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

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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
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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 milkweedCalotropis 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, tanni ns, 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 (sigmaAldrich 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 5103M aqueous silver nitrate solution in a round bottomed flask 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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+ A g N O 3

Rtn . Wi th stir rin g at RT

Latex solution Mixture after 6Hrs.

Rtn.Mixture at 0 hrs.

Rtn.

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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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Part I Constant temperature and varying latex concentration: 1) A single reaction was setup as follows20 ml each of the latex solution with varying concentrations (0.3%, 0.5%) was mixed with 20 ml of 5103M 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.

Latex conc. (%) 0.3% 0.4%

Conc. Of AgNO3 (M) 0.005 0.005

Temperature (oC) 85 85

Time (Hrs.)

4 4

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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) 85 85

Concentration of Latex (%) 0.3 0.5

Concentration of AgNO3 (M) 0.005 0.005

Colour change -

- = No colour change (AgNPs are not synthesized) laboratory Next the reaction was carried out at RT in ambience for 24 hrs. by using 0.3% latex concentration and 0.005M AgNO3 colour of Observation - After 24hrs. reaction time, the 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. (%) 0.3

Temp. (oC) RT

Time (Hrs.)

Colour change

Absorbance at 452nm 0.288

24

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 5 x 10-3M 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) 85 RT 6 6 Time (hrs.) Latex conc. (%) 0.4 0.4 16/4/2011 Conc. Of AgNO3 (M) 0.005 0.005 15

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) 85 RT

Latex conc. (%) 0.4 0.4

Time (Hrs.) 6 6

Conc. Of AgNO3 (M) 0.005 0.005

Colour Change +

- = 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. an 3ml sample was withdrawn from the reaction after interval of every 1hr, 2hr, 3hr, 4hr, 5hr & 6hrs. Absorbance of the sample was determined on UVVis Spectrophotometer and the peak was determined.

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Time (Hrs.) 1 2 3 4 5 6

Temperature (oC) RT RT RT RT RT RT

Conc. Latex (%) 0.4 0.4 0.4 0.4 0.4 0.4

Of

Conc.

Of

AgNO3 (M) 0.005 0.005 0.005 0.005 0.005 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. (%) 0.4

Conc. Of AgNO3 (M) 0.005

Temperature (oC) RT

Time (Hrs.)

1 to 6

Time in hours 1 2 3 4 5 6

Wavelength( nm) 413.6 426.4 426.4 426.4 426.4 413.6

Absorbance 0.197 0.256 0.598 0.758 0.880 0.309

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12

10

Absorbance

Wavelength(nm)

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 23 16/4/2011

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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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.
Bacterial culture Amount of Colloidal Silver nanoparticle solution (l) Tube I 200 200 Tube II Tube III 600 600

Tube I
Escherichia coli Bacillus subtilis

Tube II
400 400

Tube III

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Result: After 24hrs. incubation, it was found that broth tubes containing cultures of Bacillus subtilis -

Maximum turbidity at 200l and 400l concentrations and less or no turbidity at 600l concentration. E.coli

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

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Bacterial culture

Amount of Colloidal Silver nanoparticle solution(l) Tube I Tube II Tube III

Escherichia coli Bacillus subtilis

TUBE I

TUBE II
-

TUBEIII
-

+ = Turbidity present - = Turbidity absent

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Figure 7: Tubes inoculated with E.coli culture containing 400l & 600l 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 400l & 600l of colloidal Silver nanoparticles solutions respectively showing no turbity at a concentration of 600l and turbidity at a concentration of 400l after 24hrs. of incubation 32 16/4/2011

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 600l of colloidal Silver nanoparticles showed growth. are This shows that Colloidal Silver nanoparticles capable of inhibiting the growth of E.coli at a concentration of 600l. The MIC of colloidal silver nanoparticles for E.coli is 600l. 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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