy, (3) DPPH

Assay. These are the variety of testing used in order to determine whether the

green synthesis of copper nanoparticles is an antioxidant or it is just the

Makabuhay plant itself.

The result of the experiment was a success, for the copper

nanoparticles did attain a low level of antioxidant. However, the difference was that

the ascorbic acid gained much higher percentage of inhibition compare to the

copper nanoparticles that was presented in the DPPH Assay.

It was recommended to explore other studies, so it can add

significance to the study. The researchers recommend making sure that the

finished product that will be used for testing which is the copper nanoparticles was

in liquid form and complete made out of the chosen plant.

3
 Acknowledgement

The researchers would like to grab the opportunity to give thanks to every
person that helped them to accomplish this research.

First, to Mr. Jann Andre Padua, their research adviser and science
teacher, for trusting, guiding and helping to complete this study. For all the days of
persuading and asking. Also, for suggesting this research. Thank you for being
patient, understanding, seeing your student’s effort, and trusting them with their
abilities.
Second, to Mr. Randel Serrano, their Laboratory Technician for being their
guide in creating their product and providing them materials to complete their
solutions and experiments in this study.
Third, to Ms. Ma. Theresa D.C. Libnao, for sharing her knowledge and for
guiding us to make our research paper better. For teaching us how to do the proper
way of writing and for not giving up though with all of the wrong grammars and
punctuation we’ve made.
Fourth, to the panel of examiners during the title defense, for sharing their
expertise and suggestions for the improvement of this research.
Fifth, to their families and friends, who served as their motivation for
working on for this research and helping to provide the materials needed in the
research.
Sixth, to UST Analytical Services Laboratory especially to Ms. Junee
Linsangan and Ms. Niña Caisido for testing their product in UST and providing
the results in this study.
Seventh, to De la Salle Araneta University, for allowing their students to
conduct research and showcase their capabilities and molding them to become
better students and citizens of God.
Lastly, to Almighty God, for making all things possible and for giving us
strength and wisdom.

A.R.T.A
B.G.B
S.M.D
K.C.A.L
M.A.S.T

4
 CHAPTER 1

The Problem and its Background

Background of the Study

According to the International Journal of Engineering Science and

Technology, the field of nanotechnology is one of the most active areas of research

in modern materials science. Nanoparticles provide solutions to technological and

environmental challenges in the areas of solar energy conversion, catalysis,

medicine, and water-treatment. In addition, based on ScienceDirect, studies

recently have proved that the plant extracts act as a potential precursor for the

synthesis of nanomaterial in a non-hazardous ways. Plants and plant extracts

seem to be the best option among the biological alternative in synthesizing as

plants are the nature’s chemical factory. Nanoparticles can be synthesized using

a variety of techniques. Dry particles and nanoparticles in liquid dispersion are

synthesized by these techniques. Nanostructures can be generated by building

from atoms or by diminishing the size from microparticle to nanoparticle.

The researchers intend to make a green synthesis copper nanoparticles

that will serve as an effective safer antioxidant and antibacterial agents that will

perform in different strains of bacteria. The main purpose of this study lessen the

bacteria in the environment for people to prevent from diseases and to avoid harm

to the environment since it is completely made from plants. The researches had

the idea of producing copper nanoparticles because these have a high surface

5
area to antibacterial potency, antioxidant and catalytic activity compared to the

quality of metals such as gold and silver. Copper is able to assassinate positive

and negative Gram bacteria across all temperature and all levels of humidity

compare to other metals like silver that is needed to have a particular temperature

for a biocidal activity to be done.

In producing a copper nanoparticles, the researchers searched for a

possible product that will help in making the copper nanoparticles an antioxidant.

Thus, Makabuhay plant is the product used as it claims to have beneficial effects

for supporting immune system, metabolism, and treatment for wounds. Its extract

contains novel secondary metabolites such as alkaloids which this compound is

mainly responsible for the reduction of ionic bulk metallic nanoparticles formation.

This secondary metabolites is involved in the redox reaction to synthesize eco-

friendly nanosized particles. Using plant for synthesis of nanoparticles can be

advantageous over some environmental benign biological process as it eliminates

the elaborate process of maintaining cell cultures. Besides, plants successfully

used in the synthesis of various greener nanoparticles are metallic nanoparticles

such as cobalt, copper, silver and gold.

6
Statement of the Problem

The researchers will aim for this investigatory project to be applied in real

life and to make it a valuable thing as people can make an effective and safer

antioxidant and antibacterial agents through their own, so they have come up to

the following problems:

1. How does the range of absorbance help in determining the level of antioxidant

of the copper nanoparticles?

2. What is inside the copper nanoparticles to be an antioxidant agent and to have

scavenging activity?

3. How effective is the Makabuhay leaf extract to be used as the main product in

producing copper nanoparticles that can serve as an antioxidant agent?

Hypothesis

Null Hypotheses

1. The range of absorbance does not help in determining the level of antioxidant

of the copper nanoparticles.

2. The copper nanoparticles has no component that will serve as an antioxidant

agent and have scavenging activity.

3. The Makabuhay leaf extract is not efficient to be used as the main product in

producing copper nanoparticles.

7
Significance of the Study

The production of Copper Nanoparticles would cause health hazard to

human and animals. This study would benefit a lot of people since it is a safe way

of creating antimicrobial and antioxidant agents through production of Copper

Nanoparticles using Makabuhay plant. If this would be proven effective, people

may use this to lessen the bacteria in the environment and so people would prevent

such diseases. It can also be harmless to the environment since it is completely

made from plants.

The significance of this study to the students is that they gain knowledge on

how to make a much safer antioxidant and antimicrobial treatment. The students

would also be aware that creating this in a less safe way can cause them a really

bad health. Since students badly needed a good health, this study may benefit

them because once some bacteria are lessen, the diseases that students can get

will be prevented.

The significance of this study to the school is that since students’ second

home is the school, they are affected to the environment of it. If students would

know how to synthesize their own nanoparticle in a much safer way that will help

lessening the bacteria, the school will have a better surrounding as it will be free

from dirt that may help preventing the students from having diseases.

8
 The significance of this study to the society is that people will be able to

lessen the use of chemicals to the plants and other things. Since the research is

all about creating a green synthesis of Copper Nanoparticles, people in the society

can use it as an alternative agents that can kill bacteria without encountering any

harm since this study is eco-friendly. It also solves environmental challenges in the

areas of solar energy conversion, catalysis, medicine and others.

Scope and Limitations

The study focuses on making green synthesis copper nanoparticles using

Makabuhay (Tinospora rumphii boerl) extract as a biocompatible novel antioxidant

and antimicrobial agents. The research will only revolve in the green synthesized

copper nanoparticles and its service as an antioxidant and antimicrobial agents.

The researchers will apply factors and will perform methods to ensure the

formation of copper nanoparticles as antioxidant and antimicrobial agents. The

stated methodology and development in this research is only limited to the process

of making the green synthesized copper nanoparticles. This study will be

conducted at De La Salle Araneta University during the school year 2018-2019.

This study aims to produce safer and efficient antioxidant and antimicrobial agents

that will perform in different strains of bacteria. The research is focused on the

importance of green synthesized copper nanoparticles as a safer antioxidant and

antimicrobial agents.

9
Definition of Terms

• Distilled Water - It is used to clean off the dusts in the stem and plant of the

Makabuhay.

• Makabuhay Plant (MP) - It is the main product wherein the researchers will

create their own by making its stem and leaves.

• Green Synthesis- This is the process of making the copper nanoparticle

less hazardous.

• Whatman Filter Paper - It is used to filter the extracts from the Makabuhay

plant.

• Test Tube – It is the storage for both the precursor and reducing agent that

were mixed.

• Mortar and Pestle – It is used to crushed the washed leaves that were dried.

• Conical Flask – It is the one being used to collect filtrate using standard

sterilized filtration method.

• UV-Vis - is used to determine analytical concentration of the copper

nanoparticles in solution.

• FTIR – is used to obtain an infrared spectrum of absorption or emission of

the copper nanoparticles in solution.

• DPPH Assay – the method done in determining the percentage inhibition of

the copper nanoparticles in solution compared to the ascorbic acid.

• Copper Nanoparticles (CU NP) – It is the one being synthesized out of

Makabuhay extract.

10
 CHAPTER 2

Review of Related Literatures and Studies

Copper nanoparticles (CuNPs) have been used various fields, including

agricultural, industrial engineering and technological fields. In recent years,

research in the field of agriculture has focused on the impact of certain minor

elements on the economy of plants. Bionanotechnology utilizes biological

principles and physical and chemical approaches to yield nanosized particles with

specific functions. Although the use of nanoscience in agriculture has been

predominantly theoretical up to now, effective antibacterial activities exhibited by

CuNPs in agricultural research have increased development in the field of

nanotechnology, leading to the establishment of intensively clean, cost-effective

and efficient biosynthesis techniques of CuNPs.

Nanotechnology

In the study of Kulkarni (2013), nanotechnology had attracted many

researchers in various fields like chemistry, physics, medicine, biotechnology,

engineering, material sciences, and medicine. It really attracts researchers due to

application in wound dressings and biocidal properties, potential in gas industry,

catalytic process, high temperature superconductors and solar cells. In this study,

there is a scope to develop new methods for the synthesis of nanoparticles which

should be required inexpensive reagent, less drastic reaction condition and very

eco- friendly. Nanoparticles are synthesized by physical and chemical methods,

11
these are suffering from drawbacks like expensive reagent, hazardous reaction

conditions and takes a long period of time to isolate nanoparticles.

The advantage of using nanoparticles is that they have a far greater surface

area to volume ratio over larger particles. They revolutionize drug deliveries

wherein they allow drugs to be targeted by viruses and tissues and it will leave

healthy tissue alone. This is also known as bioavailability whereas it delivers the

molecules to where they are most needed. (Hahn, 2016).

Green Synthesis of Copper Nanoparticles

In the process of Green Chemistry, it is the process of utilization a set of

principles that reduce or eliminates the use of hazardous substances. (Anastas &

Warmer, 1998)

Nps are formed at ambient temperatures, neutral pH, low cost and

environment friendly resources. Keeping these goals in view nanomaterials have

been synthesized using various routes. Among the biological alternatives, plants

and plant extracts seem to be the best option. Plants are nature’s “chemical

factories”. They are cost efficient and require low maintenance.

The Cu Nanoparticles gains attraction to the researchers due to catalytic,

electric. optical, photonic, textile, nanofluid, and antibacterial activity depending on

its features. In this research, Copper nanoparticles using gum karaya which is a

12
natural nontoxic hydrocolloid and elaborated with its potential antibacterial

features. (Padil & Cernik, 2013).

Copper Nanoparticles were synthesized through a relatively large scale.

The Facile method was used through chemical reduction of copper sulfate with

sodium hypophosphite in ethylene glycol within the presence of a polymer

surfactant (PVP) with include to prevent aggregation and give dispersion to the

result of colloidal nanoparticles. (Lee, Choi, Lee, Stott, & Kim, 2008).

According to Cerda et al. (2016) Nanoparticles were synthesized through

the chemical reduction of aqueous solutions of Copper (II) sulfate with ascorbic

acid.

Nanoparticles were synthesized through the process of chemical reduction

of aqueous solutions of copper (II) sulfate with ascorbic acid, using different native

cyclodextrins (NCDs) (a-, b-, or c-NCD) as stabilizing agents. The Cu-NPs were

characterized by scanning electron microscopy–energy dispersive pectroscopy,

powder X-ray diffraction and transmission electron microscopy. The pattern

showed in the typical selected area electron diffraction, and lattice fringes,

evidence that the crystalline structure of Cu-NPs is face-centered cubic which can

be seen on Figure 1 with a (1 1 1), (2 0 0) and (2 2 0) lattice planes of Cu. The

analysis of the TEM images show that the size of the Cu-NPs depends on the type

of native cyclodextrin that production of cyclodextrins is relatively simple and

involves treatment of ordinary starch with a set of easily available enzymes. It was

13
also observed that the nanoparticles are spherical and with a size between 2 and

33 nm. The smaller Cu-NPs were obtained with a-NCD (mode 4 nm), while the

nanoparticles obtained with b-NCD show the narrow size distribution (mode 6.5

nm). The average particle size and particle size distribution of Cu-NPs depend upo

n the type of CDs.

Figure 1: Face Centered Cubic Structure

The Cu-NPs can be synthesized by various methods, for example metal

vapor synthesis, exploding wire method, vacuum vapor deposition, so no chemical

reduction, thermal reduction, chemical reduction, biosynthesis, laser irradiation,

and microemulsion techniques. All the method mentioned the most popular for the

synthesis of Cu-NPs is the Chemical Reduction. It is simple, inexpensive and easy

to control but it can also affect the environment due to the disposal of the toxic

solvents and hazardous chemical reducing agents. One of the examples is the

14
sodium borohydride and many more which can be caught on the surface of the

Cu-NPs increasing nanoparticle toxicity. (Suarez-Cerda et al., 2016).

In a recent research, Cu nanoparticles was achieved by using

microorganisms, plant extract of Ocinum Santum (Tulasi) which was a traditional

plant in India that has a source of bio reduction and stabilizers. For the Cu

nanoparticles synthesis, one mL of Ocimum sanctum leaf extract was added to

100 mL of one mm aqueous Copper Sulfate (II) Pentahydrate solution in a 250 ml

Erlenmeyer flask. The flask was then kept overnight at room temperature. The Cu

nanoparticles solution obtained was purified by means of repeated centrifugation

at 12,000 RPM for 15 min followed by re-dispersion of the pellet in de-ionized

water. Then the Cu nanoparticles were dried in oven at 80 OC.

Tinospora Rumphii Boerl

Panyawan (Tinospora rumphii boerl) is a vine found throughout the

Philippines. It contains a bitter principle, colombine (2.22%), traces of alkaloids,

and a glucoside. The two alkaloids present are tinosporine and tinosporidine. It

also contains an amorphous bitter principle, picroretine and traces of berberine. It

is considered febrifuge, vulnerary, tonic, antimalarial, parasiticide, and insecticidal

(Stuart 2014). In terms of constraints that hamper productivity and full development

of goat subsector, high mortality rate at weaning, development of anthelmintic

resistance, and endoparasitism were identified as the top three concerns that

15
should be addressed (PCARRD 2004). High mortality at weaning is strongly seen

in kids considering that this species is always prone to stress which more often

result to lowered immunity.

Internal parasitism is one of the biggest problems in the small ruminant

industry. Internal parasite infection of herds can cause major health issues, which

have a major effect on animal’s performance and cause great economic loss to the

producer. In fact, most of the economic losses caused by internal parasites are

actually not due to mortality ABAH Bioflux, 2018, Volume 10, Issue 1.

http://www.abah.bioflux.com.ro/ 10 but production loss (Waller 2004). Internal

parasites of goat have been a problem ever since. For this reason, new

technologies are introduced to livestock raisers to solve the internal parasites

infection.

Today, the primary control of gastrointestinal parasites is based on the over-

the counter anthelmintic. The function of anthelmintic drugs is to cause paralysis

of worms so that they are expelled in the feces of man and animals. Although

numerous advances were made in understanding the mode of transmission and

the treatment of these parasites during the past decades there are still no efficient

products to control certain internal parasites and the indiscriminate use of some

drugs has led to several cases of resistance (Nunomura et al 2006).

16
Tests

A. UV-Vis spectroscopy

Ultraviolet–Visible Spectroscopy (UV-Vis) UV-Vis Spectroscopy is used to

determine analytical concentration either at one time or often over a

desired time period. The technique measures the absorption of light

across the ultraviolet and visible light wavelengths through a liquid

sample.

B. FTIR (Fourier Transform Infrared Spectroscopy)

FTIR stands for Fourier transform infrared, the preferred method of

infrared spectroscopy. When IR radiation is passed through a sample, some

radiation is absorbed by the sample and some passes through (is

transmitted). The resulting signal at the detector is a spectrum representing

a molecular ‘fingerprint’ of the sample. The usefulness of infrared

spectroscopy arises because different chemical structures (molecules)

produce different spectral fingerprints.

C. DPPH Assay

(DPPH) free radical scavenging method offers the first approach for

evaluating the antioxidant potential of a compound, an extract or other

biological sources. This is the simplest method, wherein the prospective

compound or extract is mixed with DPPH solution and absorbance is

17
recorded after a defined period. However, with the advancement and

sophistication in instrumental techniques, the method has undergone

various modifications to suit the requirements, even though the basic

approach remains same in all of them.

18
 CHAPTER 3

Methodology

Materials

The materials that the researchers will use in able to produce a green synthesis of

copper nanoparticles are five grams leaves of Makabuhay plant which is the main

ingredient to produce copper nanoparticles, two beakers, 100ml of distilled water

to remove dust particles, mortar and pestle for crushing, Whatman No. 1 filter

paper that is use for filtration and clean and dried conical flask for the standard

sterilized filtration method. For the synthesis of copper nanoparticles, mix both

precursor and the reducing agent in a clean test tube in 1:1 proportion. Also, we

will use the 10 grams of Copper Sulfate to mix it with the Makabuhay Plant solution

to form the product.

Procedures

A. Collection of Makabuhay Leaves

First, collect Makabuhay leaves in the school garden in De La Salle Araneta

University. After that, tightly pack the collected leaves and then transfer it to a

laboratory. Then wash it with distilled water twice and keep under room

temperature for two weeks in dark condition. To make it a powder, use a blender.

Lastly, for the preparation of the plant extracts, use the collected leaves and

undergo a process.

19
B. Preparation of Plant Extracts

Weigh five gram powder of Makabuhay leaf, then dissolve it in 100ml of

distilled water and boil it for 20 min at 50°C. To remove dust particles, make sure

to do and finish this process. After that, filter the extract by using the Whatman

No. 1 filter paper. Then store the filtrate in a tight seal pack under 4°C for further

use.

C. Biosynthesis of Copper Nanoparticles

For a reaction mixture, add 80ml of 1mM cu+ and 20ml of plant leaf extract. Blank

is prepared by addition of 1000ml of distilled water to 20ml of plant leaf extract. To

indicate the reduction of Cu+, look at the color and see if it change from light color

to dark color. Change the color of the solution from blue to pale yellow when both

the solutions of leaf extract and copper sulfate are mixed in equimolecular ratio

and stirred thoroughly for 30 - 60 minutes to form homogeneous mixture. Keep the

flask at room temperature overnight and the copper nanoparticles will form

separated out and settled at the bottom of the flask. Prepare Copper sulfate

solutions in variable molarities namely 1 mM, 5 mM 10 mM and 20 mM by adding

copper sulfate to double distilled water separately. Then, mix these copper sulfate

solutions in various concentrations such as 2.5%, 5% and 7.5% with the selected

plant extract (50% concentrated). Convert the solution to the pale yellow color to

dark green in making the solution.

20
D. Characterization of Biosynthesized Copper Nanoparticles

To characterize the green synthesized copper nanoparticles, use methods

such as UV-vis spectroscopy, Fourier Transform Infrared Spectroscopy,

Transmission electron microscope, and Assessment of Antimicrobial Activity and

Hydrogen Peroxide Scavenging Assay.

a) UV-Vis spectroscopy

Monitor the reduction of pure copper by measuring the UV-Vis spectrum of

the reaction. Monitor the reduction of copper nanoparticles by UV-

spectrophotometer range of absorbance from 250-480nm. Took the measurement

of synthesized copper nanoparticles under different conditions like variation of

concentration of plant extracts (fruit rind & leaf extract of Tinospora cordifolia),

different molarity of copper sulfate solutions and incubation time at the particular

wavelength that will give maximum absorption.

b) FTIR (Fourier Transform Infrared Spectroscopy)

Investigate the characterization of functional groups of surface of Cu

nanoparticles by plant extracts by using the FTIR analysis and scan the spectra

in the range of 4000–450 cm. Pour the sample mixture into a petridish and keep it

in a hot air oven until its getting dried off. After that, scrub the dried sample and

store the powder form of sample in a sterile eppendorf. Then use it for the FT-IR

analysis from region of 400- 4000cm-1 of Cu-Nps from Makabuhay leaf extract.

21
c) DPPH Assay

The method was adapted from Clarke, G. et al. (2013), twenty microliter

(20 µL) of the sample diluted appropriately in Dimethylsulfoxide (DMSO) with 180

µL of DPPH in methanol (40 µg/mL) in wells of a 96-well plate. The plate was

kept in the dark for 15 minutes after which the absorbance of the solution was

measured at 540 nm in a plate reader. DMSO served as a blank and Ascorbic

Acid served as the standard.

22
 CHAPTER 4

Results and Discussions

After doing the experiment and testing, the researchers were able to gather this information.

Table 1: UV-Vis spectroscopy results

0.054
0.052
0.05
0.048 100% or 0 Absorbance Baseline.Correction
0.046
0.044
0.042
0.04
0.038
0.036
0.034
0.032
Absorbance

0.03
0.028
0.026
0.024
0.022
0.02
0.018
0.016
0.014
0.012
0.01
0.008
0.006
0.004
0.002

200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700
Wavelength [nm]

10

9.5
Cu NP T1.Sample
Cu NP T2.Sample
9
Cu NP T3.Sample
8.5

7.5
Absorbance

6.5

5.5

4.5

3.5

3
200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700
Wavelength [nm]

0.054
0.052
0.05
blank.Blank
0.048
0.046
0.044
0.042
0.04
0.038
0.036
Absorbance

0.034
0.032
0.03
0.028
0.026
0.024
0.022
0.02
0.018
0.016
0.014
0.012
0.01
0.008
200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700
Wavelength [nm]

23
The graphs above show the range of absorbance which means it uses light in the

visible and adjacent ranges. The absorption or reflectance in the visible range

directly affects the perceived color of the chemicals involved. The graph shows

that the copper nanoparticles is mostly presented by an orange color.

Table 2: FTIR (Fourier Transform Infrared Spectroscopy) results

24
Based on the table shown above, the copper nanoparticles is really in a liquid state

form as represented by the number of peaks. Based on an FTIR table, the copper

nanoparticles has a strong and sharp appearance and it is part of the C-H

stretching group.

Table 3: % Inhibition of Copper Nanoparticles Solution against DPPH

25
Based on the table shown above, the calculated percentage inhibition of the

copper nanoparticles is at the low level antioxidant compare to the standard

ascorbic acid. DPPH Assay will determine whether an agent is an antioxidant or

not. By comparing the two agents, the researchers concluded that the standard

ascorbic acid has more scavenging activity than the own green synthesis of copper

nanoparticles with the use of Makakuhay leaf extract.

Table 3.1: % Inhibition of Makabuhay Extract against DPPH

26
This table represents the comparison of the level of antioxidant between the

ascorbic acid and the Makabuhay Extract. Based on the results, the Makabuhay

Extract had a low percentage inhibition that only means ascorbic acid has still more

scavenging activity.

Figure 3: % Inhibition of all samples against DPPH

27
This table represents the percentage inhibition of all samples against DPPH. The

researchers have to know which of the two agents is an antioxidant and has more

scavenging activity. Based on the results above, the copper nanoparticles in

solution has higher percentage of inhibition than the Makabuhay Extract itself.

28
 CHAPTER 5

Summary of Findings, Conclusions, and Recommendations

Summary of Findings

Based from the data gathered, the following findings were discovered:

1. The copper nanoparticles has a strong and sharp appearance and it is part of

the C-H stretching group.

2. The copper nanoparticles in solution still has higher percentage of inhibition

than the Makabuhay Extract itself.

Conclusions

Based on the foregoing findings, the following conclusions are drawn:

1. The copper nanoparticles is the one that is antioxidant and not the Makabuhay

leaf extract itself.

2. The green synthesis of copper nanoparticles out of the Makabuhay leaf extract

is said to be an antioxidant agent and has scavenging activity.

Recommendations

1. Try making a solution that is in dry or paste form and see whether it affects the

antioxidant level.

2. Know the components of the main product and how it helps in making the copper

nanoparticles an antioxidant agent.

3. Explore other testing that will help determining the level of antioxidant of the

green synthesis of copper nanoparticles.

29
 Appendices

Making the Solution (1st Trial)

Date: January 11, 2019
This shows the time we cut the kept Makabuhay leaves and mix it with
copper sulfate. Thus, these photos also show the process of extraction using
water and heat.

30
Reservation for Testing in University of Santo Tomas, Analytical Services
Laboratory. This is where UV-Vis, FTIR and DPPH were tested.
Date: January 25, 2019
Scheduled Date of Testing: February 12, 2019
One of the researchers filled up the form for the reservation of testing then
afterwards proceeded to the cashier for the payment. The researchers had a talk
with the chemists and asked for some opinions for the betterment of their study.

31
Making the Solution (2nd Trial)
Date: February 9, 2019
This is the second trial of making the solution. This shows again the
process of extraction using water and heat. The researchers repeated the
extraction because their 1st solution is becoming old and some effects where
made so they repeated the extraction.

32
Proposed Flow Chart

33
 Proposed Time Table

A. Collection of Plant Leaf September 7, 2018 -

Samples September 21, 2018

Biosynthesis of Copper September 21, 2018

Nanoparticles - October 5, 2018
Green Synthesis of Copper Nanoparticles using Makabuhay
(Tinospora rumphii boerl) extract as Biocompatible Novel

B. Preparation of Plant
Extracts Biosynthesis of Copper
Nanoparticles Using Different October 5, 2018 -
Molar Solutions and
Concentrations of Copper October 12, 2018
Sulphate Solution

C. Characterization of
Biosynthesized Copper October 12, 2018
Antioxidant Agent

Nanoparticles

Makabuhay Leaf Extraction October 19, 2018

Scanning electron
microscopy (SEM) October 26, 2018
D. Testing of the Copper
Nanoparticles
FTIR (Fourier Transform
Infrared Spectroscopy) November 9, 2018

UV- Vis Spectroscopy November 16, 2018

Antioxidant Assay With November 16 - 30,

Copper 2018
E.Assessment of
Antimicrobial Activity
Antimicrobial Assay December 3 - 14,
Without Copper 2018

34
 Proposed Cost Analysis of Investigatory Project

Location
Quantity/ Subtotal (B where it will
Item Amount
Frequency X C) purchased/
undertaken
1 480 Php 480 Php UST – ASL
UV-VIS
Spectrophotometer

Scanning Electron 1 1,020 Php 1,020 Php UST – ASL

Microscopy (SEM)
FTIR(Fourier 1 960 Php 960 Php UST – ASL
Transform Infrared
Spectroscopy)
DPPH 2 2,160 Php 4,320 Php UST – ASL
Transportation 5 200 Php 1,000 Php
TOTAL 7,780 Php
1,556 Php – Per Student 7,780 / 5 Members

35
 References/Bibliography

1. Mahavidyalaya, R., Pune, R. (2013, October) Green Synthesis of Copper

Nanoparticles Using Ocimum Sanctum Leaf Extract. Retrieved from

https://www.researchgate.net/publication/258933249_Green_Synthesis_of_Copp

er_Nanoparticles_Using_Ocimum_Sanctum_Leaf_Extract

2. Barabadi, H., Eshrat, G.F., Farzaneh, N., & Soheyla, H. (2012) Green

Synthesis of copper oxide nanoparticles using penicullium arantiogriseum,

penicillium citrinum and penicillium waksmanii, Journal of Nanomaterials and

Biostructures, 7(3), 999-1005.

3. Ramasamy, S., Selvam, M. (2015, January) Green synthesis of copper

nanoparticles from Hibicus rosasinensis and their antimicrobial, antioxidant

activities. Retrieved July ,2018 from

https://www.researchgate.net/publication/281992972_Green_synthesis_of_copp

er_nanoparticles_from_Hibicus_rosasinensis_and_their_antimicrobial_antioxidan

t_activities

4. Cerda, J.S, et.al (2017) A green synthesis of copper nanoparticles using native

cyclodextrins as stabilizing agents. Journal of Saudi Chemical Society, 21(3),

341-348

36
5. Choudhary, V. (2018, June) Description and medicinal uses of Guduchi- –

Tinospora cordifolia. Retrieved August 7, 2018 from

http://natureconservation.in/description-and-medicinal-uses-of-guduchi-tinospora-

cordifolia/

6. Sharma, U. et. al. (2012) Immunomodulatory active compounds from

Tinospora cordifolia, Journal of Ethnopharmacology, 141(3), 918-926

7. Nethravathi, P. C. et. al (2015) Tinospora cordifolia mediated facile green

synthesis of cupric oxide nanoparticles and their photocatalytic, antioxidant and

antibacterial properties, Materials Science in Semiconductor Processing, 33(1),

81-88

8. Kuttan, G. & Mathew, S. (1997, Dec 16) Antioxidant activity of Tinospora

cordifolia and its usefulness in the amelioration of cyclophosphamide induced

toxicity. Retrieved August 7, 2018 from

https://www.ncbi.nlm.nih.gov/pubmed/9505214

9. Saha, H. & Ghosh, S. (2012, April – June) Tinospora cordifolia: One plant,

many roles. Retrieved August 7, 2018 from

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3644751/

37
10. Padil, V. V. T. & Cernik, M. (2013, February 28) Green synthesis of copper

oxide nanoparticles using gum karaya as a biotemplate and their antibacterial

application. Retrieved August 7, 2018 from

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3589119/

11. Hook F. (n.d.) Makabuhay. Retrieved August 7, 2018 from

http://www.stuartxchange.org/Makabuhay

12. Zhang, Y., Hsung, R. P., Tracey, M. R., Kurtz, K. C. M., & Vera, E. L. (2004,

March 2) Copper sulfate-pentahydrate-1,10-phenanthroline catalyzed amidations

of alkynyl bromides. synthesis of heteroaromatic amine substituted ynamides.

Retrieved August 7, 2018 from https://pubs.acs.org/doi/abs/10.1021/ol049827e

13. Anastas, P. T.; Warner, J. C. (1998) Green Chemistry: Theory and Practice

Retrieved August 7, 2018 from http://advancinggreenchemistry.org/green-chem-

101/what-is-gc/

14. Upadhyay, N., Ganie, S. A., Agnihotri, R. K., & Sharma, R. (n.d.) Studies on

Antioxidant Activity and Total Phenolic Content of Tinospora cordifolia (Miers.)

Stem Using in Vitro Models. Retrieved August 7, 2018 from

http://www.imedpub.com/articles/studies-on-antioxidant-activity-and-

totalphenolic-content-of-tinospora-cordifoliamiers-stem-using-in-vitro-models.pdf

38
15. Ramada, J., Soriano, M., & Abella, J. (n.d.) Retrieved January 31, 2019 from

http://www.abah.bioflux.com.ro/docs/2018.9-17.pdf

16. ABAH Bioflux, 2018, Volume 10, Issue 1. http://www.abah.bioflux.com.ro/

17. Clarke, G., Ting, K. N., Wiart, C., Fry, J. (2013). High correlation of 2,2-

diphenyl-a- picrylhydrazyl (DPPH), ferric reducing activity potential, and total

phenolic contents redundancy in use of all three assays to screen for antioxidant

activity ofextracts of plants from the Malaysian forest. Antioxidants, (2): 1-10

39