SYNTHESIS OF GOLD NANOPARTICLES FROM THE FLOWER … · To the best of our knowledge, gold...
Transcript of SYNTHESIS OF GOLD NANOPARTICLES FROM THE FLOWER … · To the best of our knowledge, gold...
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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