Impacts of Green Synthesis of

Silver Nanoparticles (AgNPs)

Using Leaf Extract of Rosmarinus
Officinalis (Rosemary) on Grow
Characteristics, Photosynthetic
Pigment Content and Anti-
inflammatory Activity of Tomato
Plant
 Acknowledgement

We sincerely expressed our gratitude to God for giving us strength, motivation and

inspiration to continue this study despite in the midst of this pandemic. Making this

proposal is difficult for us because of the issues that we are facing right now.

proposal. We want to thank her for allowing us to do research and provide us guidance

throughout this research. Her lessons were engaging, useful, and she was very patient

with everyone in our class always encouraging her students to try. Lastly, I want to thank

her for teaching us how to make a research paper, carry it out, and present the study as

clearly as possible.

Most of all, we would like to thank our family, and friends for giving us endless

guidance and support. We want to thank them for praying for us to pass this research

study. We’re very thankful for having them, their understanding, prayers and continuing

to support us to complete this research.

 Abstract

While plants are an essential living component of the terrestrial environment,

nanoparticles have distinct physicochemical features and the ability to improve plant

metabolism. Nanoparticles also exert their negative effects such as suppression of plant

growth, inhibition of chlorophyll synthesis, photosynthetic efficiency, etc. Nanoparticles

can be employed as nanofertilizers, nano-pesticides, and herbicides, which can help to

boost agricultural yield, reduce the usage of chemical fertilizers, and boost survival

against biotic stress. AgNPs have an effect on plant growth and development that is both

progressive and damaging. In plants, it enhanced plant growth; nonetheless, it reduces

root growth, root length, and biomass. The optical absorbance of the produced silver

nanoparticles was measured using a UV-visible spectrophotometer with a resolution of 1

nm between the wavelengths of 300 and 900 nm. The TEM was used to analyze the

shape and size of the AgNPs. The production of silver nanoparticles was also examined

using an X-ray diffract meter. This study aims to evaluate the effects of the biosynthesized

AgNPs using the leaf extract of rosemary on the growth characteristics, photosynthetic

pigment content, and anti-inflammatory activity of the tomato plant.

 Table of Contents

Chapter 1: Introduction Page #

Background of the Study 1
Statement of the Problem 4
Statement of the Objectives 4
Statement of the Hypotheses 5
Significance of the Study 6
Scope and Limitations 7
Chapter 2: Review of Related Literature and Studies
Related Literature 8
Related Studies 12
Chapter 3: Methodology
Collection of Materials 17
Preparation of Rosemary Extract 17
Green synthesis of Silver Nanoparticles 17
Characterization of Biosynthesized AgNPs 18
Planting and Harvesting 18
Measurement the Length of Root and the Aerial Part 19
Photosynthetic Pigment Measurement 19
Anti-inflammatory Activity 20
Statistical Analysis 21
Bibliography 22
Appendices 24
 CHAPTER 1

INTRODUCTION

Background of the Study

Nanotechnology is an appealing study subject since the application of

nanoparticles in numerous disciplines is expanding owing to specific physicochemical

properties. Nanoparticles have found widespread use in agricultural, manufacturing,

pharmaceutical, and medical disciplines.1 While plants are an essential living component

of the terrestrial environment, nanoparticles have distinct physicochemical features and

the ability to improve plant metabolism. Nanoparticles also exert their negative effects

such as suppression of plant growth, inhibition of chlorophyll synthesis, photosynthetic

efficiency, etc. Nanoparticles can be employed as nanofertilizers, nano-pesticides, and

herbicides, which can help to boost agricultural yield, reduce the usage of chemical

fertilizers, and boost survival against biotic stress.2 Despite the rapid expansion of

nanotechnology and nanomaterials over the last two decades, new attention has been

drawn to the possible toxicological consequences of metal nanoparticles on animals,

people, and the environment.

1
Abdol Miri, Elham Shakib, et.al. “Impacts of Nickel Nanoparticles on Grow Characteristics,
Photosynthetic Pigment Content and Antioxidant Activity of Coriandrum sativum L.”, February
01, 2017. http://dx.doi.org/10.13005/ojc/330329
2
Farzad Aslani, Samira Bagheri, et. al. “Effects of Engineered Nanomaterials on Plants
Growth: An Overview”, August 14, 2014. https://www.hindawi.com/journals/tswj/2014/641759/

1
 According to the previous studies, at large concentrations, nanoparticles reduce

the fresh and dry weight of both roots and aerial components. 3 In the present study, the

AgNPs will be biosynthesized using the leaf extract of rosemary to reduce and stabilize

the nanoparticles.

With the rapid expansion of nanotechnology, metal nanoparticles have become

widely utilized in a variety of applications. Silver nanoparticles (AgNPs) are commonly

employed in antibacterial activities, cosmetics, and everyday items. It is increasingly used

in various fields, including medical, food, health care, consumer, and industrial purposes,

due to their unique physical and chemical properties. Due to their peculiar properties, they

have been used as antibacterial agents, in industrial, household, and healthcare-related

products, in consumer products, medical device coatings, optical sensors, and cosmetics,

in the pharmaceutical and food industries, in diagnostics, orthopedics, drug delivery, and

as anticancer agents, and have ultimately enhanced the tumor-killing effects of anticancer

drugs. 4 AgNPs have an effect on plant growth and development that is both progressive

and damaging. In plants, it enhanced plant growth; nonetheless, it reduces root growth,

root length, and biomass.

Biological synthesis based on microbes, plant extract, and enzymes has been

proven to be a simple, cost-effective, and environmentally friendly alternative synthesis

path for metallic nanoparticles when compared to traditional synthesis. Among the several

3
Abdol Miri, Elham Shakib, et.al. “Impacts of Nickel Nanoparticles on Grow Characteristics,
Photosynthetic Pigment Content and Antioxidant Activity of Coriandrum sativum L.”
4
Wei Shen, Xi-Feng Zhang, et. al. “Silver Nanoparticles: Synthesis, Characterization,
Properties, Applications, and Therapeutic Approaches”, September 17, 2016.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5037809/

2
biological pathways explored thus far, synthesis techniques employing plant extract or

fruit extract have significant benefits over others since they do not require cell culture and

are simply scaled up for large-scale synthesis.5 Lastly, the biological synthesis of

nanoparticles is an ecofriendly and inexpensive method.

Rosmarinus Officinalis or rosemary belongs to the Lamiaceae family, and their

natural extracts have bioactive qualities. Several studies have found that rosemary

extracts exhibit biological bioactivities such as hepatoprotection, antifungal activity,

insecticide activity, antioxidant activity, and antibacterial activity. Rosemary's antioxidant

and anti-inflammatory action is mostly related to polyphenolic components such as

rosmarinic acid and carnosic acid. Because of its antioxidant properties, rosmarinic acid

is frequently used as a natural preservative to extend the shelf life of perishable goods. 6

This study aims to evaluate the effects of the biosynthesized AgNPs using the leaf

extract of rosemary on the growth characteristics, photosynthetic pigment content, and

anti-inflammatory activity of the tomato plant. As well as the toxic activities will be

investigated.

5
Sampa Maiti, Maximiliano Cledon, et. al. “Biological synthesis of metallic nanoparticles: plants,
animals and microbial aspects”, October 09, 2017.
https://link.springer.com/article/10.1007/s41204-017-0029-4
6
Jonatas de Oliveira, et.al. “Rosmarinus officinalis L. (rosemary) as therapeutic and
prophylactic agent”, January 09, 2019. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6325740/

3
Statement of the Problem
This study aims to determine the effects of green synthesis of Silver nanoparticles

using leaf extract of Rosemary on grow characteristics, photosynthetic pigment content

and anti-inflammatory activity of Tomato plant, thus the researcher wants to ask the

following questions:

1. Does Silver nanoparticles will reduce the length of root of tomato plant by mm?

2. Does the leaf extract of Rosemary will enhance the anti-inflammatory activity

of Tomato plant by the carrageenan induced paw oedema model?

3. Does biosynthesizing the Silver nanoparticles will enhance the growth

characteristics of the tomato plant?

Statement of the Objectives

To determine the effects of green synthesis of Silver nanoparticles using leaf

extract of Rosemary on grow characteristics, photosynthetic pigment content and anti-

inflammatory activity of Tomato plant.

Specific Objectives

This study aims to:

 To characterize the optical absorbance of the synthesized Silver nanoparticles

using a UV-visible spectrophotometer.

 To test the green synthesis of Silver nanoparticles using leaf extract of Rosemary

on Tomato plant.

4
  To determine the impacts of biosynthesized Silver nanoparticles on the

photosynthetic pigment content of the Tomato using the protocol described by

Lichtenthaler and Wellburn.

 To determine the effects of biosynthesized Silver nanoparticles on the anti-

inflammatory activity of Tomato using the carrageenan induced paw oedema

model.

 To measure the length of root and the aerial part of the treated and control Tomato

plant.

Statement of the Hypothesis

If the leaf extract of Rosemary is synthesized to Silver nanoparticles, then it could

significantly promote photosynthesis and anti-inflammatory activity of the tomato plant.

 If a high concentration of Silver nanoparticles will be applied to the tomato plant,

then it will reduce the root length of the plant.

 If the leaf extract of Rosemary is mostly related to polyphenolic components, then

it will enhance the anti-inflammatory activity of Tomato plant.

 If the biosynthesized Silver nanoparticles has the ability to enhanced plant growth,

then it will enhance the growth characteristics of the tomato plant.

5
Significance of the Study

It is important to investigate how plants absorb and ingest nanoparticles, as well

as how they travel inside the plant and interact with the plant cell.7 Nanomaterials can

boost agricultural production by improving fertilizer nutrient availability in the soil and plant

nutrient absorption. Silver nanoparticles (AgNPs) have been used as antibacterial

agents. AgNPs have an effect on plant growth and development that is both progressive

and damaging. In plants, it enhanced plant growth; nonetheless, it reduces root growth,

root length, and biomass.8 Synthesis techniques employing plant extract or fruit extract

have significant benefits over others since they do not require cell culture and are simply

scaled up for large-scale synthesis. This is where the Rosemary leaves happens,

biologically synthesizing the AgNPs because it is simple, low cost, non-toxic, and

environmentally friendly. This study aims to evaluate the effects of the biosynthesized

AgNPs on the growth characteristics, photosynthetic pigment content, and anti-

inflammatory activity of the tomato plant as an effective, inexpensive and eco-friendly

nanofertilizer.

7
Renato Grillo, Alejandro Perez, et.al. “Interaction of Nanomaterials with Plants: What Do We
Need for Real Applications in Agriculture?”, April 10, 2017.
https://doi.org/10.3389/fenvs.2017.00012
8
Wei Shen, Xi-Feng Zhang, et. al. “Silver Nanoparticles: Synthesis, Characterization,
Properties, Applications, and Therapeutic Approaches”,
6
Scope and Limitations of the Study

This study is focusing to evaluate the effects of the biosynthesized AgNPs

using the leaf extract of Rosemary on the growth characteristics, photosynthetic pigment

content, and anti-inflammatory activity of the tomato plant. The silver nitrate (AgNO3) will

be purchased from the University of the Philippines Diliman, Laboratory of

Electrochemical Engineering. The rosemary leaves will be collected from Puregold

Makati. This study will determine the optical absorbance of the synthesized silver

nanoparticles using a UV-visible spectrophotometer, the shape and size of AgNPs using

Transmission electron microscopy, and the formation of silver nanoparticles will be

checked by X-ray diffraction technique using an X-ray diffract meter. This study will take

around three months to complete, including the collecting and preparation of materials.

The limitation of the study is to only apply a low concentration of AgNPs to maintain the

growth characteristics of the tomato plant. This study will be divided into two groups;

experimental and control groups. The experimental group will have 3 sets, where set A

will be the leaf extract of Rosemary is applied on the tomato plant, while set B will be the

Silver Nanoparticles is applied on the tomato plant. On the other hand, set C will be the

synthesized AgNPs using the extract of Rosemary leaves and will be applied to the

tomato plant. Lastly, the control group where the commercial fertilizer will be applied on

the tomato plant.

7
 Chapter II

Review of Related Literature and Studies

Related Literature

Silver Nanoparticles

Silver is a glossy, delicate white element. Metallic silver is not soluble in water,

however metallic compounds such as AgNO3 and Silver chloride are. Metallic silver in

finely dispersed form exhibits unique properties typically associated with noble metals,

such as chemical stability, excellent electrical conductivity, catalytic activity, and other

more specific properties such as anti-bacteriostatic effects and non-linear optical

behavior, among others.9 Silver nanoparticles (AgNPs) have distinct physical and

chemical characteristics than their macroscale counterparts. This is largely owing to their

tiny size and, as a result, their remarkable surface area. Advances in the synthesis,

stabilization, and manufacturing of AgNPs have resulted in a new generation of

commercial goods as well as increased scientific research in the field of nanotechnology.

The usage of AgNPs in commercial goods is growing, although the effects on the

environment and human health are still largely understood. 10 The use of Ag NPs in

9
Preeti Rajoriya, “Green Synthesis of Silver Nanoparticles, Their Characterization and
Antimicrobial Potential” https://www.semanticscholar.org/paper/GREEN-SYNTHESIS-OF-
SILVER-NANOPARTICLES%2C-THEIR-AND-
Rajoriya/f0bf27a7f833d36f3c491b706b5fc8383b702fde
10
Monique Johnson, et. al. “Silver Nanoparticles: Technological Advances, Societal Impacts, and
Metrological Challenges” Green and Sustainable Chemistry, February 21,
2017. https://doi.org/10.3389/fchem.2017.00006

8
electronics, catalysis, medicines, and regulating microorganism development in biological

systems has made them environmentally safe.

Rosemary

Rosmarinus officinalis, L. is a fragrant plant of the Lamiaceae family native to the

Mediterranean region. Because of its hepatoprotective capacity, therapeutic promise for

Alzheimer's disease, and antiangiogenic action, rosemary extracts have been utilized in

the treatment of illnesses. On the other hand, because they inhibit oxidation and

microbiological contamination, they have been employed in food preservation. As a result,

rosemary extract may be beneficial for substituting or reducing synthetic antioxidants in

meals. As preservatives, rosemary extracts provide many technological advantages and

consumer benefits. 11 Rosemary possesses antibacterial, anti-inflammatory, anti-oxidant,

anti-apoptotic, anti-tumorgenic, antinociceptive, and neuroprotective effects. Recently,

there has been a notable increase in scientific interest in the positive medicinal effects of

various types of rosemary extracts and their major components, such as carnosic acid,

carnosol, rosmarinic acid, and so on. 12

Tomato Plant

Tomato (Solanum lycopersicum, L.) is a member of the Solanaceae family, often

known as Nightshades, which has over 3000 species. Tomatoes originated in the Andean

11
Gema Nieto, et. al. “Antioxidant and Antimicrobial Properties of Rosemary (Rosmarinus
officinalis, L.): A Review”, September 04, 2018.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6165352/
12
Hossein Hosseinzadeh, et. al. “Therapeutic effects of rosemary (Rosmarinus officinalis L.)
and its active constituents on nervous system disorders,” Iranian Journal of Basic Medical
Sciences 23, no. 9 (2020) 1100–1112. https://ijbms.mums.ac.ir/article_15705.html

9
area, which includes modern-day Chile, Bolivia, Ecuador, Colombia, and Peru; however,

the exact location of domestication remains unknown. Because of its rising economic and

nutritional importance, extensive production, and role as a model plant for research, the

tomato remains the most significant vegetable in the world. 13 Tomato is regarded a model

organism of the Solanaceae family for research purposes, and as such, it has been and

continues to be a significant crop topic of studies both in the laboratory and in the field.

Green Synthesis

Bacteria, fungi, yeast, actinomycetes, and plant extracts are all involved in the

biogenic synthesis of AgNPs. Aside from enzymes, a variety of plant components,

including flowers, leaves, and fruits, have recently been utilized in the production of gold

and silver nanoparticles. The size, shape, and stability of nanoparticles are affected by

the methods of production, solvent type, concentration, reducing agent strength, and

temperature. 14 The benefits of green nanoparticle production over physical and chemical

techniques are as follows: toxic chemicals are not employed, resulting in a clean and eco-

friendly method; the active biological component acts as a reducing and capping agent,

lowering the overall cost synthesis process. It may be utilized for large-scale nanoparticle

13
Leander Melomey, et. al. “Review on Tomato (Solanum lycopersicum, L.) Improvement
Programmes in Ghana,” July 20, 2017. https://www.intechopen.com/books/recent-advances-in-
tomato-breeding-and-production/review-on-tomato-solanum-lycopersicum-l-improvement-
programmes-in-ghana
14
Azamal Husen, et. al. “A review on biosynthesis of silver nanoparticles and their biocidal
properties,” Journal of Nanobiotechnology 16, no. 14 (2018). https://doi.org/10.1186/s12951-
018-0334-5

10
manufacturing; external experimental conditions such as high energy and high pressure

are not required, resulting in an energy-saving method. 15

Carrageenan Induced Paw Oedema Model

Inflammation is a process that happens following an infection or tissue injury, and

it is characterized by increased postcapillary venule permeability to fluid and plasma

proteins, as well as polymorphonuclear leukocyte emigration into tissues. 16 The

carrageenan-induced rat paw edema model is an appropriate test for assessing anti-

inflammatory medicines, and it has frequently been used to examine the drug's

antiedematous efficacy. Carrageenan is a powerful molecule that promotes the

production of inflammatory and proinflammatory mediators (prostaglandins, leukotrienes,

histamine, bradykinin, TNF-, and so on). 17 One of the most often used assays for

evaluating African spices and vegetables for anti-inflammatory effectiveness is

carrageenan-induced paw edema. Carrageenan-induced inflammation is effective in

15
Jagpreet Singh, et. al. “A Review On Green Synthesis and Characterization of Silver
Nanoparticles and Their Applications: A Green Nanoworld,” July 2016.
https://www.researchgate.net/publication/304693241_A_REVIEW_ON_GREEN_SYNTHESIS_
AND_CHARACTERIZATION_OF_SILVER_NANOPARTICLES_AND_THEIR_APPLICATIONS_
A_GREEN_NANOWORLD
16
Ruslan Medzhitov, et. al. “Origin and physiological roles of inflammation,” July 01, 2008.
https://www.nature.com/articles/nature07201
17
Purabi Roy, et. al. “Anti-Inflammatory Activity of Lactobacillus on Carrageenan-Induced Paw
Edema in Male Wistar Rats,” International Journal of Inflammation, February 22, 2012.
https://www.hindawi.com/journals/iji/2012/752015/

11
detecting orally active anti-inflammatory drugs, and hence has a high predictive value for

anti-inflammatory medicines working through acute inflammation mediators. 18

Photosynthetic Pigment Content

Plants contain a variety of pigments, which give birth to the many colors we see.

Flowers and fruits are obviously rich in organic compounds that absorb light. Pigments

can also be found in the leaves, stems, and roots. Anthocyanins, flavanoids, flavines,

quinones, and cytochromes are a few examples of pigment compounds. None of these,

however, should be called photosynthetic pigments. Photosynthetic pigments are the only

pigments that can collect solar energy and transfer it to the photosynthetic system. There

are two types of photosynthetic pigments found in terrestrial plants: chlorophylls and

carotenoids. 19 The photosynthetic pigment contents of the plants were tested in both

control and treatment samples using the methodology published by Lichtenthaler and

Wellburn.

Related Studies

Green Synthesis of Silver Nanoparticles Using Lysiloma Acapulcensis Exhibit

High-Antimicrobial Activity

AgNPs are utilized as an antibacterial agent in nanomedicine and biomaterials.

Green synthesis is one of the most efficient methods for producing AgNPs. Lysiloma

18
J.P. Dzoyem, et. al. “Anti-inflammatory and Anti-nociceptive Activities of African Medicinal
Spices and Vegetables,” Medicinal Spices and Vegetables from Africa (2017): 239-270.
https://doi.org/10.1016/B978-0-12-809286-6.00009-1
19
John Markwell, et. al. “Photosynthetic Pigments,” Plant Pigments and Photosynthesis,
https://passel2.unl.edu/view/lesson/920a6340a995/2

12
acapulcensis (L. acapulcensis) is a perennial tree native to Mexico that is utilized in

traditional medicine. This tree is rich in antibacterial chemicals. In terms of antibacterial

action, L. acapulcensis extracts can reduce silver to AgNPs and therefore improve its

antimicrobial activity. The FTIR and LC–MS results revealed the existence of chemical

groups that might function as either reducing agents for AgNP stabilization or

antimicrobial capping agents for AgNP antibacterial capabilities. E. coli > S. aureus > P.

aeruginosa > C. albicans had the highest antibacterial potency. Green generated

(biogenic) AgNPs were shown to have more antibacterial efficacy than chemically

manufactured AgNPs. The results show that biogenic AgNPs have a greater antibacterial

activity while being low in cytotoxicity than chemically generated AgNPs.20

Evaluation of In-vitro Anti-Inflammatory Activity of Silver Nanoparticles

Synthesised using Piper Nigrum Extract

Nanotechnology has emerged as an intriguing method in the drug development

process, and silver nanoparticles have been investigated for a range of medicinal uses.

The eco-friendly and cost-effective approach of developing silver nanoparticles with extra

characteristics imparted by the capping phytochemicals is known as phyto-assisted

synthesis of silver nanoparticles. Because of the synergistic impact of alkaloids from Piper

20
Diana Garibo, et. al. “Green synthesis of silver nanoparticles using Lysiloma acapulcensis
exhibit high-antimicrobial activity,” Scientific Reports volume 10, no. 12805 (2020): 1–11.
https://www.nature.com/articles/s41598-020-69606-7

13
nigrum extract and silver ions, the synthesised silver nanoparticles demonstrated

increased anti-inflammatory activity. 21

Impacts of Nickel Nanoparticles on Grow Characteristics, Photosynthetic Pigment

Content and Antioxidant Activity of Coriandrum sativum L.

Nanotechnology is one of the world's most rapidly growing sciences. Previous

study has shown that nanoparticles that have contaminated the environment can have a

significant impact on the characteristics of medicinal plants. The effects of nickel

nanoparticles on root and shoot elongation, relative water content (RWC), photosynthetic

pigment, total ash, and antioxidant activity of Corianderum sativum L. were studied in this

work. The results indicated that nickel nanoparticles reduced RWC, root and shoot

elongation, photosynthetic pigment content, and percent total ash. The findings of this

study indicate that nickel nanoparticles are harmful to the C. sativum L plant. This study

might be a watershed moment in the field of nanotechnology research in medicinal plants.

Other researchers can examine any element of the study on their own, with an emphasis

on molecular processes. 22

21
Aparna Mani, et. al. “Evaluation of In-vitro Anti-Inflammatory Activity of Silver Nanoparticles
Synthesised using Piper Nigrum Extract,” Journal of Nanomedicine & Nanotechnology 6, no.
268 (2015): https://doi.org/10.4172/2157-7439.1000268
22
Abdol Miri, Elham Shakib, et.al. “Impacts of Nickel Nanoparticles on Grow Characteristics,
Photosynthetic Pigment Content and Antioxidant Activity of Coriandrum sativum L.,”
14
Green Synthesis of Silver Nanoparticles Using Leaf Extract of Rosmarinus

officinalis and Its Effect on Tomato and Wheat Plants

In this study, rosemary leaf extract was employed as a reducing and stabilizing

agent in the biosynthesis of AgNPs. UV-vis spectrophotometer and X-ray diffraction

(XRD) analyses were used to validate the biosynthesized AgNPs. A high resolution

transmission electron microscope was used to examine the form and size of the

biosynthesized AgNPs (TEM). The toxicity of biosynthesized silver nanoparticles on

wheat and tomato plants was investigated by soaking wheat grains and tomato seeds in

100 mg/L AgNPs and monitoring the influence on wheat seedling growth (at 10 days)

and tomato and wheat plant vegetative growth (at 35 days). The biosynthesized AgNPs

had a notable stress impact on tomato plants, resulting in decreased chlorophyll an and

dry weight. AgNPs, in general, promote MDA buildup in tomato and wheat plants.

AgNPs had a distinct influence on soluble proteins and antioxidant enzymes such as

catalase and peroxidase in tomato and wheat plants. 23

Impact of silver nanoparticles on plant growth, some biochemical aspects, and

yield of fenugreek plant (Trigonella foenum-graecum)

Nanoparticles have the capacity to boost the development and production of

several plants, including fenugreek (Trigonella foenum-graecum) (Fabaceae). The

current study seeks to investigate the impact of silver nanoparticles (AgNPs) on

23
Fatma Farghaly, and Nivien Allam Nafady. “Green Synthesis of Silver Nanoparticles Using
Leaf Extract of Rosmarinus officinalis and Its Effect on Tomato and Wheat Plants,” Journal of
Agricultural Science 7, no. 11 (2015): 277. https://www.researchgate.net/publication/283828662

15
fenugreek plant development, certain biochemical characteristics, and yield, both

quantitatively and qualitatively. Silver nitrate was chemically reduced using trisodium

citrate to produce AgNPs. Foliar application of AgNPs at various concentrations (20, 40,

and 60 mg/l) improved fenugreek plant growth parameters (e.g., shoot length, number of

leaves/plant, and shoot dry weight) and increased some biochemical aspects such as

photosynthetic pigment (chlorophyll a, chlorophyll b, and carotenoids) and indole acetic

acid (IAA) contents, which increased yield quantity (number of pods. The most successful

therapy was 40 mg/l, which generated the greatest increases in the parameters

examined.24

24
Mervat Sh Sadak. “Impact of silver nanoparticles on plant growth, some biochemical aspects,
and yield of fenugreek plant (Trigonella foenum-graecum),” Bulletin of the National Research
Centre 43, no. 38 (2019): 1.
https://www.researchgate.net/publication/331461143_Impact_of_silver_nanoparticles_on_plant
_growth_some_biochemical_aspects_and_yield_of_fenugreek_plant_Trigonella_foenum-
graecum

16
 CHAPTER III

METHODOLOGY

1. Collection of Materials

The rosemary (Rosmarinus officinalis) leaves will be collected from Puregold

Makati, while the silver nitrate (AgNO3) will be purchased from the University of the

Philippines Diliman, Laboratory of Electrochemical Engineering. The tomato (Solanum

lycopersicum L.) will be used as a test plant and will be bought. The other materials will

be obtained from different laboratories in the Philippines.

2. Preparation of Rosemary Extract

Leaves of rosemary (Rosmarinus officinalis) will be collected, will be washed

several times with sterilized distilled water. Five grams of fresh leaves will be boiled in

100 mL distilled water for 5 min. The solution will be left to cool, and then will be filtered

with Whatman filter paper. Filtrate was centrifuged for 5 min. at 4000 rpm. The

supernatant will be used for the synthesis of silver nanoparticles.

3. Green synthesis of Silver Nanoparticles

Aqueous solution of AgNO3 (1 mM) will be added drop wise into 50 mL of rosemary

leaf extract. The mixture will be incubated for 18 h at room temperature. Control without

AgNO3 will be also kept at the same conditions. The solution will be centrifuged for 10

min. at 10000 rpm, in order to isolate the AgNPs. The nanoparticles will be washed

several times using deionized water, and then suspended in 95% ethanol prior to

characterization.

17
4. Characterization of Biosynthesized AgNPs

4.1. UV-Visible Spectroscopy

Optical absorbance of the synthesized silver nanoparticles will be performed

using a UV-visible spectrophotometer (Perkin-Elmer lambda 750

spectrophotometers) between the wavelengths of 300 and 900 nm at a

resolution of 1 nm. The reaction mixture will be first diluted 5 times with distilled

water and will be used for UV-visible analysis. The AgNPs formation by

reducing silver ions will be investigated by UV-Vis spectroscopy.

4.2. Transmission Electron Microscopy (TEM)

Transmission electron microscopy (TEM) was performed to analysis the shape

and size of the AgNPs using the JEOL TEM 100 CXII. The JEOL 100CX II TEM

is a traditional transmission electron microscope (TEM) with a tungsten

filament. The tungsten electron source is heated to a high temperature, and the

energized electrons escape the tungsten metal, are concentrated into a beam,

and accelerated down the length of the TEM column.

4.3. X-Ray Diffraction

The formation of silver nanoparticles will be checked by X-ray diffraction

technique using an X-ray diffract meter.

5. Planting and Harvesting

The tomato seeds will be planted in vases. Every other day, the seeds will be

irrigated with 25 ml water for 3 months. Then, they will be treated with 25 mL of silver

nanoparticles, rosemary leaf extract and biosynthesized AgNPs concentrations namely,

18
20, 40 and 80 ppm every other day for 22 days. The plantlets will be then collected and

will be placed in separate aluminum foils and will be stored at a -80° C freezer.

6. Measurement the Length of Root and the Aerial Part

The 6 tomato plants will be randomly selected from each of the control and treated

groups and their roots and stems length will be measured by a ruler with an accuracy of

1 mm.

7. Photosynthetic Pigment Measurement

The photosynthetic pigment contents of the plants will be tested in both control and

treatment samples using the methodology published by Lichtenthaler and Wellburn.

Three hundred mg of plant aerial parts (blank and treated samples) will be powdered

using liquid nitrogen. Then the volume will be brought to 25 ml adding 80% acetone. The

solution will be centrifuged at 4800 rpm for 20 minutes. The absorbance of supernatant

will be measured to examine the contents of chlorophyll II a, b and carotenoids. For this

purpose, the observance of the clear supernatant will be read at 663.2, 663, 646.8, 645,

and 470 nm. The following formula will be used to calculate the pigment concentrations:

Chl a (µg/ml) = 12.25 × A663.2 – 2.79× A646.8

Chl b (µg/ml) = 21.5 × A646.8 – 5.1 × A663.2

Chl a +Chl b (µg/ml) = 7.15 × A663.2 + 18.71× A646.8

b-caroten (µg/ml) = (1000 ×A470 – 1.82 ×Ca – 85.02 × Cb)/198

19
8. Anti-inflammatory Activity

The anti-inflammatory activity of tomato plant will be evaluated on a carrageenan-

induced rat paw edema model. The carrageenan induced paw oedema model in rats is

highly sensitive and has been accepted as a useful phlogistic tool to investigate new anti-

inflammatory drugs. It will be successfully used to evaluate the anti-inflammatory activity

of lycopene. Rats will be randomly allocated into 5 groups and will be subjected to daily

administration of 1 mL 0.5% CMC-Na (group 1, control), 30 mg/kg BW Trolox (group 2,

positive control) and different mixture of tomato extracts (group 3–5) by oral gavage for

14 days. The dried tomato extracts will be re-dissolved in 1 mL 0.5% CMC-Na as a

suspension and was injected to the rat’s stomach with a syringe. Rats in group 3 will

received 30 g of tomato powder treated with silver nanoparticles. Group 4 will be

administered with the extracts of 30 g tomato powder treated with leaf extract of rosemary

leaves, respectively; rats in group 5 received the extracts of 30 g tomato powder treated

with biosynthesized silver nanoparticles. The lycopene content will be analyzed by the

UHPLC method. After 14 days of administration, rats from all the groups will received a

subcutaneous injection of 0.1 mL 1% (w/v) carrageenan (prepared in 0.9% NaCl) into the

sub-plantar region of right hind paw. The paw volume will be measured by means of a

volume displacement method immersing the paw till the level of lateral malleolus at

various time intervals (0, 1, 2, 3 and 6 h) after carrageenan injection. Change in the paw

volume (g, water weight) will be recorded at above time intervals with reference to the

initial volume (0 h). Paw oedema will be expressed as a percentage inhibition of oedema

formation relative to the basal values of the control group at 6 h according to the equation:

20
where Ct is the paw volume at 6 h after carrageenan injection and C 0 is the paw volume

before carrageenan injection.

9. Statistical Analysis

Microsoft Excel 2007 program will be used to calculate and represent raw data. All

data obtained will be subjected to one-way analysis of variance (ANOVA), using the

SPSS statistical package. For comparison of the means, the Duncan’s multiple range

tests (p ≤ 0.05) will be used.

21
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 Appendices

Chapter 1 Plagiarism Report

Chapter 1 Plagiarism Report

Chapter 2 Plagiarism Report

24

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