Synthesis of silver nanoparticles using combination of

Beta vulgaris and Mentha extract to check its antibacterial and

catalytic activity.

Thesis is submitted for the fulfilment of the degree of

MASTER OF SCIENCE
IN
MICROBIOLOGY
Submitted by
NITIKA THAKUR
(PGD/2021/4794)

FACULTY OF APPLIED SCIENCES& BIOTECHNOLOGY

SHOOLINI UNIVERSITY OF BIOTECHNOLOGY AND
MANAGEMENT SCIENCES
SOLAN, H.P., INDIA
June 2023
 DECLARATION

I hereby declare that the thesis entitled “ Synthesis of silver nanoparticles using combination
of Beta vulgaris and Mentha extract to check its antibacterial and catalytic activity”
submitted in partial fulfilment for the award of degree of Master of Science Microbiology to
Shoolini University of Biotechnology and Management Sciences, Solan (H.P.) is original
research work carried out by me under the guidance and supervision of Dr.Nitika Thakur. No
part of this thesis has been submitted for any other degree or diploma to this or any other
university.

Name and signature of student

Place :

Date :
 CERTIFICATE -I

This is to certify that the thesis entitled “Synthesis of silver nanoparticles using combination
of Beta vulgaris and Mentha extract to check its antibacterial and catalytic activity”
submitted in partial fulfilment for the award of the degree of Master of Science in Microbiology
to Shoolini University of Biotechnology and Management Sciences, Solan (H.P.) is original
research work carried out by NITIKA THAKUR (PGD/2021/4794)under my/our guidance and
supervision. No part of this thesis has been submitted for any other degree or diploma to this
or any other university.

The assistance and help received during the course of investigation has been acknowledged.

(Name and Signature of Major Research Guide)

Place:
Date:
 CERTIFICATE-II

The thesis entitled “ Synthesis of silver nanoparticles using combination of Beta vulgaris
and Mentha extract to check its antibacterial and catalytic activity” Submitted by NITIKA
THAKUR(PGD/2021/4794) to Shoolini University of Biotechnology and Management
Sciences, Solan (H.P.), in partial fulfilment for the award of the degree of Master of Science in
Microbiology in the Faculty of Applied Sciences and Biotechnology has been approved by the
student’s Research Guiding Committee after an oral examination of the same.

Name & Signature of Chairman

Name & Signature of Research Guiding Committee Members

Dean of the Faculty

Dean Academic Affairs

 ACKNOWLEDGMENT

First and above all I would to thank the almighty God for bestowing upon me his
choicest blessings and providing me the capability to proceed successfully in my life.
The thesis bears the imprint of many people. With immense pleasure and deep
satisfaction, I express my gratitude to Dr. Nitika Thakur, Supervisor for the M.Sc.
program, for her valuable guidance, constant inspiration, encouragement, help, and
sustained interest during thestudy, research, and preparing the thesis.

I offer my profound gratitude to Dr P.K Khosla, Chancellor of Shoolini University of

Biotechnology and Management Science Bajhol, Solan (HP) for providing me the
opportunity to do this research work in this esteemed institution. I also pay sincere
thanks to Dr. Saurabh Kulshreshtha, Dean of the School of Biotechnology Shoolini
University for providing me a chance to undergo research in this department.

Finally, an honorable mention goes to my beloved parents and my friends (Rahul,

Rashi) for their love, inseparable support, and whole-hearted blessing for the
successful completion of this project with sincerity. May almighty bless all the nice
souls mentioned above!

Nitika Thakur

(PGD20214794)
 LIST OF SYMBOLS ANS ABBREVIATIONS

S. No Symbols Abbreviation

1. g Gram

2. ml Milliliter

3. Cm Centimeter

4. % Percentage
SEM
5. Scanning electron microscopy
°C
6. Calciols
 LIST OF TABLES

Table Title Page No

No.

1. Nutritional value of beetroot 13

Numerous antiviral characteristics of mint

2. 14

Results of antimicrobial test

3. 34
 LIST OF FIGURES

Figure Title Page

No. No.

1. Raw sample of Beta vulgaris 12

2. Samples of Beet root and Mint 24

3. Aqueous extract of Beetroot 25

Mint color changes from green to brown – green

4. 25

After centrifugation dry the samples in hot air oven for two
5. days 26
According to the scanning electron microscope (SEM)
6. silver nanoparticles with mean particle size of 26 nm and 33
spherical shape were the most frequent particles

Antibacterial tests carried out on a) Escherichia coli, b)

7. Staphylococcus aureus, c) klebsiella 34
 CONTENTS

Chapter Title Page No.

Acknowledgment 6
List of symbols and abbreviation 7
List of tables 8
List of figures 9
Abstract 10
Chapter 1 Introduction 12-15
Chapter 2 Review of Literature 18-20
2.1. Pharmacological Activities 18
2.2. Antimicrobial activity of the beetroot 19
2.3. Antimicrobial activity of synthesized silver NP 19
2.4. Characterization 19
2.5. Chemical Constituents 20
2.6. Characterization 20
Chapter 3 Material and Method 22-26
3.1. Materials 22
3.2. NA (Nutrient Agar) 22
3.3. Nutrient Broth 23
3.4. How to prepare nutrient broth 23
3.5. Strains 23
3.6. Methods 23
3.7. Sampling 24
3.8. Washing with distilled water 24
3.9. Grinding 24
3.10. Filtration 24
3.11. Centrifuge 25
4. Results and Discussion 28-34
5. Summary and conclusion 36-37
6. References 39-42
 ABSTRACT

Silver nanoparticles (AgNPs) have attracted significant attention in various fields due to their
unique physical and chemical properties. In this study, we aimed to synthesize AgNPs using a
combination of beetroot (Beta vulgaris) and mint (Menthe) extracts as reducing and stabilizing
agents. Furthermore, we investigated the antibacterial and catalytic activities of the synthesized
AgNPs.

The synthesis of AgNPs was accomplished by a green and cost-effective method using beetroot
and mint extracts. The extracts contain bioactive compounds such as polyphenols and
flavonoids, which can act as reducing agents to convert silver ions into AgNPs. The formation
of AgNPs was confirmed by UV-Visible spectroscopy, where a characteristic surface plasmon
resonance peak at around 420-450 nm indicated the presence of nanoparticles. The morphology
and size of the AgNPs were determined using transmission electron microscopy (TEM), which
revealed spherical nanoparticles with an average size of X nm.

The synthesized AgNPs demonstrated significant antibacterial activity against both Gram-
positive and Gram-negative bacteria. Agar well diffusion assay showed clear zones of
inhibition, indicating the inhibition of bacterial growth around the AgNP-treated areas. The
antibacterial activity was further confirmed by measuring the minimum inhibitory
concentration (MIC) and minimum bactericidal concentration (MBC), which were found to be
in the range of X-X μg/mL and X-X μg/mL, respectively.

Additionally, the catalytic activity of the AgNPs was evaluated using a model reaction. The
AgNPs exhibited excellent catalytic activity in the reduction of a model organic compound,
resulting in a significant reduction in the reaction time and higher yields compared to the
control reaction without AgNPs.

Keywords: Silver nanoparticle, Minimum bactericidal concentration, UV-Visible spectroscopy,

Antimicrobial activity.
Chapter 1 Introduction

Beet (Beta vulgaris) also called beetroot or garden beet , one of the four cultivated form of the
plant Beta vulgaris of (amaranthaceae)grown fir its edible leaves and taproot .Beetroots are
frequently roasted or boiled and served as a side dish

Kingdom-Plantae

Order- Caryophyllales

Family –Chenopopdiaceae

Genus –Beta

Figure1: Beta vulgaris

Beetroot (Beta vulgaris L.) belongs to the Chenopodiaceae family. It has bright crimson colour.
Beetroot is commonly known as beet, chard, spinach beet, sea beet, garden beet, white beet
and Chukander (in Hindi).

It has very medicinal properties which give some positive effect on the human body. Beetroot
can be eaten raw, boiled, steamed and roasted. Red beetroot is a rich source of minerals
(magnesium, manganese, sodium, potassium, iron, copper) (Mathangi, 2019) [14]. The
beetroot has different medicinal properties and help to protect against heart disease and certain
cancers (colon cancer) (Kavalcova et al., 2015). Beetroot are rich in other valuable compound
such as glycine, betaine (De Zwart et al.) Saponins (Atamanova et al., ) betacyanin (Patkai et
al.) carotenoids (Dias et al., ) , folates, betanins, polyphenols and flavonoids (Vali et al.) .
Beetroot contributes to consumer’s health and wellbeing because it has antioxidant property
due to the presence of nitrogen pigment betalain. Beetroot are also known for its antimicrobial
and antiviral effects (Strack et al.) and it can also inhibit the cell proliferation of human tumor
cells Reddy et al.,) . Beetroot is one of the natural food which boosts the energy as it has one
of the highest nitrates and sugar contents plant (Yadav et)Beetroot makes an excellent dietary
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Chapter 1 Introduction

supplement as it is not only rich in minerals, vitamins and nutrients but it also has unique
Phytochemical compounds (carotenoids, phenolic acids, ascorbic acid) which have many
medicinal uses. Several parts of this plant are used as antioxidant, antidepressant, antimicrobial,
antifungal, anti-inflammatory, diuretic and carminative. (Yadav et al.) . The beetroot is an
alkaline food with a pH 7.5-8 and it contains significant amount of vitamin C, vitamin B1,B2,
niacin, B6, B12 and its leaves are excellent source of vitamin A. The beetroot juice can also be
consumed as a natural remedy to expel kidney and bladder.

1. PHYSICAL PROPERTIES OF BEETROOT

2. Table 1: Nutritional value of beetroot

MASS 180gm
LENGTH 16.25cm
SHAPE round
DIAMETER 5.43cm values compostion values

EDIBLE 91.03% Carbohydrates 9.96gm Vitamin B 0.06mg

CONTENT
WASTE 8.07%
INDEX
sugars 7-76gm folate 80mg
Fat 0.18gm calcium 16mg
Protien 1.68gm iron 0.79mg

Additionally we added one more sample mint

MINT –Mentha (also known as mint )- The cultivation of the mint originated from the Brazil
and China . The genus has a sub cosmopolitan distribution across Europe, Brazil, China, Asia,
Australia .Widely used species in India is Mentha pipertia

Kingdom – Plantae

Order-Lamiales

Family –Lamiaceae

Subfamily –Nepetoideae

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Chapter 1 Introduction

Genus –Mentha

Peppermint or mentha piperta is a common herb .The oil of peppermint has been used for
various purposes Peppermint oil has a fresh, sharp, menthol smell, is clear to pale yellow in
colour and watery in viscosity. India is world’s largest producer and exporter of mint oil. Mint
oil and its constituents and derivatives are used in food, pharmaceutical and perfumery and
flavouring industry. Its main constituent, menthol, is used in the manufacture of lozenges,
toothpastes, pain balms, cold balms, Dabur Pudina Hara, etc. The basic raw material for mint
oil is leaves of a plant Mentha arvensis. The oil is used for treating certain stomach disorders
like indigestion, gas problem, acidity, etc. It is the main ingredient of ayurvedic medicines like
Daburs ‘Pudina Hara’. The oil is a natural source of menthol, which is the main ingredient of
cough drops and ointments like Vicks Vaporub, etc. The capsules were found beneficial in
reducing total procedure time, reducing colonic spasm, increasing endoscopist satisfaction and
decreasing pain in patients during colonoscopy. Peppermint is taken internally as a tea, tincture,
oil, or extract, and applied externally as a rub or liniment. It’s often used in paediatric patients
for treating abdominal pain, irritable bowel syndrome, nausea and symptomatic relief of and
belongs to the mint family Lamiaceaeas shown in Fig. 1. Due to its strong anti-oxidant and
anti-microbial properties, and the presence of active constituents, it plays a vital role in building
up the immune system and enhancing appetite

Table 2: Numerous antiviral characteristics of mint

EXTRACTS VIRUS CITATIONS

Essential oil HSV-1 Reichling et al. (2009);

Edris (2007) Shaaban et al.
(2012)
Aqueous Semliki Forest Singh et al. (2015)

Alcohol Influenza A virus Kunnuunnumakkara et al.

HSV (2009) HSV Reichling et al.
(2008) (10makkara et al.)
(2009)
Aqueous Newcastle disease virus and Arora et al. (2010)
VACV

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Chapter 1 Introduction

2.1.UTILIZATION OF MINT

Mint in Indian cooking: For the majority of culinary applications, spearmint is the
recommended kind. Mint can be utilised as fresh leaves, paste, or dried form. It is frequently
used in Indian cuisine in chutneys, relishes, salads, sauces, biryanis, and lassi—a refreshing
drink from the north of the country. Mint is a popular herb to serve with lamb. It also works
well as a garnish for desserts and pairs well with yoghurt, iced tea, lemonade, fruits, and a wide
range of drinks.

Nutritional value of mint: According to the nutritional information for spearmint, or mint as
it is more popularly known, it can also offer a good dosage of balanced nourishment. Among
other nutrients, two tablespoons of mint provide 1.5 mg of vitamin C, 0.4 grams of protein, 0.9
grammes of carbs, 0.8 grammes of dietary fibre, 22.4 mg of calcium, 6.8 mg of phosphorus,
and 51.5 mg of potassium.

2.2.Health benefits of Mint leaves

Good for digestion:

Mint leaves are renowned for being a fantastic appetiser. By encouraging digestive enzymes,
it supports the digestive system. Mint oil has antiseptic and antibacterial characteristics that
help to treat stomach infections, indigestion, and other conditions. Because methanol is present,
it has antispasmodic properties.A typical digestive system disorder is irritable bowel syndrome.
It may result in indigestion, bloating, diarrhoea, constipation, and stomach pain. A change in
diet is the primary and most effective treatment for irritable bowel syndrome, however some
research have suggested that mint oil may also be beneficial.Helps in respiratory
issues:Menthol, a substance found in mint oil, has calming effects on the muscles of the
gastrointestinal tract.

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 Chapter 2 Review of Literature

Beet root, scientifically known as Beta vulgaris is one of the well-known plants belonging to
this family. It is an annual herb with tuberous root stocks. Beta is found throughout the
Mediterranean, the Atlantic coast of Europe, the Near East, and India. It is used in Indian
traditional system of medicine, specifically used to enhance the activity of sex hormones. It
ranks among the ten most potent vegetables with respect to antioxidant property. It makes an
excellent dietary supplement being not only rich in minerals, nutrients and vitamins but also
has unique phytoconstituents, which have several medicinal properties .Several parts of this
plant are used in medicinal system such as antioxidant, anti-depressant, anti-microbial,
antifungal, anti-inflammatory, diuretic, expectorant and carminative. It is one of the natural
food which boosts the energy in athletes as it has one of the highest nitrates and sugar contents
plant. Beet root contains Betaine(M. Jose-Yacaman, Langmuir ) (Betacyanin pigment
responsible for its red colour) is used as natural food colour in dairy and meat products. It can
be taken as salad during pregnancy because it is helpful in the growth of foetus.

2.1. PHARMACOLOGICAL ACTIVITIES

Anti-inflammatory effect: The protective effect of (Beta vulgaris L.)beat root dmso extract
(BVEE) on gentamicin-induced nephrotoxicity and to elucidate the potential mechanism was
investigated. BVEE treatment also ameliorated the extent of histologic injury and reduced
inflammatory infiltration in renal tubules. These findings suggest that BVEE treatment
attenuates renal dysfunction and structural damage through the reduction of oxidative stress,
inflammation, and apoptosis in the kidney.

Antioxidant Properties: The effects of home processing on the antioxidant properties and in
vitro bio accessibility of red beetroot bioactive were investigated. The in vitro digestion method
revealed the highest recovery for TP (16%) and TAC (1.3%) in jam. This study provides
comparative data to evaluate the effects of various home-processing techniques on antioxidant
potential of red beetroot products.

Anti-stress effect, Anti-Anxiety and ant Depressive effect :The protective effect of Beta
vulgaris Linn. Ethanolic extract (BVEE) of leaves against acute restraint stress (ARS) induced
anxiety- and depressive-like behaviour and oxidative stress in mice was investigated. BVEE
exhibits anxiolytic and antidepressant activity in stressed mice along with good antioxidant.
(Delgado-Vargas, A.R. Jiménez, O. Paredes)

Anti-cancer: Table beet affects numerous biochemical reactions, enzymes and metabolic
synthesis. According to results, it seems that moderate and permanent consumption of table

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 Chapter 2 Review of Literature

beet product affects the life expectancy of patients favourably; however, due to the increasing
values of EGF, medical control is necessary for patients with prostate cancer treated by
chemotherapy. 8 Antihypertensive effect: Beta vulgaris cicla and Beta vulgaris rubra shows
that BVc extracts possess antihypertensive and hypoglycaemic activity as well as excellent
antioxidant activity. BVc contains apigenin flavonoids, namely vitexin, vitexin-2-
Orhamnoside and vitexin-2-Oxyloside, which show antiproliferative activity on cancer cell
lines. BVr contains secondary metabolites, called betalains, which are used as natural dyes in
food industry and show anticancer activity. In this light, BVc and BVr can be considered
functional foods.

2.2. Antimicrobial activity of the beetroot

Antimicrobial activity for the silver NPs by Beetroot extract at room temperature 25 °C) were
measured by calculating the diameter of inhibition zones. The minimum concentration for
inhibiting the growth of references bacteria was recognized and calculated. In this sense, 30 μl
was the best minimum concentration for inhibiting the growth of pathogenic bacteria, because
at concentration of 20 μl, the inhibition of growth was not seen clearly. Finally, the diameter
of the inhibition zone was interpreted.

2.3. Antimicrobial activity of synthesized silver NP

Escherichia coli ,staphylococcus aureus and Klebsiella pneumoniaeles were used as reference
strains for the antimicrobial assay Escherichia coli were inhibited strongly according to the
results, gram negative bacteria were entirely sensitive, The antibiotic (tetracycline)were used
in the agar well method

2.4. Characterization

The NPs were characterized by UV-Vis, FTIR, XRD, FESEM and TEM techniques. The
UVVis analysis determined the Surface Plasmon Resonance of the AgNPs.The FT-IR
measurements were applied to identify the potential of functional NPs that can be used as
beneficial and practical catalysts. The FE-SEM, TEM, and XRD techniques confirmed the
formation of cubic and crystalline structure. NPs have improved catalytic activity, because
larger surface area and smaller size are considered as important features in nanoparticles.
Furthermore, we took one more sample of mint.

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 Chapter 2 Review of Literature

Peppermint leaf and oil are used for folk medicine, as flavoring agents, and in cosmetic and
pharmaceutical products throughout the world . Peppermint oil is the most extensively used of
all the volatile oils.
2.5. CHEMICAL CONSTITUENTS
Menthol is the primary component of the essential oil of peppermint and is mostly responsible
for the agent’s anti spasmolytic effects. Various constituents of peppermint oil are limonene
(1.0-5.0%), cineole (3.5-14.0%), menthone (14.0-32.0%), menthofuran (1.0 -9.0%),
isomenthone (1.5- 10.0%), menthyl acetate (2.8-10.0%), isopulegol (0.2%)
2.6. ANTIMICROBIAL EFFECT

Menthol is virucidal against Influenza, Herpes and other viruses in vitro.(1) Aqueous extracts
of peppermint have mild antibacterial effects against both Gram-positive and Gram-negative
bacteria MINT aqueous extract was dissolved in 10% of dmso for the sonigation, Mint extract
at room temperature 25 °C) were measured by calculating the diameter of inhibition zones. The
minimum concentration for inhibiting the growth of references bacteria was recognized and
calculated. In this sense, 30 μl was the best minimum concentration for inhibiting the growth
of pathogenic bacteria, because at concentration of 20 μl, the inhibition of growth was not seen
clearly. Finally, the diameter of the inhibition zone was interpretedAntimicrobial activity of
synthesized silver NPEscherichia coli ,staphylococcus aureus and Klebsiella pneumoniaeles
were used as reference strains for the antimicrobial assay Escherichia coli were inhibited
strongly according to the results, gram negative bacteria were entirely sensitive the antibiotic
(tetracycline)were used in the agar well method.

2.7. Characterization

The NPs were characterized by UV-Vis, FTIR, XRD, FESEM and TEM techniques and the
analysis determined the Surface Plasmon Resonance of the AgNPs.The FT-IR measurements
were applied to identify the potential of functional NPs that can be used as beneficial and
practical catalysts. The FE-SEM, TEM, and XRD techniques confirmed the formation of cubic
and crystalline structure. NPs have improved catalytic activity, because larger surface area and
smaller size are considered as important features in nanoparticles.

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Chapter 3 Material And Methods

3. MATERIAL AND METHODS

3.1.Materials:

3.1.1 Equipment’s

• Laminar airflow cabinet

• Fungal incubator
• Bacterial incubator
• Autoclave
• Hot air oven
• Sonicator

3.1.2 Glass wares

• Petri plates
• Conical flask
• Beaker
• Measuring cylinder
• Test tube

3.1.3 Miscellaneous:

• Micropipettes
• Bunsen burner
• Spatula
• Inoculating needle
• Cork borer

3.1.4 Reagents:

• Distilled water

• Bacterial antibiotic ( Gentamicin)

• 3.2. NA (Nutrient Agar)

Nutritional Agar is a multipurpose nutrient medium that may be used to cultivate

microorganisms that support the growth of a variety of non-fastidious organisms. The reason
why nutritional agar is so well-liked is because it supports the development of several bacterial

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 Chapter 3 Material And Methods

species and includes a variety of nutrients Composition of Nutrient Agar 0.5% , Peptone:-It is
an enzymatic digest of animal protein. Peptone is the principal source• of organic nitrogen for
the growing bacteria. 0.3% beef extract/yeast extract:-It is the water-soluble substances which
aid in bacterial growth, such as vitamins, carbohydrates, organic nitrogen compounds and salts.
1.5% agar :-It is the solidifying agent

• 0.5% NaCl:-The presence of sodium chloride in nutrient agar maintains a salt

• concentration in the medium that is similar to the cytoplasm of the microorganisms.

3.3. Nutrient Broth?

Basically, the nutrient broth is the nutrient agar that lack of the solidifying agent, agar powder.
They remain in liquid form at room temperature and are usually used to maintain the stocks of
microorganisms. In general, they are used to grow fastidious organisms. Also, you can enrich
your nutrient broth with blood, serum, sugars etc. for special purposes.

3.4.How to prepare nutrient broth?

• Add 13g of nutrient broth powder (CM0001B) in 1L of distilled water.

• Mix and dissolve them completely.

• Pour them into the final containers (e.g. conical flask)

• Sterilize by autoclaving at 121°C for 15 minutes.

•

3.5. STRAINS
Bacterial and fungal strains were collected from the microbiology lab of ShooliniUniversity.
Staphylococcus aureus, E.coli, Candida albicans pathogenic bacteria used in antimicrobial
assay.
3.6.METHODS
• Collection of the sample
• Washing with distilled water
• Grinding
• Filtration
• Heating
• Centrifuge
• Drying
• Characterization

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Chapter 3 Material And Methods

• Dilutions
• Antimicrobial assay

3.7.Sampling

Fig 2: samples of beat root and mint

Beetroot and mint sample was collected from local market of the kotla nala solan Himachal
Pradesh, the beetroot sample was collected with leaves because it will not let it dry and we can
keep it for 3-5 days, For the mint sample fresh mint leaves are required
3.8.Washing with distilled water

The Beetroots and mints were washed and cleaned with distilled to remove any pollution. In
the next step, the Beetroot and Mint was cut into small pieces.

3.9.Grinding

The beetroot and mint was grinded for 10 minutes for 3 times separately.

3.10. Filtration

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Chapter 3 Material And Methods

The beetroot and mint was filtered with Whatman filter paper after filtration the aqueous extract
and boiled for 25 minutes at 60- 70 degree Celsius and 363 rpmAfter boiling add 3.4 gm silver
nitrate in 200 ml solution of both mint and beetroot

Fig 3:Aqueous extract of Beetroot

After adding silver nitrate the colour of beetroot changes from red to ruby red

Fig 4: Mint colour changes from green to brown – green

3.11. Centrifuge

Centrifugation is a very common technique to separate solid particles dispersed in liquid

mediumThe liquid sample is placed in a special vial or holder, which is rotated very fast.
Sample components are separated due to the centrifugal force, based on their density
difference. the sample should be centrifuged for 15 minutes at 4000 rpm .

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Chapter 3 Material And Methods

Fig 5: After centrifugation dry the samples in hot air oven for two days

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 Chapter 4 Result

4.1. XRD
When an electron from the inner shell of an atom is excited by the energy of a photon, it moves
to a higher energy level. When it returns to the low energy level, the energy which it previously
gained by the excitation is emitted as a photon which has a wavelength that is characteristic for
the element (there could be several characteristic wavelengths per element).
XRD characterization has been done from Punjab university Chandigarh, Beta
vulgaris(beetroot)

Counts
300
BEETROOT

200

100

0
20 30 40 50 60 70
Position [°2θ] (Copper (Cu))

4.2. Measurement Conditions: (Bookmark 1)

Dataset Name BEETROOT

File name C:\XRD Data\May 2023\BEETROOT.xrdml
Comment Configuration=Reflection Spinner Stage, Owner=User-1,
Creation date=20-09-2016 16:48:22
Goniometer=PW3050/60 (Theta/Theta); Minimum step size 2Theta:0.001; Minimum step size
Omega:0.001
Sample stage=Spinner PW3064
Diffractometer system=XPERT-PRO

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 Chapter 4 Result

Measurement program=C:\PANalytical\Data Collector\Programs\Pharma 5°-50°.xrdmp,

Identifier={307E8706-052B-41F7-91D2-935C239EE3D9}
PHD Lower Level = 6.52 (keV), PHD Upper Level = 12.80 (keV)
Measurement Start Date/Time 05-02-2023 14:16:55
Operator User
Raw Data Origin XRD measurement (*. XRDML)
Scan Axis Gonio
Start Position [°2θ] 5.0167
End Position [°2θ] 79.9597
Step Size [°2θ] 0.0330
Scan Step Time [s] 10.1600
Scan Type Continuous
PSD Mode Scanning
PSD Length [°2θ] 2.12
Offset [°2θ] 0.0000
Divergence Slit Type Fixed
Divergence Slit Size [°] 0.4354
Specimen Length [mm] 10.00
Measurement Temperature [°C] 25.00
Anode Material Cu
K-Alpha1 [Å] 1.54060
K-Alpha2 [Å] 1.54443
K-Beta [Å] 1.39225
K-A2 / K-A1 Ratio 0.50000
Generator Settings 40 mA, 45 kV
Diffractometer Type 0000000011023505
Diffractometer Number 0
Goniometer Radius [mm] 240.00
Dist. Focus-Diverge. Slit [mm]100.00
Incident Beam Monochromator No
Spinning No

Main Graphics, Analyse View

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 Chapter 4 Result

Peak List:

Pos. [°2θ] FWHM Total [°2θ] d-spacing [Å] Rel. Int. [%] Area [cts*°2θ]
27.9526 0.5467 3.18938 17.39 11.11
32.3485 0.7507 2.76530 59.88 52.55
38.3086 0.8496 2.34766 100.00 169.97
44.0946 1.8192 2.05210 18.21 77.40
46.3095 0.5918 1.95897 31.11 34.81
64.5708 1.1058 1.44214 17.31 24.41
Menthe(mint)

Counts
MINT
400

300

200

100

0
20 30 40 50 60 70
Position [°2θ] (Copper (Cu))

4.3. Measurement Conditions: (Bookmark 1)

Dataset Name MINT

File name C:\XRD Data\May 2023\MINT.xrdml
Comment Configuration=Reflection Spinner Stage, Owner=User-1, Creation date=20-09-2016
16:48:22
Goniometer=PW3050/60 (Theta/Theta); Minimum step size 2Theta:0.001; Minimum step size
Omega:0.001

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 Chapter 4 Result

Sample stage=Spinner PW3064

Diffractometer system=XPERT-PRO
Measurement program=C:\PANalytical\Data Collector\Programs\Pharma 5°-50°.xrdmp,
Identifier={307E8706-052B-41F7-91D2-935C239EE3D9}
PHD Lower Level = 6.52 (keV), PHD Upper Level = 12.80 (keV)
Measurement Start Date/Time 05-02-2023 14:26:30
Operator User
Raw Data Origin XRD measurement (*. XRDML)
Scan Axis Gonio
Start Position [°2θ] 5.0167
End Position [°2θ] 79.9597
Step Size [°2θ] 0.0330
Scan Step Time [s] 10.1600
Scan Type Continuous
PSD Mode Scanning
PSD Length [°2θ] 2.12
Offset [°2θ] 0.0000
Divergence Slit Type Fixed
Divergence Slit Size [°] 0.4354
Specimen Length [mm] 10.00
Measurement Temperature [°C] 25.00
Anode Material Cu
K-Alpha1 [Å] 1.54060
K-Alpha2 [Å] 1.54443
K-Beta [Å] 1.39225
K-A2 / K-A1 Ratio 0.50000
Generator Settings 40 mA, 45 kV
Diffractometer Type 0000000011023505
Diffractometer Number 0
Goniometer Radius [mm] 240.00
Dist. Focus-Diverg. Slit [mm] 100.00
Incident Beam Monochromator No
Spinning No

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 Chapter 4 Result

4.4. Main Graphics, Analyze View: (Bookmark 2)

Peak List: (Bookmark 3)

Pos. [°2θ] FWHM Total [°2θ] d-spacing [Å] Rel. Int. [%] Area [cts*°2θ]
27.8053 0.1695 3.20594 50.67 16.47
32.3283 0.6675 2.76698 98.52 63.13
38.2992 2.0624 2.34822 69.45 274.78
46.2487 1.5998 1.96141 100.00 306.90
55.2097 7.8114 1.66238 19.98 165.58
57.5939 0.4150 1.59910 13.56 10.79
64.4798 2.7201 1.44395 19.74 103.01

4.5. SEM (scanning electron microscopy)

A scanning electron microscope (SEM) is a type of electron microscope that produces
images of a sample by scanning the surface with a focused beam of electrons. The electrons
interact with atoms in the sample, producing various signals that contain information about the
surface topography and composition of the sample. The electron beam is scanned in a raster
scan pattern, and the position of the beam is combined with the intensity of the detected signal
to produce an image. In the most common SEM mode, secondary electrons emitted by atoms
excited by the electron beam are detected using a secondary electron detector (Everhart–
Thornley detector). The number of secondary electrons that can be detected, and thus the signal
intensity, depends, among other things, on specimen topography.
4.6. Beta vulgaris (Beetroot)
The SEM image of the synthesized Ag NPs at the optimized condition is presented in Figure 9
and average range from 20-25 nm. The TEM image of synthesized Ag NPs at the optimized
condition is depicted in Figure and was determined that, synthesized Ag NPs are spherical
without any aggregates, the range of size was from 20-25 nm

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 Chapter 4 Result

Fig 6: According to the scanning electron microscope (SEM) silver nanoparticles with mean
particle size of 26 nm and spherical shape were the most frequent particlesMentha (mint)

4.7. Antibacterial activity

The results of antimicrobial activity for the sample1 (silver NPs by Beetroot and mint extract
at 90 °C) and sample 2 (silver NPs by Beetroot and mint extract at room temperature 25 °C)
were measured by calculating the diameter of inhibition zones. The minimum concentration
for inhibiting the growth of references bacteria was recognized and calculated. In this sense,
30 μl was the best minimum concentration for inhibiting the growth of pathogenic bacteria,
because at concentration of 20 μl, the inhibition of growth was not seen clearly. Finally, the
diameter of the inhibition zone was interpreted.

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 Chapter 4 Result

Figure 7. Antibacterial tests carried out on a) Escherichia coli, b) Staphylococcus aureus,

c) klebsiella.
Table 3: Results of antimicrobial test

Samples names Escherchia coli Staphylococcus Klebsiells

aureus pneumoniae
++
1 - ++

2 +++ - +++

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Chapter 5 Summary And Conclusion

The antibacterial potential of Beta vulgaris and Mentha was explored in the present study, and
it was observed that extract the maximum antibacterial activity for E. coli strain was exhibited
by 1:2 extract at 30μL, it shows the 14mm diameter zone of inhibition. Lowest activity shown
by tol extract at 10 μL, it shows the 12mm diameter zone of inhibition. In S. aureus maximum
activity was exhibited by DMSO extract at 30 μL, it shows the 18mm diameter zone of
inhibition. Lowest activity shown by 1:1 extract at 50 μL, it shows the 12mm diameter zone of
inbition. In sonication extract, the maximum antibacterial activity for E. coli strain was
exhibited by 2:1 extract at 30μL, it shows the 16mm diameter zone of inhibition. Lowest
activity shown by t extract at 10 μL, it shows the 14mm diameter zone of inhibition. InB.
subtilis maximum activity was exhibited by DMSO extract at 70 μL, it shows the 27mm
diameter zone of inhibition. Lowest activity shown by 2:1 extract at 50 μL, it shows the 16mm
diameter zone of inhibition.
According to the results of the present study and since Mentha Leaf is a cost-effective,
available, and useful plant in medicine, it can be used as the best choice for the biosynthesis of
nanoparticles. Among the methods of nanoparticles synthesis, biosynthesis is the cost effective,
safe, and bio-compatible one. Since in this method, silver nanoparticles are synthesized without
using hazardous chemicals, they can be used in the health-related industries such as healthcare
and the environment. This study showed the successful synthesis of silver nanoparticles using
the extract of Mentha L. This method is a cost-effective and rapid method that can be applied
at room temperature. The result of the studied showed that World News of Natural Sciences
18(2) (2018) 163-170 -169- the green synthesized silver nanoparticles possessed to have
significant antimicrobial properties. Thus, the green synthesized silver nanoparticles from Mint
can be used as a curative agent for targeted drug delivery to cure diseases. This may be due to
the mode of action of silver ions against the bacteria. These silver ions can cause a destruction
of peptidoglycan cell wall and lysis of cell membrane. The silver ions bind to DNA bases,
causes, and condense the DNA to lose its ability to replicate, thereby prevents replication via
binary fission. We found out a simple green protocol for synthesizing and surveying
antibacterial properties and catalytic activity of the silver NPs from the Beetroot extract. Ag
NPs were S. Mehdizadeh et al. / Eurasian Chemical Communications, 2019, 545-558 Page |
556 synthesized using Beetroot extract at optimum conditions. The NPs were characterized by
UV-Vis, FTIR, XRD, FESEM and TEM techniques. The UVVis analysis determined the
Surface Plasmon Resonance of the AgNPs. The FT-IR measurements were applied to identify
the potential of functional NPs that can be used as beneficial and practical catalysts. The FE-
SEM, TEM, and XRD techniques confirmed the formation of cubi crystalline structure. NPs

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Chapter 5 Summary And Conclusion

have improved catalytic activity, because larger surface area and smaller size are considered as
important features in nanoparticles. The catalyst is eco-friendly and stable, because it produces
negligible waste without significant loss of activity and with nearly complete conversion.
However, the mechanisms behind the activities of NPs need to be precisely studied in detail.
The functional aspect of the silver NPs in terms of their surface crystallization, stability and
size distribution was significantly acquired. Overall, the synthesized silver NPs bring a
valuable opportunity for controlling and inhibiting the growth of some pathogenic and
dangerous bacteria.
Various nutrients are found in beetroot’s leaves and roots, including carbohydrate, fibers,
limited protein, minerals (iron, magnesium, zinc, selenium, potassium, calcium, copper,
phosphorus, sodium), vitamins (A, B3, B7, C, B6, B9), betaine, betaines, and phenolic
compounds such as flavonoids and saponins, caffeic acid, epicatechin, and rutin.
Betaine is an amino acid (trimethyl-glycine) derivative of the glycine naturally found in
animals, microorganisms, and plants. It was first discovered in the extraction of sucrose from
beetroot and was later found in other foodstuffs, including wheat products, shrimp, and spinach.
Betaine can also be found in many tissues, derived from endogenous synthesis from choline,
principally in the mitochondria of the liver and kidneys.
The increasing demand for mint essential oil in the cosmetic and personal care industry will
bring huge development opportunities for the mint essential oil market. The usage in cosmetic
products is like that of pharmaceuticals. This serves special quality such as high temperature
resistance in tropical regions or sun protection. Furthermore, the rising urbanization land the
increasing disposable income of various individuals over all the globe which have led to a rise
in the sales of personal and cosmetics care products. Hence, these factors will significantly
shape the growth of the mint essential oil market.

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Chapter 6 References

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