SYNTHESIS OF GOLD NANOPARTICLES FROM THE

FLOWER EXTRACTS OF TABEBUIA ARGENTIEA AND THEIR ANTICANCER ACTIVITY

PROJECT REFERENCE NO.: 40S_BE_2020

COLLEGE : SHRIDEVI INSTITUTTE OF ENGINEERING AND TECHNOLOGY,

TUMAKURU

BRANCH : DEPARTMENT OF BIOTECHNOLOGY ENGINEERING

GUIDE : DR. C P CHANDRAPPA

STUDENTS : MR. VINAY Y G

MS. PALLAVI T

MR. VINAY W

MR. SIDDALING REDDY

Key words Gold nanoparticles, Chloroauric acid, SEM, EDX, Antioxidant activity.

ABSTRACT Biosynthesis of nanoparticles by plant extracts is currently under exploitation. Plant

extracts are very cost effective and eco-friendly and thus can be an economic and efficient

alternative for the large-scale synthesis of nanoparticles. The current study revealed that the

aqueous flower extracts of Tabebuia argentiea were used and compared for their extracellular

synthesis of gold nano-particles. Stable gold nanaoparticles were formed by treating aqueous

solution of AuCl3 with the plant flower extracts. The formed Au NPs were characterized by

energy-dispersive X-ray spectroscopy (EDX), and scanning electron microscopy (SEM) analyses

were performed. Different time intervals for the reaction with aqueous chloroauric acid solution

increase in the absorbance with time. The complete reduction of auric chloride was observed

after 48hours of reaction at 30˚C. The characteristic colour changes from pale yellow to dark

brown during the formation of gold nanoparticles in the reaction due to their scientific properties

was observed. The flower extracts acts as reducing as well as encapsulating agent for the gold

nanoparticles. The SEM images shown the obtained samples have spherical morphology with

the average particles size of 56 nm. And the Anticancer activity was done using Hepatic cells

(Hep G2).

Chapter 1

INTRODUCTION

Nanotechnology is gaining tremendous impacts in the present century due to its capability of

modulating metals into their nano size. Plant/Flower extracts are very cost effective and eco-

friendly and thus can be an economic and efficient alternative for the large scale synthesis of

nanoparticles. Nagaraj.B(2012) (1)

With the advancement of technologies and superior scientific understanding paved a way for

research and development in the plant biology towards intersection of nanotechnology.

Nanoparticles are of numerous scientific intrest as they are effectively a bridge between bulk

materials and atomic or molecular structures. It is cost effective and less tedious purification

steps. B.S.Bahu (2015).(2)

To the best of our knowledge, gold nanoparticle synthesis from Tabubea argentea is reported for

the first time by reducing a solution of gold chloride. In our study we report a yellow method for

the synthesis of gold nanoparticles at room temperature by using flower extracts of Tabubea

argentea as readucing / stabilizing agents and the probable mechanism for the formation of

nanoparticles. B.S. Bahu (2015).(2)

1.1. Tabebuia argentiea :

Tabebuia is a genus of flowering plants in the family Bignoniaceae. The common name “roble”

is sometimes found in English. Tabebuias have been called “trumpet trees”, but this name is

usually applied to other trees and has become a source of confusion and misidentification.

Tabebuia consists almost entirely of trees, but a few are often large shrubs. A few species

produce timber, but the genus is mostly known for those that are cultivated as flowering trees.

Tabebuia is native to the American tropics and subtropics from Mexico and the Caribbean to

Argentina. Most of the species are from Cuba and Hispaniola. It is commonly cultivated and

often naturalized or adventive beyond its natural range. It easily escapes cultivation because of

its numerous, wind-borne seeds.

Fig 1: Tabebuia argentiea

Table 1: Scientific classification of Tabebuia argentiea

Scientific classification

Kingdom Plantae

(unranked)

Angiosperms

(unranked)

Eudicots

(unranked)

Asterids

Order

Lamiales

Family

Bignoniaceae

Tribe

Tecomeae

Genus

TabebuiaGomes exA.P. de

Candolle

Common name:Yellow Tabebuia, Golden Bell, Silver Trumpet Tree.

Regional name: Tabebuia yellow.

Light:Sun glowing.

Water:Normal, can tolerate less.

Primarily grown for:Flowers.

Flowering season:February, March, April.

Flower or Inflorescence color:Yellow.

Foliage color:Green, Blue Grey or Silver.

Plant spread or width:8 to 12 meters.

Plant spread or width:6 to 8 meters.

Plant form:Irregular, Upright or Erect.

Estimated life span:Very long lived.

Special character:

Good for screening

Attracts bees

Quick growing trees

Suitable for road median planting

Suitable for avenue planting

Hanging or weeping growth habit

Good on seaside

Generally available in India in quantities of: Over hundreds

Plant Description:

- One of the best tropical yellow flowering trees.

- Origin - Brazil

- 10 m tall.

- Completely leafless tree during early summer.

- Showy tropical flowering tree with crooked trunk and corky bark, to 8 m high, covering

itself in the leafless stage with a profusion of rich yellow trumpet flowers 5-8 cm long.

- Foliage appears after the bloom.

- Leaves palmately divided into 5-7 narrow leaflets to 15 cm long, and covered with

silvery scales oblong woody dark brown fruit 15 cm long.

- Leaves arise after first flowering and a second but minor flush of flowers occurs after

emergence of leaves.

- The tree is often damaged by strong wind or uprooted in cyclonic weather due to its

shallow root system.

Growing tips:

- Can be effectively used in small as well as large gardens.

- Tree will grow in any well drained soil.

- Some support and training is required when the plants are small.

- Regular irrigation in the dry period in the first two years will help the tree establish itself.

1.2. Cancer : Cancer (medical term : malignant neoplasm) is a large, heterogeneous class of diseases in which

a group of cells display uncontrolled growth, invasion that intrudes upon and destroys adjacent

tissues and often metastasizes, where the tumor cells spread to other locations in the body via the

lymphatic system or through the bloodstream. These three malignant properties of cancer

differentiate malignant tumors from benign tumors, which do not grow uncontrollably, directly

invade locally or metastasize to regional lymph nodes distant body sites like brain, bone, liver, or

other organs.

Researchers divide the causes of cancer into two groups: those with an environmental cause

and those with a hereditary genetic cause. Cancer is primarily an environmental disease, through

genetics influence the risk of some cancers [3]. Common environmental factors leading to cancer

include: tobacco use, poor diet and obesity, infection, radiation, lack of physical activity and

environmental pollutions. These environmental factors cause or enhance abnormalities in the

genetic material of cells (Kenneth et al., 2002). Cell reproduction is an extremely complex

process that is normally tightly regulated by several classes of genes, including oncogenes and

tumor suppressor genes. Hereditary or acquired abnormalities in these regulatory genes can lead

to the development of cancer. A small percentage of cancers, approximately to ten percent, are

entirely hereditary [4].

The presence of cancer can be suspected on the basis of clinical signs and symptoms or

findings after medical imaging. Definitive diagnosis of cancer, however, requires the

microscopic examination of a biopsy specimen. Most cancer can be treated, with the most

important modalities being chemotherapy, radiotherapy and surgery. The prognosis in cancer and

the extent of diseases. While cancer can affect people of all ages and a few types of cancer are

more common in children than in adults, the overall risk of developing cancer generally

increases with age, at least up to age 80-85year. In 2007, cancer caused about 13% of all human

deaths worldwide (7.9 million). Rates are rising as more people live to an old age as mass

lifestyles changes occur in the developing world [4].

1.2.1. Treatment of cancer : Here the list of the methods for treating the cancer:

For tumors that are still inside the prostate, radiation therapy (using X-rays that kill the cancer

cells) and a surgery called radical prostatectomy are common treatment options.

Chemotherapy (for example cisplatin are carboplatin)

Hormone therapy (for example, tamoxifen)

Another option currently being tested in clinical trials is biologic therapy, which uses the

patient’s immune system to fight cancer.

1.2.2. Side effects of chemotherapy : Chemotherapy acts by killing cells that divide rapidly, one of most cancer cells. This means that it also

harms cells that divide rapidly under normal circumstances like cells in the bone marrow, digestive tract

and hair follicles. This results in the most common side effects of chemotherapy like myelosuppression

(decreased production of blood cells, hence also immunosuppression), mucositis (inflammitio of the

lining of the digestive tract) and alopecia (hair loss) [5&6].

1.3. Liver cancer : Liver cancer, also known as hepatic cancer and primary hepatic cancer, is cancer that starts in the

liver. Cancer which has spread from elsewhere to the liver, known as liver metastasis, is more

common than that which starts in liver. Symptoms of liver cancer may include a lump or pain in

the right side below the ribcage, swelling of the abdomen, yellowish skin, easy bruising, weight

loss, and weakness.

1.3.1. Synonyms:

Hepatic cancer

Primary hepatic malignancy

Primary liver cancer.

The leading cause of liver cancer is cirrhosis due to hepatitis B, hepatitis C, or alcohol. Other

cause include aflatoxin, non-alcoholic fatty liver disease, and liver flukes. The most common

(HCC), which makes up 80% of cases and cholangiocarcinoma. Less common types include

mucinous cystic neoplasm. The diagnosis may be supported by blood tests and medical imaging

with conformation by tissue biopsy.

Preventive efforts include immunization against hepatitis B and treating those infected with

hepatitis B or C. screening is recommended in those with chronic liver disease. Treatment

options may include surgery, targeted therapy, and radiation therapy. In certain cases ablation

therapy, embolization therapy, or liver transplantation may be simply closely followed.

Primary liver cancer is globally the sixth most frequent cancer (6%) and the second leading

cause of death from cancer (9%). In 2012 it occurred in 782,000 people and resulted in 746,000

deaths. In 2013, 300,000 deaths from liver cancer were due to hepatitis B, 343,000 to hepatitis

C, and 92,000 to alcohol.

1.3.2. Treatment:

Treatment for liver cancer is based on the cancer and overall health but may include surgery,

radiation, chemotherapy or ablation therapy, according to MedicineNet. Embolization and liver

transplant may also be options.

Surgery is sometimes the best option for liver cancer, according to MedicineNet. Surgery is only

recommended when the tumor is small, since it involves removing the part of the liver where the

cancer is found. Chemotherapy and radiation therapy are also used to kill cancer cells in the

liver, and both can be used in combination with surgery for a total treatment plan. Similarly

ablation therapy can use heat, acid or laser therapy to kill cancer cells.

Objectives

Collection and identification of sample

Synthesis of gold nanoparticles using flower extracts

Characterization of gold nanoparticles : SEM-EDS

Studying of Antioxidant activity

Studying Anticancer activity

Chapter 2

Review of Literature

Nagaraj et al. (2012)conducted the experiment on environmental being synthesis of gold

nanoparticles from the flower extracts of Plumeria albaLinn (Frangipani) and evaluation of their

Biological activities. This was the first report on the Synthesis of gold nanoparticles using

extracts of Plumeria albaLinn flower samples. They confirmed that the gold nanoparticles were

present by the color change. And it was characterized by UV-Visible Spectrometer. It appears to

have significant Antimicrobial capacity resembling a broad spectrum of Antibiotics against

different microorganisms. Bhau et al. (2015)they conducted the experiment on green synthesis of gold nanoparticles from

the leaf extract of Neponthes khasina andantimicrobial assay. They developed a eco-friendly,

simple and efficient method for the synthesis ofgold nanoparticles using leaf extracts of

Neponthes khasina. They confirmed the shape and size of AuNPs by SEM and TEM. The

outcome was Positive concluding that the AuNPs synthesized shows good antimicrobial

properties. They also concluded that rate of reduction of metal ions using plant agents is found to

be much faster.

Mukundan et al. (2014)prepared gold nanoparticles using leaves extract of Bauhinia tomentosa

Linnand evaluated their in vitro anticancer activity. Metal raw particles have several applications

such as Optics, biomedical sciences, drug delivery, and catalysis. They performed the Qualitative

Photochemical analysis of the leaves Extracts to show the presence of saponins, flovonoids,

alkaloids, proteins, steroids, and quinines. They characterized using UV-Visible Spectrometer,

Surface Plasmon Resonance (SPR), FTIR, FESEM-EDAX, HR-TEM, XRD, MTT, and HEp-2

assy.

Padma Vankar and Dhara Bajpai (2010) worked on preparation of gold nanoparticles from

Mirabiles jalapa flowers. They worked on the reductivity of Au3+

ions by M. Jalapa flower

extract resulted in the formation of stable NPs with multi-shaped morphologies. They recognized

that gold nanoparticles synthesized by the green chemistry will be having more biomedical and

pharmaceutical applications. They characterized using UV, X-RAY, Diffraction, FT-IR, Energy

dispersive X-ray, Transmission Electron Microscopy (TEM).

Neda Ramezani et al. (2008)described synthesis of gold nanoparticles by medicinal plant

extracts with their reducing potential. The potential ability of different plants extracts for the

reduction of Au3+

to gold nanoparticles was investigated. Characterized by UV-Vis, TEM, and

EDS techniques which confirmed the reducing of gold ions to gold nanoparticles. As per their

literature survey that was the first report on the synthesis of gold nanoparticles using total

extracts of Pelargonium roseum.

Avnika Tomas and Garima Gong (2013) conducted short review on application of gold

nanoparticle, As per their conclusion , Gold Nanoparticles emerge as promising carriers of bio

molecules like protein peptides, nucleic acid and insulin. Gold Nanoparticlescan be

functionalized with protein , peptides, nucleic acid and insulin, so these have a great application

not only in biosensing delivery but also in drug, gene and protein delivery.

Jae Yong Song et al. (2009) Biological synthesis of gold nanoparticles using magnolia korus

and Diopyros kaki leaf extracts. They proposed on Ecofriendly method for gold nanoparticles

synthesis using plant extracts. This method can be applied in various products that directly

comes in contact with human body, such as Cosmetics, Foods and Consumer goods ,besides

medical application.

Khan et al. (2014) Gold Nanoparticles : synthesis and application in drug delivery. They have

concluded that gold nanoparticles have wide spread application integrated such as drugs

delivery , imaging , diagnosis and therapeutics due to their extremely small size and high surface

area. Side effects of conventional drug have been minimized by conjunction with gold

nanoparticles and they increase the quality life of patients.

Thirumurugan et al. (2010) Biotechnological Synthesis of Gold Nanoparticles of Azadirachta

medical leaf extract. They confirmed the synthesis by its color change and they characterized by

UV-Visible spectroscopy. They concluded that according to analysis the major bioactive

compounds are salving, nimbin, in the Azadirachota indica plant leaf extract with this we can

conclude that it may be one of the reason for the reduction of the gold nanoparticles.

Prathap Chandran et al. (2006) Synthesis gold nanoparticles and silver nanoparticles using

Aloe vera plant extract. The slow rate of reducing of gold ions by the bio molecules aided by the

shape directing ability of the carbonyl compounds of the Aloe vera extract are belived to be

responsible for the formation of the single crystalline gold nanoparticles. They found the

interesting application in the field of cancer hyperthermia and optical coatings.

Chapter 3

Materials and Methods

3.1. Collection and identification of the sample: Tabebuiea argentiea was collected in our college Shridevi Institute of Engineering and

Technology, Tumkuru, Karnataka, India. It was authenticated by Dr. C.P. Chandrappa Professor

and Head, Department of Biotechnology, Shridevi Institute of Engineering and Technology

Tumkuru.

The collected flowers are washed in distilled water and boiled using 100ml double distilled water

about 15-20 minutes and then it was filtered through whatmans filter paper.

3.2. Synthesis of gold nanoparticles using flower extracts Then prepare the gold auric chloride solution then add it to the 300ml of double distilled water.

Then take 270ml of the gold solution prepared and 30ml of the sample and mixed keep it in a

dark place about one day. Then it should be mixed well and centrifuged at 10,000rpm for 10

minutes. The supernatant should be discard and the pellet should be mixed with little double

distilled water and centrifuge it at 10,000rpm for 10 minutes. This procedure should be repeated

for three times then the pellet should kept for drying under shadow until the water molecules are

completely dried. After the completion of drying the sample was taken and crushed with the help

of pestle and mortar. Now the sample is in powder form and it is fed for various tests.

3.3. Phytochemical analysis The leaf extracts are used for the phytochemical analysis qualitatively and quantitavely for the

detection of primary and secondary metabolites.

Qualitative phytochemical screening:

Phytochemical analysis of each extract has been carried out according to standard protocols.

(chandrappa et al., 2012)

3.3.1. Screening for alkaloids:

0.5g of the extract was stirred in 5ml of 1% hcl on a steam bath and filtered while hot. Distilled

water was added to the residue and 1ml of the filtrate was treated with a few drops of Wagner’s

reagent. A reddish brown precipitate indicates the presence of alkaloids.

Wagner’s reagent- 2g of iodine and 6g of KI in 100ml of distilled water.

3.3.2. Screening for Flavonoids:

2ml of sodium hydroxide was added to 2ml of the extract the appearance of a yellow color

indicates the presence of flavonoids.

3.3.3. Screening for Saponins:

1ml of distilled water added to 1ml of the extract and shaken vigorously. A stable persistent froth

indicated the presence of saponins.

3.3.4. Screening for Phenols:

Equal volume of extract and iron chloride were mixed. A deep bluish green solution gave an

indication of the presence of phenols.

3.3.5. Screening for Tannins:

About 0.5g of dried powdered sample was boiled in 20ml of water in a test tube and then filtered.

A few drops of 0.1% ferric chloride was added and observed for brownish green or a blue black

coloration.

3.3.6. Screening for Aanthraquiononens:

0.5g of the extract was shaken with 10ml of benzene and filtered, 10% of ammonia solution was

added to filtrate and the mixture was shaken. The formation of a pink, red or violet color on the

ammonical phase indicates the presence anthraquinones.

3.3.7. Screening for cardiac glycosides:

0.5g of the extract was dissolved in 2ml glacial acetic acid containing one drop of ferric chloride

solution. This was under layered with 2ml of concentrated sulphuric acid. A brown ring

formation at the inter phase indicates the presence of deoxy sugar characteristics of cardiac

glycosides.

3.4. Antioxidant activity 3.4.1. Reducing power assay:

Substance which have reduction potential, react with potassium ferricyanide (Fe3+) to form

potassium ferricyanide (Fe2+), which then reacts with ferric chloride to form ferric ferrous

complex that has an absorption maximum at 700 nm (Jayanthi and Lalitha 2011).

0.2 M sodium phosphate buffer (pH 6.6 ) :

First, prepared phosphate buffer A by diluting 31.2 grams NaH2PO42H2O to 1000ml. second ,

prepared phosphate buffer B by diluting 53.61 grams Na2HPO47H2O to 1000ml. Then, mixed

62.5% of buffer A with 37.5% of buffer B. adjusted the pH using NaOH and H2PO4.

1% potassium ferricyanide :

1% potassium ferricyanide was prepared by dissolving 1g of ferric cyanide in 100ml of distilled

water.

10% trichloroacetic acid (w/v):

10% TCA was prepared by dissolving 10ml of 100% TCA in 100ml of distilled water.

Stock solution of Tabebuia argentia gold nanoparticles (1mg/ml)

1mg of extract was dissolved in 1ml of phosphate buffer.

Stock solution of ascorbic acid (1mg/ml):

1mg of ascorbic acid was dissolved in 50ml of phosphate buffer

0.1gm of ferric chloride was dissolved in 100ml of distilled water

The reducing capacities of sample extract of Tabebuia argentea was determined by (Oyaizu et

al., 1986) with some experimental modifications. The reaction mixture consists of 1ml distilled

water with different concentrations (200µg - 600µg) of the sample of Tabubea argentia, 1.0ml of

0.2M sodium phosphate buffer (pH 6.6) and 1.0ml of 1% potassium ferricyanide (w/v). The

mixture was incubated at 50˚C for 20 min. after cooling at room temperature 1.0ml of 10%

trichloroacetic acid (w/v) was added and the mixture was centrifuged at 3000rpm for 10min. The

upper layer (1.0 ml) was mixed with 1.0ml of distilled water and 1.0ml of 0.1% ferric chloride,

and the absorbance was measured at 700nm. Ascorbic acid was used as a standard. Blank as

phosphate buffer and control was prepared without adding standard or test compound. Higher

absorbance indicates higher reducing power of the sample. The relative percentage reducing

power of the sample was calculated by using the formula (Xican and Chan, 2012).

3.4.2. Determination of total antioxidant capacity by Phospomolybdenum method

The method is based on the reduction of Mo(VI)-Mo(V) by the test sample and subsequent

formation of a green phosphate /Mo(V) complex at acidic pH.

2.6MH2SO4

33.33 ml of concentrated (18N) sulfuric acid (Rankem) was added to distilled water to make up

the final volume of the reagent to 1 L.

28 mM sodium phosphate

It was prepared by dissolving 3.35g of sodium phosphate (SRL) in 1L of distilled water.

Stock solute of alcoholic extract of Tabubea argentea (20mg)/ml)

20mg of alcoholic extract was dissolved in 1ml of phosphate buffer.

Stock solution of ascorbic acid (50mg/ml)

50mg of ascorbic acid was dissolved in 50 ml of phosphate buffer.

The total antioxidant capacities of ethanol extract of Tabebuia agentieawas evaluated by the

phosphomolybdnem method of reducing transition metal ions reported by Prieto et al.,(1999)

with some experimental changes. 1.0ml alcoholic extract Tabebuia agentieawith various

concentrations (50micro gram-400 micro gram) were added to 2.0ML of reagent solution (0.6M

sulfuric acid, 2 Mm sodium phosphate and 4Mm ammonium molybdate). The reaction mixture

were capped and incubated in a water bath at 95 0

C for 90 minutes. After cooling the mixture to

room temperature, the absorbance was used as the blank. Ascorbic acid was used as the standard

and the total antioxidants capacity is expressed as equivalents of ascorbic acid. The calibration

curve was prepared by ascorbic acid with methanol. All assays were done in triplicate.

3.5. Anticancer activity MTT Assay:

An assay is an investigative or analytical procedure used in laboratories for qualitatively

assessing or quantitatively measuring the presence or amount or functionality of an analyte

(target substance e.g. drug or biochemical substance) in an organism or organic sample.

MTT Assay is a colorimetric

assay used in assessing viability and

cell proliferation. It can also be used to

determine cytotoxicity of agents since

the agent would stimulate or inhibit

cell viability. Viable cells depend on

intact mitochondria for chemical

reactions to take place and for the cell

to stay alive. The mitochondria of a

cell contain dehydrogenases which can

be used to identify toxic agents in the cell. When added, Dimethyl thiazolyl diphenyl tetrazolium

bromide (MTT) is reduced from a yellow salt to purple insoluble formazan by dehydrogenases

belonging to the mitochondrial respiratory chain. These dehydrogenases are only active in viable

cells. The amount of formazan can be quantified by dissolving it in an organic solvent and

reading the optical density (OD) value under a spectrophotometer.

The directly proportional relationship between viable cells and formazan produced can be

used to measure the effectiveness of an anti-cancer drug on a cancer cell line such as HeLa. The

more effective the drug, the more apoptosis that takes place. Since only viable cells have active

dehydrogenases there is a negative correlation between the amount of purple formazan produced

(or viability) and the efficacy of the drug. If the drug is effective, more apoptosis (cell death)

takes place and thus less formazan is produced thus resulting in a lower OD value when read on

the spectrophotometer and vice versa.

The IC50 value (half maximal inhibition concentration) is the measure of the concentration

of a drug (compound) which when applied results in 50% biological inhibition in vitro. This

value is very critical in drug testing. Using the IC50 value, we can determine how much of the

drug would be required to provide an effective dose in an in vivo system and eventually on a full

human body. From this basic value itself, it can be gauged whether further testing should be

pursued or the drug scrapped as a preferential IC50 value would occur in the Nano scale.

Sample Preparation:

500mg of sample (labeled Tabebuia argentiea) was dissolved in 1mL of distilled water with

contact vortexing. To make get rid of the undissolved particles the sample was centrifuged at

10000rpm for 15mins. The supernatant was taken and filtered through 0.22µm pre wet filter and

collected in a sterile MCT tube. Recovered sample volume was 710µL. Therefore the stock was

considered as 500mg in 710µL.

Procedure:

Day 1

MTT Salt Reduced to Purple Insoluble Formazan(Jenpen, 2006)

Culture dish is taken from the incubator and medium was discarded. Washed with saline

to remove the trace amount of globular protein and other compounds in the dish.

Discarded the saline and add trypsinization for 2 to 3 mins

Media was added to inhibit the activity of trypsin

Centrifuged for 10 to 15 minutes at 1500 rpm and pellet was formed.

The pellet was collected and re-suspended in the media.

And cell count was done using hemocytometer.

100 µl of cell suspension/well was added to each well of a 96 well micro titer plate.

Incubated for 24 hrs.

Day 2

The cells were observed under inverted microscope.

The various drug concentrations (70mg/100µL, 35mg/100µL, 17.5mg/100µL,

8.75mg/100µL, 4.37mg/100µL and 2.18mg/100µL) along with appropriate controls

were prepared by serial dilution method.

96 well plate was taken from the incubator and the medium was discarded.

The controls and concentration of drugs also added in the 96 well plates.

Incubated for 24 and 48 hrs.

Day 3 / Day 4: (24 hrs and 48 hrs)

20µl of MTT dye was added per well.

Incubated 4 hours at 37°C in a CO2 incubator and then the formazan crystals were

observed under microscope.

The content of the wells was discarded.

100µl of DMSO was added to each well to dissolve formazan crystals.

Incubated for 1 hour.

Absorbance was measured at 545 nm.

Chapter 4

RESULTS AND DISCUSSION

4.1. Synthesis of gold nanoparticles :

(A) (B)

Fig2: Flower extract Fig3: Before adding the sample

(C)

Fig4: After adding the sample

4.2. Phytochemical analysis:

Fig 5. Phytochemical analysis: A) Alkaloids B) Flavonoids C) Saponins D) Phenols

E) Tannins F) Anthaquinones G) Cardiac glycosides

Table 2: Phytochemical screening of Tabebuia argentiea

Phytochemicals

Tabebuia argentiea

Alkaloids

+++

Flavonoids +++

Saponins -

Phenols ++

Tannins +++

Anthraquinones -

Cardaic glycosides +++

+ : Indication, ++: Present, +++: Confirms, - :Absent

4.3. Scanning Electron Microscopy : Energy Dispersive X-ray spectrometry

(SEM : EDX) analysis

The scanning electron microscopy (SEM) image further ascertains that the Gold nanoparticles

are pre-dominantly spherical in morphology with their sizes ranging from 50 to 60nm and have

an average size of about 56.77nm.Energy-dispersive X-ray spectroscopy (EDX) illustrated the

chemical nature of synthesized gold nanoparticles using Tabebuia argentiea flower extract . The

peak was obtained at the energy of 3 keV, for gold, and also some of the weak peaks for C, O,

Cl, Au, Mg, Si,T,S and K were found. The emission energy at 3 keV indicates the reduction of

gold ions to element of gold. The quantitative analysis using EDX showed high gold content of

52.27%. The spectrum also showed the presence of carbon,oxygen, and silicon of 8.17% ,2.28%

and 1.16 %, respectively.

Fig 6: SEM

Fig 7: EDX

4.4. Antioxidant activity

4.4.1. Phosphomolybdenum method

Table 3: Determination of % of inhibition of AuCl3 and Ascorbic acid by

Phosphomolybdenum method

Concentration

(µg/ml)

Absorbance at 695 nm Relative %

inhibition

Aucl3 Ascorbic acid Aucl3 Ascorbic

acid

Control 0.280 0.280 - -

100 0.408 0.493 20.38 33.91

200 0.491 0.569 33.59

46.01

300 0.584 0.604 38.85 51.59

400 0.612 0.741 52.86 73.40

500 0.731 0.908 71.81 100

Fig 8: Antioxidant assay using Phosphomolybdenum method

4.4.2.Reducing power assay

0

20

40

60

80

100

120

0 100 200 300 400 500 600

rela

tiv

e %

in

hib

itio

n

concentration µg/ml

sample

ascorbic acid

Table 4: Determination of % of inhibition of AuCl3 and Ascorbic acid by

Reducing power assay

Concentration

(µg/ml)

Absorbance at 700 nm Relative % inhibition

AgNPs Ascorbic acid AgNPs Ascorbic

acid

control 0.642 0.642 - -

100 0.823 0.865 26.17 27

200 0.876 0.901 31.51 32.13

300 0.915 0.956 37.59 38.95

400 1.020 1.201 46.89 69.35

500 1.218 1.448 71.46 100

Fig 9:Antioxidant assay using Reducing power assay

4.5. Anticancer activity

0

20

40

60

80

100

120

0 100 200 300 400 500 600

rela

tive

% in

hib

itio

n

concentration µg/ml

sample

ascorbic acid

Table 5: Readings and calculation:

Concentration

OD

value

(AVG) Corrected % Viability

70mg/100µL 1.112 0.691 76.77777778

35mg/100µL 1.198 0.777 86.33333333

17.5mg/100µL 1.205 0.784 87.11111111

8.75mg/100µL 1.225 0.804 89.33333333

4.37mg/100µL 1.296 0.875 97.22222222

2.18mg/100µL 1.308 0.887 98.55555556

Media 1.321 0.9

Vehicle (Water) 1.311 0.89 98.88888889

Positive control 0.463 0.042 4.666666667

Blank 0.421

The MTT assay values indicate that the highest concentration 70mg/100µL has only

23.23% of cell death. Therefore IC50 value cannot be determined.

CONCLUSION

The present work indicates the Synthesized AuCl3 using Tabebuia argentiea flower extract was

done and confirmed by SEM and EDX techniques. The SEM images suggested that the particles

are spherical shaped with average size of 56.77nm. The antioxidant indicates the higher

absorbance of nanoparticles. This synthesized method is rapid, facile, convenient, less time

consuming, environmentally safe, and can be applied in variety of existing applications. This

plant flower extract compounds can be extended to the synthesis of the other metal and non

metal oxide nanoparticles. Here we report extracellular biosynthesis of gold nanoparticles using flower extracts of Tabubiea

argentea as redusing agent. Antioxident activity and medicinal values of Tabubiea argentea

fascinated us to utilize it for biosynthesis of gold nanoparticles.

Chapter 5

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