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“GREEN SYNTHESIS OF SILVER NANOPARTICLES FROM THE LATEX OF CALOTROPIS PROCERA & EVALUATION OF ITS ANTIMICROBIAL EFFICACY” PRESENTED BY: Rashmi Deepak Shetty, MSc II, Microbiology GUIDED BY: Dr. Satishchandra. B. Ogale Scientist, NCL, Pune. Internal Guide- Mr. Vivek .N. Bobade 16/4/2011 1

Rashmi Presentation 999

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“GREEN SYNTHESIS OF SILVER NANOPARTICLES FROM THE LATEX

OF CALOTROPIS PROCERA & EVALUATION OF ITS ANTIMICROBIAL

EFFICACY”

PRESENTED BY: Rashmi Deepak Shetty,MSc II, Microbiology

GUIDED BY: Dr. Satishchandra. B. OgaleScientist, NCL, Pune.

Internal Guide- Mr. Vivek .N. Bobade16/4/2011

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ABSTRACT Biologically synthesized nanoparticles have been widely used in the

field of medicine. Biogenesis of nanoparticles have proven to be better methods due to slower kinetics and they offer better manipulation and control over crystal growth and their stabilization. The Silver nanoparticles were successfully synthesized from AgNO3 through a simple green route by using latex of the common Indian Milkweed- Calotropis procera as reducing agent as well as capping agent. Nanoparticles were characterized using Double beam UV-Vis Spectrophotometer. Further their Antimicrobial activity was investigated by Broth Dilution Method and the MIC was determined. These biologically synthesized colloidal solutions of Silver nanoparticles were found to inhibit the growth of Gram negative E.coli. Extracellular synthesis of nanoparticles using the latex of Calotropis procera is conventional, eco-friendly, cost effective and a novel approach towards weed utilization.

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INTRODUCTION

• Nanotechnology - electronics, catalysis, chemistry, energy and medicine.

•Nanoparticles exhibit completely new or improved properties based on specific characteristics such as size, distribution and morphology.

•Synthesis – Physical, chemical & biological. •Synthesis of Inorganic nanoparticles - biological

methods makes nanoparticles more biocompatible and environmentally benign.

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• The milkweed- Calotropis gigantea are native to Indonesia, Malaysia, Philippines, Thailand, Sri Lanka, India and China. • Shrub growing to 4 m tall. • Clusters of waxy flowers that are either white or lavender in

colour. • Milkweed is a common folk remedy used for the clotting of

small wounds and the removal of warts.

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• Latex is a complex emulsion consisting of proteins, alkaloids, starches, sugars, oils, tannins, resins and gums that coagulate on exposure to air. • It is usually exuded after tissue injury. In most plants, latex is white, but some have yellow, orange, or scarlet latex. • Since the 17th century, latex has been used as a term

for the fluid substance in plants. • It serves mainly as a defense against herbivorous insects. Many people are allergic to latex. .

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In the present Green synthesis –• Latex of the common Milkweed- Calotropis gigantea

as reducing as well as capping agent for the synthesis of Silver Nanoparticles.

• The AgNPs obtained were characterized using Double beam UV-Vis Spectrophotometer.

• Further their Antimicrobial activity was investigated by Broth Dilution Method and the MIC was

determined. • These biologically synthesized colloidal solutions of

Silver nanoparticles were found to inhibit the growth of Gram negative E.coli.

• Extracellular synthesis of nanoparticles using the latex of Calotropis gigantea is conventional, eco-

friendly, cost effective and a novel approach towards weed utilization.

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MATERIALS AND METHODS

CHEMICALS & GLASSWARES: For Ag Nanoparticle synthesis-

• Latex of Calotropis gigantea• Silver nitrate (AgNO3) analytical grade (sigma-Aldrich Chemical Pvt. Ltd.)• Triply distilled de-ionized water• Glass wares- round bottomed flask, Micropipettes,

Conical flasks• Magnetic stirrer

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METHODS:

Green synthesis of Silver Nanoparticles:

Crude Latex was obtained and stored in the refrigerator until use. (All Aqueous solutions were prepared using triply distilled de-ionized water).

0.4% latex solution was prepared

0.005M Silver Nitrate (AgNO3) solution was prepared

20 ml of 0.4% latex solution + 20 ml 5×10−3M aqueous silver nitrate solution in a round bottomed flask

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The Mixture - kept at RT in laboratory ambience for 6 hrs on a magnetic stirrer with constant stirring

Same type reactions were also performed with various concentrations of AgNO3 and latex.

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+ AgNO3Rtn. With stirring at RT

Latex solution Rtn.Mixture at 0 hrs. Rtn. Mixture after 6Hrs.

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REACTION STANDARDISATION:

As no background information was available regarding the biosynthesis of silver nanoparticles from latex of the common Indian milkweed- Calotropis gigantea, it is essential to determine the concentration of latex, Silver nitrate (AgNO3), optimum temperature and time required for synthesis of Silver Nanoparticles.

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Latex conc.

(%)

Conc. Of

AgNO3 (M)

Temperature

(oC)

Time (Hrs.)

0.3% 0.005 85 4

0.4% 0.005 85 4

Part I – Constant temperature and varying latex concentration:

1) A single reaction was setup as follows-20 ml each of the latex solution with varying concentrations (0.3%, 0.5%) was mixed with 20 ml of 5×10−3M aqueous silver nitrate solution in a round bottomed flask.2) The Reaction Mixture was maintained at 85oC in an oil bath with constant stirring for 4 hrs. on a magnetic stirrer.

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RESULT:

UV-Vis Specrophometric measurements revealed that none of the latex concentrations were capable of synthesizing Silver nanoparticles 85oC. This might be because of the denaturation of proteins present in the latex, which are responsible for the reduction of silver ions to form Silver nanoparticles. Hence it was decided to perform the reaction at room temperature (RT) in laboratory ambience.

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Temperature

(oC)

Concentration

of Latex (%)

Concentration

of AgNO3 (M)

Colour change

85 0.3 0.005 -

85 0.5 0.005 -

‘-‘ = No colour change (AgNPs are not synthesized)

• Next the reaction was carried out at RT in laboratory ambience for 24 hrs. by using 0.3% latex concentration and 0.005M AgNO3

• Observation - After 24hrs. reaction time, the colour of the reaction mixture changed from Colourless to Brown. The UV-Vis Spectrophotometric

measurements after 24hrs. showed an absorbance of 0.288 at 452nm.

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Latex

conc. (%)

Temp.

(oC)

Time

(Hrs.)

Colour

change

Absorbance

at 452nm

0.3 RT 24 + 0.288

Figure 5: Gradual colour change of the reaction mixture with 0.3% latex conc. and 0.005M AgNo3 at RT (from colourless to Brown in 24Hrs.)

Since the reaction time was longer, we decided to use 0.4% latex concentration and 0.005M AgNO3 for the further reactions.

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Part II – Constant Latex concentration and varying temperature:

A single reaction was setup as follows-• 20ml of 0.4% latex solution + 20ml of

aqueous Silver nitrate solution in a round bottomed flask.• First set of the Reaction Mixture was maintained at 85oC

in an oil bath and second set of the reaction mixture was maintained at room temperature in laboratory ambience with constant stirring for 6 hrs. on a magnetic stirrer.

Temperature

(oC)

Time (hrs.) Latex conc.

(%)

Conc. Of

AgNO3 (M)

85 6 0.4 0.005

RT 6 0.4 0.005

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5 x 10-3M

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RESULT:

Part II- Constant latex concentration and varying temperature:

During the reaction, the colour of reaction mixture changed from Colourless to Brown at RT, whereas no colour change was found at 85oC. The UV Spectrophotometric measurements of the resultant reaction mixtures showed that, Silver nanoparticles are formed at RT, but not at the temperature of 85oC.

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Temp.

(oC)

Latex

conc. (%)

Time

(Hrs.)

Conc. Of

AgNO3 (M)

Colour

Change

85 0.4 6 0.005 -

RT 0.4 6 0.005 +

‘-‘ = No colour change‘+’ = Colour changes to Brown

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Part III – Optimization of Reaction Time:

A single reaction was setup as follows-• 20ml of 0.4% latex solution + 20ml of 5 x 10-3 M

aqueous Silver nitrate solution in a round bottomed flask.• The reaction mixture was maintained at room temperature in laboratory ambience with constant stirring on a magnetic stirrer.• 3ml sample was withdrawn from the reaction after

an interval of every 1hr, 2hr, 3hr, 4hr, 5hr & 6hrs. • Absorbance of the sample was determined on UV-

Vis Spectrophotometer and the peak was determined.

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Time

(Hrs.)

Temperature

(oC)

Conc. Of

Latex (%)

Conc. Of

AgNO3 (M)

1 RT 0.4 0.005

2 RT 0.4 0.005

3 RT 0.4 0.005

4 RT 0.4 0.005

5 RT 0.4 0.005

6 RT 0.4 0.005

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RESULT:

Part III- Optimization of Reaction Time

From UV-Vis Spectrophotometric measurements, it is observed that the intensity of SPR bands at 426.4nm increases as the reaction time progresses i.e., from 2hrs to 5hrs of the reaction and after initial one hour and after 6 h of reaction time a considerable intensity of the SPR bands is achieved at 413.6nm. Higher intensity of SPR band is a measure of higher concentration of particles.

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Latex

Conc. (%)

Conc. Of

AgNO3 (M)

Temperature

(oC)

Time (Hrs.)

0.4 0.005 RT 1 to 6

Time in hours

Wavelength(nm)

Absorbance

1 413.6 0.1972 426.4 0.2563 426.4 0.5984 426.4 0.7585 426.4 0.8806 413.6 0.309

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426.4nm 413.6nm0

0.5

1

1.5

2

2.5

3

0.256

0.598000000000001

0.758000000000003

0.880000000000002

0.1990000000000010.309000000000001

1hr 2hr 3hr 4hr 5hr 6hrWavelength(nm)

Abs

orba

nce

Figure 6: Time variation study at 0.4% latex concentration and 0.005M AgNO3

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Figure 6: Gradual change in the color of reaction mixture with 0.4% latex conc. and 0.005M AgNO3 at RT from Colorless to Brown with time (from left Latex solution-0.4%, at 0hr of rtn., at 1hr of rtn., at

4hrs. of rtn., at 6hrs. of reactiotn.)16/4/2011

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APPLICATION

Silver nanoparticles synthesized by various techniques can have various applications. Additionally, silver nanoparticles possess an excellent biocompatibility and low toxicity. The major application that has been worked on is its use as an Antibacterial agent.

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Application as Antibacterial agent:

• Silver nanoparticles have been known to exhibit strong toxicity to wide range of microorganism.

• Antibacterial property of colloidal solution of AgNPs against Bacillus subtilis and Escherichia coli has been investigated. Silver nanoparticles were found to be cytotoxic to E. coli.

• It was showed that the antibacterial activity of silver nanoparticles was size dependent.

• Silver nanoparticles mainly in the range of 1 -10 nm attach to the surface of cell membrane and drastically disturb its proper function like respiration and

permeability. • The general understanding is that silver nanoparticles

get attached to sulfur containing proteins of bacterial cell and causes the death of bacteria.

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Chemicals & Glass wares:

• Muller-Hinton broth medium• Muller-Hinton agar medium• Silver Nanoparticle colloidal solution• Pure cultures of E. coli & Bacillus• Saline • Glass wares- Petri plates(4), Bumper tubes(8), Micropipettes

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Bacterial culture Amount of Colloidal Silver nanoparticle

solution (µl)

Tube I Tube II Tube III

Escherichia coli 200 400 600

Bacillus subtilis 200 400 600

Tube I Tube II Tube III

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Procedure:

The colloidal silver nanoparticles solution synthesized using Calotropis gigantea latex was tested for antimicrobial activity against the Gram negative E. coli and Gram positive bacillus by Broth Dilution method.

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Result:

After 24hrs. incubation, it was found that broth tubes containing cultures of –

• Bacillus subtilis -

Maximum turbidity at 200µl and 400µl concentrations and less or no turbidity at 600µl concentration.

• E.coli –

Turbidity at 200 µl concentration and no turbidity at 400 µl and 600 µl concentrations.

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Bacterial

culture

Amount of Colloidal Silver nanoparticle

solution(µl)

Tube I Tube II Tube III

Escherichia

coli

+ - -

Bacillus

subtilis

+ + -

‘+’ = Turbidity present‘-‘ = Turbidity absent

TUBE I TUBE II TUBEIII

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Figure 7: Tubes inoculated with E.coli culture containing 400µl & 600µl of colloidal AgNP solutions respectively showing no

turbidity after 24 hrs. of incubation

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Figure 4: Tubes inoculated with Bacillus subtilis containing 400µl & 600µl of colloidal Silver nanoparticles solutions respectively showing no turbity at a

concentration of 600µl and turbidity at a concentration of 400µl after 24hrs. of incubation

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After plating 0.1ml broth culture on Muller-Hinton agar plates, the plates are incubated for further 24hrs.

Observation - All the plates other than the only plate inoculated with E.coli broth culture containing 600µl of colloidal Silver nanoparticles showed growth. • This shows that Colloidal Silver nanoparticles are capable of inhibiting the growth of E.coli at a concentration of 600µl. • The MIC of colloidal silver nanoparticles for E.coli is 600µl.• This shows that Silver nanoparticles synthesized

by using Latex of milkweed- Calotropis gigantea can be used an effective Antibacterial agent.

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CONCLUSION:

• Present green synthesis shows that the environmentally benign and renewable latex of Calotropis gigantea can be used as an effective capping as well as reducing agent for the synthesis of AgNPs. • The reduction & stabilization being accomplished by latex proteins and metabolites. • Also the observations show that AgNPs can be synthesized better at Room temperature in laboratory ambience and at 0.4% latex concentration. • AgNPs synthesized by the above method are quite stable

and no visible changes are observed even after10 to 15 days, if the nanoparticle solutions are kept in light proof conditions. • Synthesis of AgNPs from the Latex of the common Milkweed- Calotropis gigantea can be a novel approach towards Weed Utilization and is simple, cost effective and requires only a small amount of latex for synthesis.

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REFERENCES1) Zygmunt Sadowski, Wroclaw University of Technology Poland, “ Biosynthesis and Application of Silver and Gold Nanoparticles”,2) M. Linga Rao et al, ” Biological Synthesis of Silver Nanoparticles using Svensonia Hyderabadensis Leaf Extract and Evaluation of their Antimicrobial Efficacy”, J. Pharm. Sci. & Res. Vol.3(3), 2011,1117-11213) K. Govindaraju, S.Tamilselvan, V. Kiruthiga and G. Singaravelu, “Biogenic silver nanoparticles by Solanum torvum and their promising antimicrobial activity”, Journal of Biopesticides 3(1 Special Issue) 394 - 399 (2010)

4)Monali Gajbhiye, MSc, Jayendra Kesharwani, MSc, Avinash Ingle, MSc, Aniket Gade, MSc, Mahendra Rai, PhD, “Fungus-mediated synthesis of silver nanoparticles and their activity against pathogenic fungi in combination with fluconazole”, Nanomedicine: Nanotechnology, Biology, and Medicine 5 (2009) 382–3865) G.Thirumurugan*, S.M.Shaheedha, M.D.Dhanaraju, “In-vitro evaluation of anti-bacterial activity of silver nanoparticles synthesised by using phytophthora Infestans”, International Journal of ChemTech Research Vol.1, No.3 , pp 714-716

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6) Hong-Juan Bai Zhao-Ming Zhang Jun Gong, “Biological synthesis of semiconductor zinc sulfide nanoparticles by immobilized Rhodobacter sphaeroides”, Biotechnol Lett (2006) 28:1135–11397) Kalimuthu Kalishwaralal, Venkataraman Deepak, Sureshbabu Ram Kumar Pandian, Sangiliyandi Gurunathan, “Biological synthesis of gold nanocubes from Bacillus licheniformis” Bioresource Technology 100 (2009) 5356–53588) Anal K. Jhaa, K. Prasadb, A.R. Kulkarni, “Synthesis of TiO2 nanoparticles using microorganisms”, Colloids and Surfaces B: Biointerfaces 71 (2009) 226–2299) Kornphimol Kulthong1, Sujittra Srisung2, Kanittha Boonpavanitchakul1, Wiyong Kangwansupamonkon1 and Rawiwan Maniratanachote, “RDeseeatrcehrmination

10) of silver nanoparticle release from antibacterial fabrics into artificial sweat”, Particle and Fibre Toxicology 2010, 7:811) Harekrishna Bar, Dipak Kr. Bhui, Gobinda P. Sahoo, Priyanka Sarkar, Sankar P. De, Ajay Misra, “Green synthesis of silver nanoparticles using latex of Jatropha curcas”, Colloids and Surfaces A: Physicochem. Eng. Aspects 339 (2009) 134–13912) S. Anil Kumar, Majid Kazemian Abyaneh, S. W. Gosavi, Sulabha K. Kulkarni, Renu Pasricha, Absar Ahmad ,M. I. Khan, “Nitrate reductase-mediated synthesis of silver nanoparticles from AgNO3”, Biotechnol Lett (2007) 29:439–445

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13) K. Prasad a,*, Anal K. Jha b, “Biosynthesis of CdS nanoparticles: An improved green and rapid procedure”, Journal of Colloid and Interface Science 342 (2010) 68–7214) Dabin Yu and Vivian Wing-Wah Yam, “Controlled Synthesis of Monodisperse Silver Nanocubes in Water”, J. AM. CHEM. SOC. 2004, 126, 13200-1320115) Jha, Anal K. and Prasad, K. 'Green Synthesis of Silver Nanoparticles Using Cycas Leaf', International Journal of Green Nanotechnology: Physics and Chemistry, 1:2, 110 – 11716) Naheed Ahmad,1 Seema Sharma,2 V. N. Singh,3 S. F. Shamsi,4 AnjumFatma,5 andB. R.Mehta3, “Biosynthesis of Silver Nanoparticles from Desmodium triflorum: A Novel Approach TowardsWeed Utilization “,Biotechnology Research International Volume 2011, Article ID 454090, 8 pages17) Eric C. Njagi, Hui Huang, Lisa Stafford, Homer Genuino, Hugo M. Galindo, John B. Collins, George E. Hoag, and Steven L. Suib, “Biosynthesis of Iron and Silver Nanoparticles at Room Temperature Using Aqueous Sorghum Bran Extracts”, Langmuir, 2011, 27 (1), pp 264–27118) en.wiikipedia.org/wiki/Latex19) James JF. (1887). The milkweeds. Am. Nat. 21:605–15. 

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20) Cristina Buzea, Ivan Pacheco, and Kevin Robbie, "Nanomaterials and Nanoparticles: Sources and Toxicity", Biointerphases 2 (4): MR17–MR71.21)  http://www.pharmainfo.net/vishnupriya22) http://www.pharmainfo.net/umadevia23) Majid Darroudi, Mansor Bin Ahmad, Abdul Halim Abdullah, Nor Azowa Ibrahim, “Green synthesis and characterization of gelatin-based and sugar-reduced silver nanoparticles “, International Journal of Nanomedicine 2011:6 569–57423) N. Saifuddin, C. W. Wong and A. A. Nur Yasumira, “Rapid Biosynthesis of Silver Nanoparticles Using Culture Supernatant of Bacteria with Microwave Irradiation”, E-Journal of Chemistry 2009, 6(1), 61-7024) en.wikipedia.org/wiki/Nanotechnology25) Agrawal AA, Konno K. (2009), “Latex: A Model for Understanding Mechanisms, Ecology, and Evolution of Plant Defense Against Herbivory”, Annu. Rev. Ecol. Evol. Syst. 40:311–326) James JF. (1887), “The milkweeds”, Am. Nat. 21:605–15.27) Manish Hudlikar, Shriram Joglekar, Mayur Dhaygude and Kisan Kodam, “Latex mediated synthesis of ZnS Nanoparticles: Green synthesis Approach”, [email protected] pages: 1 to 11. 

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