Synthesis of Gold Nanoparticles from the Flower Extracts of Tabebuia Argentiea and Their Anticancer Activity

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 0C 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 MTT Salt Reduced to Purple Insoluble Formazan(Jenpen, 2006) 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  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

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 Absorbance at 695 nm Relative % (µg/ml) 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 71.81 100 120

100 sample 80 ascorbic acid

20 relative % % inhibition relative

0 0 100 200 300 400 500 600 concentration µg/ml

Fig 8: Antioxidant assay using Phosphomolybdenum method

4.4.2.Reducing power assay Table 4: Determination of % of inhibition of AuCl3 and Ascorbic acid by Reducing power assay Concentration Absorbance at 700 nm Relative % inhibition (µg/ml) 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

120

100 sample 80 ascorbic acid

40 relative % inhibition % relative

0 0 100 200 300 400 500 600 concentration µg/ml

Fig 9:Antioxidant assay using Reducing power assay

4.5. Anticancer activity Table 5: Readings and calculation:

OD value Concentration (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 REFERENCES Nagaraj et al. (2012). Environmental Benign Synthesis of gold nanoparticles from the flower extracts of Plumeria alba Linn and evalution of their Biological activities, International Journal of drug development and research, 4(1), 144-150. Bhau et al. (2015). Green Synthesis of Gold Nanoparticles from the leaf extract of Nepenthes khasiana and Antimicrobial Assay, VBRI Press in 2015, 6(1), 55-58. S Anjali andS Sheetal. (2013).Analyphyto chemical analysis and free radical scavenging potential of herbal and medicinal plants. Journal of pharmacogenesis and phytochemistry. 2(4): 22-29. N Krithiga and A Jayachitra (2012).antioxidant and antibacterial study on coleus aromaticus and lawsonia internals .international journal of pharmacy and life sciences.vol 3(9):1-5 K S Girish, K D Machiah, Ushanandini S, Harish Kumar K, Nagaraju S, Govindappa M,Vedavathi M and Kemparaju k (2006). Antimicrobial properties of a non toxic glycoprotein (WSG) Govindappa M, Salman dinakar (2015). pharmocology of low molecular protein in cancer: A review. plant science feed.5(1):17-21. Mukundan et al. (2014). Green Synthesis of gold nanoparticles using leaves extract of Bauhinia tomentosa linn and in vitro anticancer activity, International journal of Innovation research in science and engineering, 2347-3207. Padma S Vankar and Dhara B. (2010). Preparation of gold nanoparticles from Mirabiles jalapa flowers, Indian Journal of Biochemistry and Biophysics, 47, 157-160. Neda Ret al. (2008). Screening of medicinal plant methanol extracts for the synthesis of gold nanoparticles by their reducing potential, verlag der Zeitschrift fiir Naturforschung , Tibingen , 63(7), 903-908. Avnika T and Garima G. (2013). Short Review on Application of gold nanoparticles, Global journal of application of pharmacology, 7(1), 34-38. Jae Yong Song et al. (2009). Biological Synthesis of gold nanoparticles using mangnolia kobus and Diopyros kaki leaf extracts, Process Biochemistry, 44, 1133-1138. Khan et al. (2014). Gold Nanoparticles : Synthesis and application in drug delivery, Tropical Journal of Pharamaceutical Research, 13(7), 1169-1177. Prathap Cet al. (2006). Synthesis of Gold Nanoparticles and Silver Nanoparticles using Aloe vera plant extract, Biotechnol prog,2006, 22 : 577-583. Thirumurugan et al. (2010). Biotechnological Synthesis of gold Nanoparticles using of Azadirachta midica leaf Extract, International Journal of Biological Technology, 1(1) :75-77.