SYNTHESIS, CHARACTERIZATION, AND SUSCEPTIBILITY TEST OF

THE NANOEMULSIFIED Canarium ovatum LEAF EXTRACTS

AND Cocos nucifera OIL

A THESIS MANUSCRIPT
Submitted to the Faculty of the Department of Chemistry,
College of Mathematics and Natural Resources,
Caraga State University
Butuan City

In partial fulfillment
of the requirements for the Degree
BACHELOR OF SCIENCE IN CHEMISTRY

CONIE GRACE C. OTAZA

June 2022
 ii

CERTIFICATE OF ACCEPTANCE

The Faculty of the Department of Chemistry, College of Mathematics & Natural

Sciences ACCEPTS THE UNDERGRADUATE THESIS entitled:

SYNTHESIS, CHARACTERIZATION AND SUSCEPTIBILITY TEST OF

THE NANOEMULSIFIED Canarium ovatum LEAF EXTRACTS
AND Cocos nucifera OIL

submitted by CONIE GRACE C. OTAZA in partial fulfillment of the requirements

for the degree of BACHELOR OF SCIENCE IN CHEMISTRY.

TEMMY P. VALES, Ph.D. ELIZABETH P. PARAC, Ph.D.

Panel member Panel member
Date: ___________ Date: ____________

FELMER S. LATAYADA, Ph.D.

Adviser
Date: ___________

TEMMY P. VALES, Ph.D. ESAMEL M. PALUGA, Ph.D.

Chair, Department of Chemistry Dean, CMNS
Date: ____________ Date: ____________

F-CMNS-006-f
Rev: 1
Effective: 02/01/21
 iii

ABSTRACT
The growing prevalence of life-threatening bacterial and fungal infections,
as well as pathogens' ability to develop resistance to current treatment strategies,
necessitates the discovery and development of new compounds to combat them.
Nanoparticles ranging from 1 to 200 nm in diameter, are used to boost plant growth
and protect them from harmful pests like insects and pathogens like bacteria, fungi,
and viruses. An oil-in-water nanoemulsions were synthesized using Canarium
ovatum leaf extracts and Cocos nucifera oil, water, and Tween 80. The
nanoemulsions were prepared by an ultra-sonication method. Thirty minutes of
sonication time delivered a long-term stable C. ovatum-based and C. nucifera-based
nanoemulsions which were then characterized by Fourier Transform Infra-Red
(FTIR) spectroscopy and Dynamic Light Scattering (DLS). FTIR analysis of
nanoemulsions observed a presence of hydroxyl group and broad intensity assigned
to a phenolic group O-H stretching. The mean diameter of the synthesized
nanoemulsions gives the particle size distribution of <200 nm. The prepared
nanoemulsions were subjected to susceptibility test against Escherichia coli,
Staphylococcus aureus, S2K3Y, and Stenotrophomonas maltophilia via disc
diffusion assay and was found to be partially active only against S. maltophilia
bacteria.

Keywords: Nanoemulsion, S. maltophilia, C.ovatum, C.nucifera, biopesticides

 iv

ACKNOWLEDGEMENT

I would like to extend my warmest gratitude to the Omnipotent One whom

always been there throughout my thesis journey.

And to all the people who contributed to the success of this undergraduate

thesis. To my adviser, Dr. Felmer S. Latayada, for all the guidance, concerns and

suggestions. To Dr. Temmy P. Vales and Dr. Elizabeth P. Parac for sharing their

expertise and giving comments, suggestions and corrections.

To my family and batch mates who had helped me gathered my plant

samples. To my sister as my helping hand who supported financially to keep my

undergraduate thesis going.

And to all the people who had helped me in little ways, I couldn’t be more

thankful and proud. God bless us all.

 v

TABLE OF CONTENTS

PAGE
TITLE PAGE
APPROVAL SHEET - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - ii
ABSTRACT - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - iii
ACKNOWLEDGEMENT - - - - - - - - - - - - - - - - - - - - - - - - - - - - iv
TABLE OF CONTENTS - - - - - - - - - - - - - - - - - - - - - - - - - - - - - v
LIST OF FIGURES - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - vii
LIST OF SCHEMES - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - viii
LIST OF APPENDICES - - - - - - - - - - - - - - - - - - - - - - - - - - - - - ix

CHAPTER
1. INTRODUCTION - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1

1.1 Background of the Study - - - - - - - - - - - - - - - - - - - - - - - 1

1.2 Objectives of the Study - - - - - - - - - - - - - - - - - - - - - - - - 3
1.3 Significance of the Study - - - - - - - - - - - - - - - - - - - - - - 4
1.4 Scope and Limitation of the Study - - - - - - - - - - - - - - - - 4

2. REVIEW OF RELATED LITERATURE - - - - - - - - - - - - 6

2.1 Canarium ovatum - - - - - - - - - - - - - - - - - - - - - - - - - - - 6

2.2 Cocos nucifera - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 7
2.3 Stenotrophomonas maltophilia - - - - - - - - - - - - - - - - - - - 8
2.4 Related Studies in C. ovatum - - - - - - - - - - - - - - - - - - - - 8
2.5 Related Studies in C. Nucifera - - - - - - - - - - - - - - - - - - - 9
2.6 Mechanism of Action of Nanoparticles - - - - - - - - - - - - - 10
2.7 Biosafety of Nanoparticles - - - - - - - - - - - - - - - - - - - - - 11
2.8 Developments of Pesticide Nanoemulsions - - - - - - - - - - 12

3. METHODOLOGY - - - - - - - - - - - - - - - - - - - - - - - - - - - - 14

3.1 Isolation of Plant Extract and Essential Oil - - - - - - - - - - 14

3.2 Preparation of Canarium ovatum-based nanoemulsion - - - 15
3.3 Preparation of Cocos nucifera-based nanoemulsion - - - - - 16
3.4 Characterization of Nanoemulsions - - - - - - - - - - - - - - - 18
3.4.1 Evaluation of Droplet Size - - - - - - - - - - - - - - - - - 18
3.4.2 Fourier Transform Infra-Red Spectroscopy (FTIR) - 18
3.4.3 Susceptibility Test via Disc Diffusion Method - - - - 18

4. RESULTS AND DISCUSSIONS - - - - - - - - - - - - - - - - - - 20

 vi

4.1 Dynamic Light Scattering Analysis - - - - - - - - - - - - - - - 20

4.2 Fourier Transform Infra-Red (FTIR) Analysis - - - - - - - - 21
4.3 Evaluation of Susceptibility Test - - - - - - - - - - - - - - - - - 23

5. SUMMARY, CONCLUSIONS AND

RECOMMENDATIONS - - - - - - - - - - - - - - - - - - - - - - - - 26
REFERENCES - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 28
APPENDICES - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 33
CURRICULUM VITAE - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 38
 vii

LIST OF FIGURES
Figure Page

2.1 Photo of Canarium ovatum leaves - - - - - - - - - - - - - - - - 6

2.2 Photo of Cocos nucifera fruit - - - - - - - - - - - - - - - - - - - 7
4.1 Size distribution by the intensity of 4% C. nucifera oil
Nanoemulsion - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 20
4.2 Size distribution by the intensity of 2% C. ovatum leaf
extract nanoemulsion - - - - - - - - - - - - - - - - - - - - - - - - - 21
4.3 IR Spectra of 4% C. nucifera oil nanoemulsion - - - - - - - 22
4.4 IR Spectra of 2% C. ovatum leaf extract nanoemulsion - - 23
4.5 Antimicrobial activity assays of the Canarium ovatum
based and C. nucifera-based nanoemulsions against (A) E.
coli; (B) S. aureus (C) S2K3Y (D) S. maltophilia.- - - - - - 24
 viii

LIST OF SCHEMES

Scheme Page

3.1 Schematic diagram of C. ovatum nanoemulsion methodology - - - 16

3.2 Schematic diagram of C. nucifera oil nanoemulsion methodology - 17
 ix

LIST OF APPENDICES
Appendix Page

A Antimicrobial Test Result of Nanoemulsions - 33

B Documentations on Plant Collection - - - - - - - 34
C Certifications
Certificate of Plant Verification - - - - - - - 36
MSCRC Certification - - - - - - - - - - - - - 37
1
 CHAPTER 1

INTRODUCTION

1.1 Background of the Study

The growing prevalence of life-threatening bacterial and fungal infections,

as well as pathogens' ability to develop resistance to current treatment strategies,

necessitates the discovery and development of new compounds to combat them 1.

Similarly, the problem of antimicrobial resistance is further aggravated because of

similarities in resistance mechanism and genetic linkages2. In order to minimize the

negative impact, there exist an accumulative interest in bio-ecology studies of

pathogen and a worldwide trend to explore new alternatives to synthetic bio

controls. Among the alternative methods, the use of natural compounds as essential

oils is one which can be characterized by less toxicity for humans and environment

safety, selectivity, biodegradable liability and a variety of chemical secondary

metabolites3. One of the potent antimicrobial compounds isolated from essential

oils are lipophilic monoterpenes such as thymol, carvacrol, linalool, citral, geraniol

and 1,8-cineole4. The application of nanoemulsion as an antimicrobial agent is a

promising and new innovation. Nanoemulsions have attracted much attention due

to their applications especially in plant protection field5. Essential oils as well as

compounds derived from them possess wide range of antimicrobial activities.

Essential oil nanoemulsions are preferred due to higher solubility capacity and

thermodynamic stability as compared to unstable emulsions and suspensions6.

 2

Canarium ovatum Engl., commonly known as pili, is

a species of tropical tree belonging to the genus Canarium. It is one of

approximately 600 species in the family Burseraceae7. The large tropical trees are

common in Malaysia, the Philippines, and are also found in Africa and northern

Australia8. The medicinal properties have been attributed to the presence of several

bioactive compounds such as flavonoids, alkaloids and phenolic compounds9.

Cocos nucifera L. palm of the family Arecaceae10. The parts of its fruit like coconut

kernel and tender coconut water have numerous medicinal properties such as

antibacterial, antifungal, antiviral, antiparasitic, antidermatophytic, antioxidant,

hypoglycemic, hepatoprotective, immunostimulant11.

Nanoemulsion is defined as a colloidal dispersion of two immiscible liquids

that is thermodynamically unstable12. High energy methods are extensively used to

formulate nanoemulsion13. High mechanical energy is used that provide strong

disruptive forces, which break up large droplets to nano-sized droplets and produce

nanoemulsions with high kinetic energy. The disruptive forces are created by using

mechanical devices such as ultra-sonicators, microfluidizer, and high-pressure

homogenizers14. Nanotechnology is now frequently used in agricultural operations

to improve efficacy and reduce the amount of pesticides required. It can benefit

pesticides by lowering toxicity, extending shelf life, and enhancing the solubility

of pesticides that are poorly water-soluble, all of which could have a favorable

environmental impact15. Modern agriculture is one of the many applications of

nanotechnology. Nanoparticles, which have a particle size ranging from 1 to 200

 3

nm in diameter16, are used to boost plant growth and protect them from harmful

pests like insects and pathogens like bacteria, fungi, and viruses. Because these

Nano-based insecticides are often delivered in small doses and promptly up taken

hence, taken by cells, they can inhibit the development of resistance in targeted

pests. The primary benefit of utilizing nanocides (nanotechnology-based

pesticides) is that they have little impact on non-targeted insects and are

environmentally friendly17. This study will attempt to synthesize nanoemulsions

from Canarium ovatum and Cocos nucifera and will be subjected to susceptibility

assay.

1.2 Objectives of the Study

This study aims to synthesize separate nanoemulsions containing C. ovatum

leaf extracts and C. nucifera oil with a non-ionic surfactant Polysorbate 80 (Tween

80). This specifically aims to:

(i) prepare C. ovatum leaf extracts and C. nucifera oil;

(ii) separately synthesize nanoemulsions using the plant extract and oil with

non-ionic surfactant Polysorbate 80;

(iii) characterize the resulting nanoemulsions in terms of particle size using

Dynamic Light Scattering (DLS) and FTIR to identify organic

functional groups.
 4

(iv) determine the susceptibility of the prepared nanoemulsions against

selected microorganisms Escherichia coli, Staphylococcus aureus,

S2K3Y, and Stenotrophomonas maltophilia.

1.3 Significance of the Study

In the last few decades, a major problem faced by the agricultural industry

is the use of conventional agrochemicals that contribute broad-spectrum effects

towards the environment and organisms18. For insect pest control, plant-based

extracts and essential oils have emerged as viable alternatives to synthetic

insecticides. Because they are derived from plants and contain a variety of bioactive

compounds, these pesticides are naturally occurring19. As a result of this issue,

researchers are currently developing various formulations using different

nanotechnology approaches. Thus, innovations of plant extracts based

nanoemulsions is an important alternative for plant protection.

1.4 Scope and Limitation of the Study

This study focuses only on the synthesis of nanoemulsions containing C.

ovatum leaf extracts and C. nucifera crude oil and their antimicrobial efficacy

against E. coli, S. aureus, S2K3Y, and, S. maltophilia. The preparation of

nanoemulsions was done at the Material Science and Polymer Chemistry

Laboratory and the antimicrobial assay was done at the College of Agriculture and
 5

Agri-Industries laboratory of the Caraga State University-Main Campus, Ampayon,

Butuan City.
 6

CHAPTER 2

REVIEW OF RELATED LITERATURE

2.1 Canarium ovatum Engl.

Figure 2.1 Photo of Canarium ovatum leaves

Canarium ovatum Engl. belongs to the Sapindales Burseraceae family,

which is part of the Eudicotyledoneae order. Burseraceae is a tropical family with

16 genera and roughly 550 species found in both sides of the world. Canarium is a

genus of roughly 100 species that are mostly found in tropical Asia and the Pacific,

as well as Africa. Approximately 50% of the Canarium spp. are native to Southeast

Asia's ancient world tropics. Around 75 tree species are found in tropical Asia and
 7

the Pacific, as well as a few species in tropical Africa, under the Canarium class

(named after the Malay title 'Canari.' for one of the species). Around 53 species

were thought to be found in the Philippines, but that number has since been reduced

to nine20.

2.2 Cocos nucifera L.

Figure 2.2 Photo of Cocos nucifera fruit

The "coconut tree," or Cocos nucifera L., is the most widely grown natural

product plant on the planet. People have used medicinal plants for healing

throughout history, and minerals, plants, and animals have traditionally been the

most common sources of medications. The elements that make up C. nucifera has

a variety of natural properties, including anti-inflammatory, antioxidant, antifungal,

 8

antibacterial, and anticancer properties. C. nucifera L. is a member of the Arecaceae

(palm) family, and is commonly referred to as coconut, coco, coco-da-bahia, or

coconut-of-the-beach21.

2.3 Stenotrophomonas maltophilia

Apart from indirect biological control, there has been little research

investigation towards finding directly relevant biocontrol agents for bacterial wilt

management. Microorganisms which are able to grow in the rhizosphere are ideal

for controlling root-borne pathogens22. Stenotrophomonas maltophilia is a

common microorganism in the rhizosphere of cruciferous plants, and has also been

found in association with corn and beets23. Excretion of sulphur-containing amino

acids such as methionine by roots of cruciferous plants may favour the growth of

this species. S. maltophilia is also quite dominant in the rhizosphere of cereal

crops24. However, it was proven to be an effective biocontrol agent for the control

of various fungal and oomycetous plant pathogens25.

2.4 Related Studies on C. ovatum

Based on the analyses of Pili pulp and Pili oil contain several carotenoids

in the form of lycopene, lutein, and zeaxanthin 26. The extraction of Pili oil at

different stages of maturity shows that the level of this pigment decreases as the

seed matures, but it remains a substantial source of carotenoids. Carotenoids also

play an important potential role in human health by acting as biological antioxidants

protecting cells and tissues from the damaging effects of free radicals and singlet
 9

oxygen. The sterol fraction is the highest minor component. It is higher in the Pili

pulp than in Pili nut. Stigmasterol is the dominant sterol in the Pili nut oil, whereas

campesterol is dominant in Pili pulp oil. The presence of these phytosterols such as

campesterol, b-silosterol, and stigmasterol make Pili pulp and nut oils potential

products for the nutraceutical industry because of the pharmacological properties

these phytosterols27.

2.5 Related Studies on C. nucifera

Presently, VCO and its MCFAs have been used broadly against fungi and

most of the research were focusing on Candida albicans, the most common and

frequently isolated fungus from the human body. An in vitro study by Arnfinnsson

et al.28 showed that capric acid and LA had a strong ability to inhibit the growth of

C. albicans. The result reported that even at low concentration, LA was able to

inhibit the yeast cell but still, it required a longer than usual incubation time. The

reduction in infectivity titers suggested some fungicidal activity by capric acid and

LA29.

Huang et al.30 stated that different kinds of fatty acids displayed different

patterns of inhibition against oral bacteria. Their study demonstrated that MCFAs

had a significant anti-Candida activity while SCFAs and LCFAs showed limited

bioactivity against oral fungal species. According to the report of Ogbolu et al., C.

albicans, C. glabrata, C. tropicalis, C. parapsilosis, C. stellatoidea, and C. krusei

that had been isolated from the surrounding environment of Ibadan, Nigeria were
 10

sensitive to coconut oil. An agar well diffusion technique was used to test the

susceptibilities of Candida species to coconut oil and the results revealed that at

100% concentration of coconut oil, all the Candida species were sensitive to

coconut oil while at the lowest concentration (0.79%), only 35% of Candida species

were affected by coconut oil. Among all the species tested, C. albicans showed the

highest susceptibility to coconut oil while C. krusei demonstrated the utmost

resistance to coconut oil.

2.6 Mechanism of Action of Nanoparticles

Nanoparticles have been discovered to serve an important function in

inhibiting the growth and activity of certain fungus31. The use of

agronanochemicals in crop production is fundamentally based on the control of

certain plant diseases. Similarly, to nanosensors, research in this area has only lately

begun. Despite this, few studies on the use of agronanofungicides in the

management of fungal diseases have been conducted in the laboratory or in the

field. Plants produce essential oils primarily to protect themselves from harmful

microbes. Essential oils provide a number of advantages over conventional

fungicides, including fast breakdown, a broad antifungal spectrum, minimal

toxicity, and bioaccumulation32. Nanoencapsulation is a method of encapsulating

bioactive compounds using a nanocarrier technology. Because of the small size,

which enhances cellular uptake, it can boost the antifungal efficacy of bioactive

substances (essential oils) by increasing cell interactions among bacteria and

 11

nanoparticles. Because of their surface-to-volume proportions, physical properties,

sizes, degrees of selectivity, and chemical stabilities, the constituents of EOs can

access to the routes of the cell membrane, allowing them to arrive at their target

locations33.

2.7 Biosafety of Nanoparticles

Although nanoformulations are thought to be safer and more

environmentally friendly for disease control, a high amount of NPs toxicity

accidentally released into the environment could have detrimental consequences

for other bacteria and humans34. Nanomaterials' toxicological effects on soil

microorganisms and plants have been extensively researched. Despite this, little is

known about the nanotoxicity effects of plant–soil interaction systems. This is a

major concern when it comes to using nanotechnology to treat fungal diseases in

plants. There are several unknowns about the long-term effects of nanofungicide

formulations on human health and the environment. As a result, it is necessary to

examine the risk of agricultural laborers inhaling the nanofungicide during

spraying35.

2.8 Developments of Pesticide nanoemulsions

Oil-in-water nanoemulsions are thermodynamically unstable colloidal

dispersions that usually consist of emulsifier-coated oil droplets dispersed in water.

In any case, nanoemulsions have been defined as systems containing droplets with

diameters <200 nm, whereas conventional emulsions have been defined as systems
 12

containing >200. The small size of the droplets in nanoemulsions gives them some

advantages over conventional emulsions: higher optical clarity; higher stability to

droplet aggregation and gravitational separation; and higher bioactivity of

encapsulated components. Nanoemulsions are distinguished from microemulsions

based on their thermodynamic stability, rather than on their particle size

characteristics. Nanoemulsions are thermodynamically unstable, whereas

microemulsions are thermodynamically stable. Nevertheless, nanoemulsions can

be formulated to have good long-term kinetic stability36.

The biological activity of pesticides is a critical requirement for the

performance of pesticide nanoemulsions and is crucial for disease, insect, and weed

management. Because waxy insect and plant leaf surfaces have low hydrophilicity,

pesticides applied to them may readily come off and have a higher tendency to

evaporate, reducing their efficacy37. Emulsifiable concentrates and other classic

pesticide formulations have low wetting and rain wash resistance, which makes it

difficult for pesticides to absorb and penetrate, severely limiting their biological

activity. In practical applications, reducing the droplets size can significantly

improve their adhesion and permeability on the target organisms, thereby

enhancing the biological activity of pesticides and/or reducing their utilization

levels. A nanoemulsion with smaller spray droplets, excellent wetting performance,

rapid diffusion, and penetration at the target surface is conducive to improve the

biological activity of pesticides38.

 13

CHAPTER 3

METHODOLOGY

3.1 Isolation of Plant Extract and Essential Oil

C. ovatum leaves was collected in the low-lying bush-land area, around the

oval ground, inside the Caraga State University main campus, Ampayon, Butuan

City. The fresh leaves of C. ovatum were thoroughly washed with water and shade

dried in a dust-free condition. C. nucifera was purchased in Ampayon public market

Ampayon, Butuan City, Philippines.

The shade dried leaves of the C. ovatum were pulverized using an electrical

blender. The powdered plant materials were soaked in 96% Hexane 39 with a 100 g:

200 mL ratio for 48 hours. The containers that were used as storage jars were

covered with aluminum foil to prevent the penetration of light. The solution used

for the extraction was filtered using Whatman filter paper. The solvent was

evaporated using a rotary evaporator (HB4 basic IKA ® - WERKE) at 80°C at 70

rpm. The extracts obtained were transferred to a small beaker and were then left in

the fume hood for the complete evaporation of the solvent until the sample became

viscous.

The C. nucifera oil was extracted using the fermentation method as

described by40. The purchased coconut meat was then grated. Two matured coconut

fruit was fermented for the extraction of their oil. The coconut meat was mixed with

warm water with a 1:1 equivalent ratio. The mixture was wrung often to create a
 14

creamier mixture. And then the wringing was done again until the creamy juice of

the coconut meat was squeezed out. The resulting coconut milk was placed in a

shallow jar container and was left at rest for 48 hours in a closed container. After

48 hours’ time, the formation of curd was visible and the separation of oil from the

water was observed. The recovered coconut oil was filtered using Whatman filter

paper to remove impurities. The C. nucifera oil was set aside for the preparation of

the C. nucifera oil-based nanoemulsion.

3.2 Preparation of Canarium ovatum-based nanoemulsion

C. ovatum-based nanoemulsion was prepared using an oil-in-water

nanoemulsion. From the recovered extracts of C. ovatum (2%) concentration was

formulated. In a 50 mL beaker, 50 ml composition, 1 ml of the C. ovatum extract

was mixed together with 2 mL of Tween 80 surfactant (1:2 ratio), in a hot plate set

to 800 rpm equipped with a magnetic stirrer for 30 minutes. Thereafter, 47 mL of

distilled water was added dropwise into the mixture with a flow rate of 3.5 mL per

minute and was left stirring for 2 hours. Finally, the 50 mL total mass emulsions

were submitted to sonication (QSonica Sonicator- Ultrasonic Processor) at 50%

amplitude for 30 minutes’ time. As probe sonication may damage samples, so to

mitigate the ultrasound thermal effect of probe sonicator, all the samples of pre-
 15

emulsions were kept in an ice bath throughout the experiment. The formed

nanoemulsions of C. ovatum were kept for further analysis.

Scheme 3.1 Schematic diagram of C. ovatum nanoemulsion

methodology

3.3 Preparation of Cocos nucifera oil-based nanoemulsion

C. nucifera oil-based emulsion was prepared using oil-in-water

nanoemulsion. In order to fabricate oil-in-water nanoemulsion, in a 50 mL beaker,

50 ml composition, 4% of the C. nucifera essential oil was mixed together with 2

mL of Tween 80 surfactant (2:2 ratio) concentration in a hot plate set to 800 rpm

equipped with a magnetic stirrer for 30 minutes. After the 30 minutes, 46 mL of

 16

distilled water was added dropwise to the mixture with a flow rate of 3.5 mL per

minute and was then left for stirred for 2 hours until the emulsions were formed. It

was then sonicated (QSonica Sonicator- Ultrasonic Processor) at 50% amplitude

for 30 minutes’ time. As probe sonication may damage the sample, so to mitigate

the ultrasound thermal effect of probe sonicator, all the samples of pre-emulsions

were kept in an ice bath throughout the experiment. And the generated

nanoemulsion was then stored at room temperature prior to further application and

evaluation.

Scheme 3.2 Schematic diagram of C. nucifera oil

nanoemulsion methodology
 17

3.4 Characterization of Nanoemulsions

3.4.1 Evaluation of Droplet Size

The particle size of the samples was measured using an instrument Litesizer

500. Each sonicated nanoemulsion (6000 µl) was centrifuged for 10 minutes at

9000 rpm. An aliquot of a sample (1000 µl) was measured for droplet particle size

by a Dynamic Light Scattering device.

3.4.2 Fourier Transform Infra-Red Spectroscopy (FTIR)

The FTIR analysis of the nanoemulsions was performed using PerkinElmer

UATR Two. The nanoemulsions test samples were scanned in the transmittance

mode of 4000-400 cm−1 with a resolution of 4 cm−1 and an auto baseline correction

was performed before the chemometric analysis.

3.4.3 Susceptibility Test via Disc Diffusion Method

The potential antimicrobial efficacy of the C. ovatum and C. nucifera oil

nanoemulsions were determined via the disc diffusion method for susceptibility to

test microbes41. Before experimental use, cultures from a solid medium were sub-

cultivated in liquid media and then incubated. A 24 hours’ culture was diluted with

a sterile physiological saline solution with reference to the 0.5 McFarland standards

to achieve an inoculums size of approximately 10 6 colony-forming unit mL-1. A

population of each strain was inoculated on triplicate plates of (MHA) Muiller

Hinton Agar by using sterile cotton swabs. All bacterial cultures were standardized
 18

at 0.5 on the McFarland scale. The bacterial culture was then inoculated by

spreading on the surface of the three Petri dishes containing the solidified MHA as

a culture medium42. Immediately, the sterile paper discs were impregnated with the

C. ovatum nanoemulsion and C. nucifera oil nanoemulsion samples to be tested

were then applied to the inoculum medium in triplicate. All Petri plates were

allowed to dry, inverted, and incubated under the aerobic conditions at 36 ± 0.5 °C

for 24 h 43. After this time period, the plates were visually inspected for observation

(or not) of any growth inhibition halos.

 19

CHAPTER 4

RESULTS AND DISCUSSION

4.1 Dynamic Light Scattering analysis

The nanoemulsions droplet size was examined using a particle size

analyzer. In general, nanoemulsions droplet size range from 1-200 nm 44.

Figure 4.1. Size distribution by the intensity of 4%

C. nucifera oil nanoemulsion.

Figure 4.1 and 4.2 shows the particle size distribution of 4% C. nucifera oil

nanoemulsion and 2% C. ovatum leaf extracts nannoemulsion respectively. The

prepared nanoemulsions show a low mean diameter with a particle size distribution
 20

of <200 nm. The size distribution of the synthesized nanoemulsion from the C.

nucifera with 4% concentration showed the size distribution of the particles with

the maximum intensity at 111.41 nm. While the size distribution of the 2% C.

ovatum leaf extracts nanoemulsion was found to have a sharp peak with a maximum

intensity of 23.92 nm.

Figure 4.2 Size distribution by the intensity of 2%

C. ovatum leaf extract nanoemulsion

4.2 FTIR Analysis

FTIR analysis allow to identify functional groups, since each functional

group absorbs radiation in a characteristic frequency of the infrared spectrum 45.

 21

Figure 4.3 shows the infrared spectra of the 4% C. nucifera oil

nanoemulsion. As observed, a strong absorption band assigned to the saturated

hydrocarbon groups (2925 cm−1, corresponding to a methyl group (C–H3), The

bands at 1639 cm−1 belonging to the stretching vibration of C=O stretch and bands

at 1097 cm−1 corresponding to the bending vibration of C–O stretch groups can

indicate the presence of hydroxyl group. At 3350 cm−1 corresponds to O–H stretch.

The IR region between 1542 to 965 cm-1 is usually referred to as the “fingerprint”

Figure 4.3 IR Spectra of 4% C. nucifera

oil nanoemulsion

region, and the IR bands, including those corresponding to the vibration of the C–

O, C–C, C–H, and C–N bonds,occur in this region. This area provides important

information regarding organic compounds such as sugars, alcohols, and organic

acids that are probably present in the extract. The 1639 cm-1 absorption band may

be attributed to the C=O in-plane deformation in alkenes (nonconjugated)46.

 22

Figure 4.4 depicts the IR Spectra of the 2% leaf extract nanoemulsion of the

C. ovatum. At 3339 cm-1, an absorption band was observed with a robust and broad

intensity assigned to a phenolic group O-H stretching. The band at 1639 cm-1 has

the mode of vibration C=O stretching corresponding to the aldehydes, ketones and

ethers.

Figure 4.4 IR Spectra of 2% C. ovatum leaf

Extract nanoemulsion

4.3 Evaluation of Susceptibility Test

The Kirby-Bauer disc diffusion susceptibility test was performed to

determine the sensitivity or resistance of the pathogenic bacteria to the test

microbes41.
 23

Figure 4.5 Antimicrobial activity assays of the Canarium ovatum-

based and C. nucifera-based nanoemulsions against
(A) E. coli (B) S. aureus (C) S2K3Y (D) S. maltophilia.

Antimicrobial activity was assessed using the following rating system: (1)

<10 mm zone of inhibition maybe expressed as inactive, (2) 10-13 mm zone of

inhibition is partially active: (3) 14-19 mm zone of inhibition is active and (4) >19

mm zone of inhibition is very active 47. The average zones of inhibition (in mm)

produced by C. ovatum and C. nucifera nanoemulsions against E. coli, S. aureus,

S2K3Y and S. maltophilia was measured after 24 hours.

Figure 4.5 shows the antimicrobial activities of the 4% C. nucifera oil-based

and 2% C. ovatum-based nanoemulsions. It is revealed that the 4% C. nucifera oil-

based and 2% C. ovatum-based nanoemulsions does not exhibit any antimicrobial

activity against all test microorganisms. On the other hand, 4% C. nucifera oil-
 24

based and 2% C. ovatum-based nanoemulsions exhibited a slight inhibition against

S. maltophilia bacteria only as shown in the figure 4.5, D.

 25

CHAPTER 5

SUMMARY, CONCLUSION, AND RECOMMENDATION

The growing prevalence of life-threatening bacterial and fungal infections,

as well as pathogens' ability to develop resistance to current treatment strategies,

necessitates the discovery and development of new compounds to combat them.

Modern agriculture is one of the many applications of nanotechnology. Identifiable

endophytic bacteria with plant growth-promoting traits promise to ensure

sustainable agriculture. Stenotrophomonas maltophilia is one of the most prevalent

opportunistic bacteria causing nosocomial infections. Nanoparticles, which range

in diameter from 1 to 200 nm, are used to enhance plant growth and protect them

against damaging pests such as insects and pathogens such as bacteria, fungi, and

viruses. The Canarium ovatum leaf extracts nanoemulsion was formulated 2%

concentration and Cocos nucifera oil nanoemulsion was formulated 4%

concentration. The nanoemulsions developed were characterized using Dynamic

Light Scattering (DLS) for the size distribution intensity and FTIR analysis to

identify organic functional groups and was tested for their antibacterial activities.

The synthesized nanoemulsions show a low mean diameter with a particle size

distribution of <200 nm. The 4% C. nucifera-oil nanoemulsions showed a

maximum intensity at 111.41 nm. The C. ovatum leaf extracts nanoemulsions 2%

concentration gives a size distribution with a maximum intensity at 23.92 nm. The

FTIR analysis of the 4% C. nucifera oil nanoemulsion observed a strong absorption

 26

band assigned to the saturated hydrocarbon groups and a stretching vibration

indicating the presence of alcohol and ether in general. The spectra of the 2% C.

ovatum leaf extracts nanoemulsions displayed an absorption band observed with a

robust and broad intensity assigned to a phenolic group O-H stretching. The band

at 1639 cm-1 has the mode of vibration C=O stretching corresponding to the

aldehydes, ketones and ethers. Current findings of the antimicrobial assay of the

2% C. ovatum leaf extracts and 4% C. nucifera oil nanoemulsions against four

different test microorganisms (E. coli, S. aureus, S2K3Y and S. maltophilia)

examined to be partially active only against S. maltophilia bacteria.

It is further recommended that the study will be continued and explore the

possibility of using other solvents for the extraction of the plant constituents.
 27

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 32

APPENDICES

Appendix A

Table 1. Diameter of zones of inhibition (mm) of plant extracts

nanoemulsions against test organisms.
Test Nanoemulsions
Organisms
4% C. nucifera oil 2% C. ovatum
nanoemulsion nanoemulsion
E. coli 8 8

S. aureus 8 8

S2K3Y 10 8

S. maltophilia 12 14
 33

Appendix B

Documentations

Canarium ovatum collection

Preparation of Nanoemulsions

(pulverizing plant sample using blender) (weighing and soaking using Hexane solvent)

(filtration of extracts after soaking for 48 hours) (evaporation of the excess solvent)
 34

(C. nucifera oil extraction using fermentation method)

Antimicrobial Assay

(Impregnating paper discs with synthesized nanoemulsion in triplicates)

 35

Appendix C

Certificate of Plant Verification

 36

MSCRC Certification
 37

CURICCULUM VITAE

Personal Information
Name: Conie Grace C.Otaza
Address: P-Rose, San Agustin, Talacogon, ADS

Age: 24 years old

Birthday: December 04, 1997
Status: Single

Citizenship: Filipino
Mother’s Name: Melita C. Otaza
Father’s Name: Salvador B.Otaza

Educational Attainment

College Level: Caraga State University

Ampayon, Butuan City
2015-2022

Secondary Level: Talacogon National High School

Talacogon, Agusan del Sur
2011-2015

Elementary Level: Talacogon Central Elementary School

Talacogon, Agusan del Sur
2005-2011