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GREEN SYNTHESIS, OPTIMIZATION, CHARACTERIZATION OF SILVER NANOPARTICLES USING CASUARINA EQUISETIFOLIA BARK EXTRACT AND ITS ASSAYS

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GREEN SYNTHESIS, OPTIMIZATION, CHARACTERIZATION OF

SILVER NANOPARTICLES USING

CASUARINA EQUISETIFOLIA

BARK EXTRACT AND ITS ASSAYS

Saranya V. T. K.1 and S. Uma Gowrie*

1

Research Scholar, Department of Plant Biology and Plant Biotechnology, Ethiraj College for

Women, Chennai – 600 008.

*Associate Professor, Department of Plant Biology and Plant Biotechnology, Ethiraj College

for Women, Chennai – 600 008.

ABSTARCT

Casuarina equisetifolia, an exotic tree species, which serves as store

house of several potential phytoconstituents. The present study aims to

explore the role of these potential biomolecules as an effective

reducing and capping agent, in the synthesis of Silver nanoparticles

using aqueous bark extract of Casuarina equisetifolia. Further,

optimization of various parameters such as temperature, time, pH for

incubation and stability was also standardized for enhanced synthesis

of Silver nanoparticles. Characterization of the optimized silver

nanoparticles were carried out by different methods such as UV-Vis

spectroscopy, FTIR analysis TEM micrograph study and by X-ray

diffraction. Thus the optimized condition for the effective synthesis of

silver nanoparticles using aqueous bark extract of Casuarina equisetifolia revealed that the

required temperature was 80·C, time for incubation was 20 mins at neutral pH, which was

found stable for 30 days. FTIR study was carried out to identify the functional groups that

were responsible for capping and stabilization of the synthesized silver nanoparticles. TEM

result shows that the synthesized Silver nanoparticles were round in shape and the size varied

between 20nm -24nm. The average crystalline size was found to be 74 Å using Scherrer’s

formula. Maximum antibacterial activity of the biosynthesized silver nanoparticles was

observed against Bacillus sp., staphylococcus and E.coli strains. Silver nanoparticles

synthesized biogenically with the help of bark extract of Casuarina equisetifolia had positive

implications on germination percentage, mean germination time and germination rate This

Volume 6, Issue 3, 797-814. Research Article ISSN 2277– 7105

*Corresponding Author Dr. S. Uma Gowrie

Associate Professor,

Department of Plant Biology

and Plant Biotechnology,

Ethiraj College for Women,

Chennai – 600 008. Article Received on 26 Dec. 2016,

Revised on 16 January 2017, Accepted on 06 February 2017

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biogenic silver nanoparticles effectively degrade the methylene blue dye, with the

degradation percentage of 56.18% at the end of fifth hour. Thus the synthesized silver

nanoparticles using aqueous bark extract proves to be an effective tool to control pathogenic

bacterial strains, germination of seeds and degrade methylene blue efficiently.

KEYWORDS: Casuarina equisetifolia, bark extract, optimization, temperature, pH, TEM.

INTRODUCTION

Nanotechnology refers to the engineering and construction of the functional systems at very

micro level i.e. at atom level. Nanotechnology has various innovative application in the field

of energy, medicine, drugs and especially dealing with environmental issues.[1]

Microbe-mediated synthesis of nanoparticles, requires sophisticated set up for culture and maintenance

of the microorganism, which turns to be highly expensive.[2] Among various biological

methods used in the silver nanoparticle synthesis, plant extract proves to be simple, nontoxic,

ecofriendly and also feasible.[3]

Casuarina equisetifolia is a tree species that contains several phytochemical constituents like

glycosides, quercetin, triterpenoids, tannin and rutin.[4] The presence of the potential

secondary metabolites proves the tree species as an efficient source to be used as astringent,

diuretic, as a remedy for cough, diarrhea, beri-beri, colic and toothache.[5] Biological

properties like anticancer[6], hypoglycemic activity[7], has been reported from this tree

species.

Nanoparticles are involved in the field of agriculture with an ultimate aim to impart

stratergical improvement in growth of the plant and to control the plant disease.[8, 9] Studies

dealing with the effect of biologically synthesized nanoparticles on seed germination are very

limited.[10] Hence in the present study an attempt has been taken to test the effect of silver

nanoparticles synthesized from the aqueous bark extract of Casuarina equisetifolia on seed

germination.

The presence of vital phytoconstituents like Phenolic compounds has been reported in this

tree species which proves to be an efficient tool to degrade complex organic chemical

constituents under visible light illumination. There are no reports till date about the synthesis

of silver nanoparticles, using aqueous bark extract of, Casuarina equisetifolia, belonging to

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Hence, present study is focused on i) Synthesis of silver nanoparticles using aqueous bark

extract of Casuarina equisetifolia ii) Optimization of the synthesized particles for various

parameters like temperature, time, pH required for the synthesis and stability iii)

Characterization using UV-Vis spectrophotometer, FTIR,TEM and XRD iv) Application -

antibacterial efficacy, seed germination study and photocatalytic degrading potential of the

synthesized nanoparticles using methylene blue dye.

MATERIALS AND METHODS

COLLECTION OF PLANT MATERIAL

Casuariana equisetifolia Bark samples were collected from, Pudhucherry union territory, Tamilnadu (Latitude: 12.10·, Longitude 79.9·). The plant Materials has been taxonomically

identified and authenticated by botanical survey of India. The collected bark materials were

washed thoroughly in running tap water to remove adhered debris and then with distilled

water. After which the materials were shade dried at room temperature for more than a week,

finely powdered and were stored for analysis.

PREPARATION OF AQUEOUS PLANT EXTRACT

Finely powdered bark was used for extract preparation. About 10 g of powdered bark was

boiled in 100 ml of distilled water for the formation of extract. The extract was then filtered

using whatman No 1 filter paper and centrifuged at 5000 rpm for 15mins. Supernatant was

collected and stored at 4·C for further analysis.

SYNTHESIS OF SILVER NANOPARTICLE

1mM of AgNO3 (Silver nitrate) solution was prepared using distilled water, to synthesis silver

nanoparticle from aqueous bark extract of Casuariana equisetifolia. 10 ml of bark extract was

added to 90 ml of 1mM AgNO3 solution for the synthesis of silver nanoparticles,. The

mixture was incubated in dark for 24 hours. The reduction of aqueous silver ions by aqueous

bark extract, to form stable silver nanoparticles, were indicated by the formation of brown to

black colour solution.[11]

OPTIMISATION OF THE SILVER NANOPARTICLES

Temperature: Reaction temperature plays a vital part in controlling the nucleation process of

nanoparticles. Temperature ranges, 20·C, 40·C, 60·C, 80·C and 100·C were fixed for the

reaction trial to standardize the optimium temperature for the synthesis of silver

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Time: Synthesis of nanoparticles of perfect morphology and internal properties depends on

time given for incubation. The optimization of time was studied at various time intervals viz

10 mins, 20 mins, and 30 mins. Absorbance was measured spectrophotometrically.

pH: The role of pH is to control the shape of the silver nanoparticles synthesized. pH for the

interaction was adjusted as 4.0,7.0 and 9.0, using NaOH solution (0.1N) and HCl solution

(0.1N). The results were read using UV-Vis Spectrophotometer.

STABILITY

The stability of the synthesized nanoparticles at the optimized condition, was checked

periodically at regular time interval for 30 days.

CHARACTERIZATION OF SILVER NANOPARTICLES

UV-Vis Spectrophotometer: The reduction of Ag + ions were monitored spectrometrically

using double beam UV-Vis spectroscopy (UV 1650pc, Shimazdu) by dissolving a small

aliquot of the reaction solution into distilled water. The wavelength 350nm -500nm was

observed and peak was recorded.

FT-IR Spectroscopy: The synthesized silver nanoparticles were mixed with potassium

bromide in the ratio of 1:100 and the pellet was prepared. This spectrum analysis was done

using Shimazdu IR - FTIR instrument with diffuse reflectance mode (DRS- 8000). The

measurement range was 650 - 4500 cm-1 and DRS operating at resolution 4 cm-1. FTIR study

was carried out to identify the biomolecule responsible for capping and stabilization of the

synthesized nanoparticles.

Transmission Electron Microscope: The optimized silver nanoparticles were centrifuged at

20,000 rpm for 15 mins and the pellet was formed. The pellet was washed to be free from

debris. The pellet thus obtained was air dried and suspended in ethanol. The same was

observed in Transmission Electron Microscope (Techai 10 Philips) and photographed.

XRD ANALYSIS

Powder X-ray Diffraction

The powdered silver nanoparticles, were deposited on a microscopic slide and air dried

overnight at room temperature using Cu-Kα1radiation source in powder diffractometer (XRD

– Rich siefert 300 diffractometer). X-ray diffraction (XRD) measurements were carried out

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XRD patterns were recorded in the 2θ range 10–70◦ at a scan speed of 10◦ min-1 at room

temperature.

ANTIBACTERIAL ACTIVITY

In vitro antibacterial activity of the silver nanoparticles synthesized using the bark extract of

Casuarina equisetifolia was studied. Three pathogens viz., Bacillus subtilis., Staphylococcus

aureus, Escherichia coli strains were used for antibacterial studies, obtained from the

Department of Microbiology, Ethiraj college for women, Chennai. Bacterial test organisms

were grown in nutrient broth for 24 hours. These cultures were used to prepare the bacterial

lawn on the Muller Hinton Agar medium used for plating. Four wells of diameter 8mm were

made on each bacterium inoculated plate. Wells were loaded with 10μl, each with the

concentration of 25μg, 50μg, 75μg and 100 μg of silver nanoparticles synthesized using

aqueous bark extract of Casuarina equisetifolia. Well ‘S’ was loaded with 10 μl of freshly prepared silver nitrate solution. The plates were incubated for 24 hours at 37·C. 10μl of 100 μg Gentamycin was used as standard. Triplicates were maintained.

EFFECT OF SILVER NANOPARTICLES ON GERMINATION OF Vigna radiata

Seeds of Vigna radiata were collected from Tamilnadu horticulture society, Chennai. Seeds

were surface sterilized by immersing them in 5% of Sodium hypochlorite solution for 10

mins.[12] After two hours, the seeds were transferred to distilled water and rinsed well. Then

the seeds were soaked in various concentration (0.5mg/l, 1.00mg/l and 2mg/l) of silver

nanoparticles synthesized using bark extract of Casuarina equisetifolia. Whatman No 1 filter

paper was placed into a petridish with ten seeds per plate for one concentration. 5ml of test

solution was added to the plate.[13] Triplicates were maintained for each concentration. The

plates were incubated for five days. Control was maintained, in which the test solution was

only distilled water.

GERMINATION MEASUREMENT

The measurements were carried out according to the International Rules for Seed Testing.[14]

Germination parameters were calculated using the following equations.[15, 16]

Germination Percentage (GP %) = (Gf/n) × 100

Gf-total number of germinated seeds at the end of experiment n-total number of seed used in

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Mean Germination Time (MGT) = Ʃ NiDi/n

Ni-number of germinated seeds until the ith day

Di-number of days from the start of experiment until the ith counting n - the total number of

germinated seeds.

Germination Rate (GR) = Σ Ni/ΣTiNi

Ni - number of newly germinated seeds at time Ti.

PHOTOCATALYTIC DEGRADATION OF DYE

Methylene blue dye solution was prepared by dissolving 0.1mg of the methylene blue dye in

100 ml of water. To this about 10mg of silver nanoparticles synthesized from the aqueous

bark extract was added and mixed well for 20 minutes using magnetic stirrer. Control was

maintained without addition of the silver nanoparticles. This was subjected to exposure of

sunlight for 5 hours, with a temperature ranging between 36ºC - 38ºC. At the end of each

hour, 3ml of suspension was taken to analyse the dye degradation percentage. This was done

by measuring the absorbance spectrum at 660nm, specific for methylene blue dye, using

UV-Vis Spectrophotometer. Percentage of dye degradation was calculated using the formula:

(𝐶0− 𝐶)

%Decolorisation = --- X 100 𝐶0

where

𝐶0is the initial concentration of dye solution and

𝐶 is the concentration of dye solution after photocatalytic degradation.

RESULTS AND DISCUSSION

SYNTHESIS OF SILVER NANOPARTICLES

To confirm the formation of the silver nanoparticles visual observation of change of colour to

reddish wine, on addition of Silver nitrate to bark extract was observed (Fig1a). The

formation of AgNP’s were confirmed spectrometrically, by the SPR (Surface Plasma

Resonance Band). It was observed that there was an absorbance peak at 422nm (Fig 1b),

which confirmed the formation of Silver nanoparticles, since, the characteristic SPR

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[image:7.595.145.452.70.258.2]

Fig 1a: Synthesized AgNP’s using aqueous bark extract of Casuarina equisetifolia

Fig 1b: UV-Vis spectra of Silver nanoparticles

OPTIMISATION OF SILVER NANOPARTICLES

Temperature: Temperature plays an important aspect in effective formation of Silver

nanoparticles. Increase in absorbance was recorded with increase in temperature from 20·C to

100·C (Fig 2). In the present study, the most effective temperature for the formation of silver

nanoparticles, using aqueous bark extract of Casuarina equisetifolia was recorded at 80·C,

that has second maximum absorbance value at 424nm. Though the maximum absorbance was

observed at 100·C, the analysis of the Peak pattern reveals that agglomeration of

nanoparticles would have taken place at higher temperature. In the previous reports of the

synthesis and optimization of silver nanoparticles using Hippophae rhamnoides Linn, the

[image:7.595.151.424.308.486.2]
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[image:8.595.145.454.70.249.2]

Fig 2: Uv-Visible Spectra of AgNP’s showing effect of different reaction temperature

Time: Increase in incubation period showed increase in absorbance value. The optimum time

period required for the synthesis of silver nanoparticles, using aqueous bark extract of

Casuarina equisetifolia was found to be 20 mins (Fig 3), agglomeration of silver

nanoparticles was observed after the optimum duration that results in the formation of larger

sized particles.[18]

Fig 3: UV-Visible Spectra of AgNP’s showing effect of different reaction time.

pH: To investigate the effect of pH in the formation of silver nanoparticles, using aqueous

bark extract of Casuarina equisetifolia a wide range of pH viz 4.0,7.0 and 9.0 was studied. It

was noticed that, maximum absorbance was recorded (optimized temperature of 80·C and

incubation period of 20 mins) when the pH condition of the reaction Mixture was at neutral

pH-7 (Fig 4). Increase in pH was marked by decrease in absorbance. In acidic state (low pH)

[image:8.595.144.456.406.587.2]
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of larger sized particles than nucleation[19], whereas basic state (higher pH) results in the

[image:9.595.144.453.125.311.2]

synthesis of smaller sized AgNP’s, that results in agglomeration.[18]

Fig 4: Uv-Visible Spectra of Agnp’s showing effect of different reaction pH

STABILITY

The AgNP’s synthesized at temperature 80·C, an incubation period of 20 mins and pH 7 was

found to be stable upto the period of 30 days without the incorporation of any stabilizing

agent, after which it leads to reduction in stability due to agglomeration of the nanoparticles.

CHARACTERIZATION OF SILVER NANOPARTICLES

UV-Vis Spectrophotometer: The characteristic colour of silver nanoparticles synthesized

was mainly due to the excitation of surface plasma vibration of the AgNP’s.[5] The SPR band

that confirms the presence of silver nanoparticles lies between 400nm – 475nm. Thus,

throughout the experiment during synthesis and optimsation of silver nanoparticles, Surface

Plasma absorption was maximum and was close to 420 nm, which indicates the dispersed

state of the particles in the solution and also the absence of aggregation.[11]

FTIR Analysis: FTIR analysis was done to identify the potential biomolecules present in the

aqueous bark extract of Casuarina equisetifolia responsible for the reduction and capping of

the bioreduced silver nanoparticles (Fig 5a, b). The presence of absorption band at 3616.53

cm-1, 3564.45 cm-1 is the characteristic feature of phenol OH stretch. The sharp band at

1035.77 cm-1, 3419.79 cm-1, 3396.64 cm-1 corresponds to amine stretch and the band at

1512.19 cm-1 corresponds to the aromatic ring stretch, 779.24 cm-1 represents aromatic CH

plane. Vibrational stretch is also seen at 1649.14 cm-1 which relates to C=C stretch. Bark of

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glycosides, steroids, saponins, tannins and flavonoids.[20] Thus, the FTIR report serves as an

evidence for the presence of effective biomolecules in the aqueous bark extract of Casuarina

equisetifolia(Fig 5b) which would have been responsible for the reduction of Ag+. These

biomolecules possibly prevents agglomeration of particles and also helps in the stabilization

of the nanoparticles.

Fig 5(a): FTIR spectrum of the Silver nanoparticles synthesized using aqueous bark

extract of Casuarina equisetifolia

Fig 5(b): FTIR spectrum of aqueous bark extract of Casuarina equisetifolia

TEM analysis: Fig 5 shows the TEM micrographs of the silver nanoparticles synthesized.

The micrograph shows variation in the particle size. The size of the particles varies between

20nm - 24nm (Fig 6). It is clear from the micrograph that most of the particles are spherical

in shape. The micrograph shows few agglomerated silver nanoparticles which are seen that

[image:10.595.145.453.192.372.2] [image:10.595.141.460.436.619.2]
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of silver nanopaerticles using Plukenetia volubili L.,[17] where the particles was spherical in

[image:11.595.144.452.130.307.2]

Shape and the maximum size of the particles was 25nm.

Fig 6: TEM micrograph

XRD ANALYSIS

Powder XRD Analysis: To confirm and identify the crystalline nature of SNPs using X-ray

diffraction Pattern was recorded from the 2θ upto 10 to 70 degrees (Fig 7). The figure shows

that, the characteristic 2θ values are 24.29º, 29.65º and 57.48º. The average crystalline size

was found to be 74 Å using using Scherrer’s formula, d = 0.9 λ / B cosθ. The characteristics

of nanocrystals were indicated with a slight shift in the peak positions leading to strain in the

crystal structure.[21]

[image:11.595.158.440.521.690.2]
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ANTIBACTERIAL ACTIVITY

Antibacterial potential of synthesized silver nanoparticles against E.coli., S. aureus and

B.subtilis, were studied based on the zone of inhibition and was compared with the standard.

Zone of inhibition increases with the increase in the concentration of the synthesized silver

nanoparticles. S.aureus showed minimum level of susceptibility when compared with the

standard Gentamycin. E.coli and B.subtilis., were found to be highly susceptible to silver

nanoparticles synthesized from bark extract of Casuarina equisetifolia, which showed the

maximum zone of inhibition of 26±0.43mm at 100 µg concentration (fig 8&9). Potential

inhibition of E.coli and S.aureus was exhibited by the silver nanoparticles using Papaya fruit

extract[22] and Mangosteen leaf.[19]

Chemical property of silver supports strong antimicrobial activity, which on combination

with phytoconstituents of plants extracts as silver nanoparticles, gains efficient dual activtiy

which serves as an efficient antimicrobial agent.[23] The cell wall of both gram positive and

gram negative bacteria possess negative charge that creates a strong electrostatic force of

attraction with the positively charged nanoparticles[24], which contributes to an effective

antibacterial activity of silver nanoparticles. In addition, silver is a non-toxic chemical

substance to animal cell, that prevents bacterial growth, by inactivating the bacterial DNA

and enzyme, thus leading to the cellular death of bacteria.[25]

EFFECT OF SILVER NANOPARTICLES ON GERMINATION OF Vigna radiata

The usage of nanoparticles in our daily products has increased tremendously, yet its impact

on environment is still unknown. Therefore, the present study investigates, the impact of

Silver nanoparticles on seed germination of Vigna radiata. The germination percentage of the

treated seeds were in par with the control. There was an effective impact of treatment, on

mean germination time, which was 1.2 day when treated with 2mg/l of AgNP’s, whereas the

most extended germination time was observed on treatment with 0.5mg/l of AgNP’s. The

highest germination rate of 1.8 seeds/day was observed after the exposure of seeds to 2mg/l

of AgNP’s from bark extract of Casuarina equisetifolia, which is an increased germination

rate compared to the control and minimum germination rate (less than one seed per day) was

seen in seeds treated with 0.5mg/ml of AgNP. Thus, there was an increase in germination

percentage, mean germination time and germination rate with the increase in concentration of

the silver nanoparticles. Similar results were observed[21] during the studies on seed

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Euphorbia hirta and it also eliminated the microbial contamination during the germination

process. The ability of the silver nanoparticles to increase the absorption of water by the

seeds[26], increases the enzymatic activity of superoxide dismutase, ascorbate peroxidase,

guaiacol peroxidase, and catalase[27] which leads to positive impact on enhanced seed

germination.[28]

[image:13.595.142.450.189.346.2]

s

Fig 8: Antibacterial activity of synthesized nanoparticles

E.coli S.aureus B. subtilis

Fig 9: Antibacterial activity of synthesized nanoparticles

PHOTOCATALYTIC DEGRADATION OF DYE

Photocatalytic degradation of methylene blue dye was evaluated using biosynthesized silver

nanoparticles spectrometrically. Degradation of methylene blue dye was observed visually by

change in the colour of the Silver nanoparticles-dye solution.

There was a gradual decrease in the absorption peak at 660nm, which was followed by

simultaneous increase in the absorption of silver nanoparticles at 420nm, with increase in

time period of exposure to sunlight (Fig 9). The percentage of degradation efficiency of silver

nanoparticles synthesized using aqueous bark extract of Casuarina equisetifolia, was

[image:13.595.110.491.387.519.2]
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dye degradation was Found to be increased with constant increase in exposure time of

AgNP-dye solution to sunlight.

The photocatalytic degradation of methylene blue dye was found to be 95 percent on 72

hours of exposure to sunlight which was mediated by silver nanoparticles successfully

synthesized by using Morinda tinctoria leaf extract.[30]

Thus the synthesized silver nano particles were proved to be the stable photocatalyte, that can

degrade organic compounds and dyes under suitable temperature of visible illumination[28]

which can be due to the property of Surface Plasma Resonance excitation of the synthesized

[image:14.595.144.455.300.467.2]

silver nanoparticles.[31]

Fig 10: Variation of dye degradation at different exposure time

Fig 11: UV Spectra indicating photocatylytic degradation of methylene blue dye by the

silver nanoparticles synthesized from aqueous bark extract of C.equisetifolia with

[image:14.595.145.454.512.687.2]
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CONCLUSION

In this study, simple and environmental friendly approach was adapted to green synthesis of

silver nanoparticles from aqueous bark extract of C.equisetifolia, at 80·C, within 20 mins at

pH 7 and was found to be stable for 30 days. Synthesized nanoparticles were characterized by

UV spectra, FT-IR, TEM and XRD studies. This experimental results suggest the application

of silver nanoparticles synthesized using aqueous bark extract of C. equisetifolia, as an

effective antibacterial agent in pharmaceutical industries. Germination studies using Vigna

radiata has proved the potential usage of silver nanoaparticle without having adverse effect

in the field of agriculture for crop improvement and food production. This could also be

implemented for efficient photo catalytic degradation of organic dyes present in textile

effluents in the presence of sunlight, which serves as an efficient mode for a cost effective

treatment of textile industrial effluents.

ACKNOWLEDGEMENT

The authors thank Mrs. Prema Sampathkumar, Associate Professor and Head, the Faculty

Members and supporting staff of the Department of Plant Biology and Plant Biotechnology,

Ethiraj college for Women (Autonomous), Chennai – 600008. We would also like to express

our thanks for the facilities extended by the Central instrumentation centre, Ethiraj college

for Women (Autonomous), and Dr. Mrs. A. Nirmala, Principal, Ethiraj college for Women

(Autonomous) for her valuable support, encouragement throughout the entire period of

research.

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Figure

Fig 1a: Synthesized AgNP’s using aqueous bark extract of Casuarina equisetifolia
Fig 2: Uv-Visible Spectra of AgNP’s showing effect of different reaction temperature
Fig 4: Uv-Visible Spectra of Agnp’s showing effect of different reaction pH
Fig 5(b): FTIR spectrum of aqueous bark extract of Casuarina equisetifolia
+4

References

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