FORM 2
THE PATENT ACT 1970
&
The Patents Rules, 2003
COMPLETE SPECIFICATION (See section 10 and rule 13)
1. TITLE OF THE INVENTION:
METHOD FOR SYNTHESIS OF BIO STABILIZED GOLD NANOPARTICLES
USING WATER HYACINTH
2. APPLICANT:
Name Nationality Address
1. Dr. Rajendra S. Zunjarrao Both Indian Modern College of Arts,
Nationals Science and Commerce,
2. Dr. Rebecca S. Thombre Shivajinagar, Pune-5,
1 Maharashtra, India.
3. PREAMBLE TO THE DESCRIPTION:
COMPLETE
The following specification particularly describes the Invention and the manner in which it is to be performed.

METHOD FOR SYNTHESIS OF BIO STABILIZED GOLD NANOPARTICLES USING
WATER HYACINTH
FIELD OF THE INVENTION
The present invention relates to preparation of gold nanoparticles, and more particularly, to a method for synthesis of bio stabilized gold nanoparticles using water hyacinth.
BACKGROUND OF THE INVENTION
There is an increasing commercial demand for nanoparticles due to their wide applicability in various areas such as electronics, catalysis, chemistry, energy, and medicine.
Nanobiotechnology deals with the synthesis of nanostructures using living organisms. Among the use of living organisms for nanoparticle synthesis, plants have found application particularly in metal nanoparticle synthesis. Use of plants for synthesis of nanoparticles could be advantageous over other environmentally benign biological methods as this eliminates the elaborate method of maintaining cell cultures. Biosynthetic methods for nanoparticles would be more useful if nanoparticles were produced extracellularly using plants or their extracts and in a controlled manner according to their size, dispersity and shape. Plant use can also be suitably scaled up for large-scale synthesis of nanoparticles.

Water hyacinth [Eichhornio crossipes] is an aquatic plant found in lakes and ponds in India. It is originally a native to South America. Water hyacinth is considered a nuisance plant because it covers lakes and ponds entirely which dramatically impacts water flow, blocks sunlight from reaching native aquatic plants, and starves the water of oxygen, often killing aquatic biodiversity. The plants also create good conditions for mosquito that spreads diseases. Water hyacinth contains phytochemical constituents and plant bioactive molecules that cause the reduction of gold tetrachloride to gold nanoparticles and its effective stabilization. The unwanted water hyacinth is successfully used for the preparation of biostabtlized gold nanoparticles.
OBJECT OF THE INVENTION
An object of the present invention is to provide gold nanoparticles which are prepared from plant growth of which if not controlled pollutes rivers.
SUMMARY OF THE INVENTION
Accordingly, the present invention provides a Method for synthesis of bio stabilized gold nanoparticles using water hyacinth. The method includes collecting water hyacinth plants. Further, the method includes separating the swollen petioles from the water hyacinth plant and washing the swollen petioles with sterile distilled water. Furthermore, the method includes crushing the swollen petioles to get the extract from the petioles of water hyacinth and filtering the extract. Thereafter, the method includes adding the prepared extract of water hyacinth petioles to equal volumes of 10 mM gold tetrachloride (AuCI4) to form

mixture. The reaction mixture thus formed is incubated for 4 hours at 37°C until the color of the mixture is changed.
BRIEF DESCRIPTION OF DRAWINGS
Figure 1 shows bioreduction of gold tetrachloride by water hyacinth in accordance with the method of present invention;
Figure 2 shows UV-Vis spectra analysis of nanoparticles produced by extract of water hyacinth, in accordance with the present invention;
Figure 3 shows FTIR spectra of gold nanoparticles produced using a method in accordance with the present invention;
Figure 4a shows FEG-SEM of gold nanoparticles produced using a method in accordance with the present invention; and
Figure 4 b shows EDS of gold nanoparticles produced using a method in accordance with the present invention;
DETAILED DESCRIPTION OF THE INVENTION
The foregoing objects of the present invention are accomplished and the problems and shortcomings associated with the prior art, techniques and approaches are overcome by the present invention as described below in the preferred embodiment.

The present invention provides a method for synthesis of bio stabilized gold nanoparticles using water hyacinth. The aqueous extract of water hyacinth is used for synthesis of biologically stabilized gold nanoparticles.
The method includes collecting water hyacinth plants. In the present case, the water hyacinth is collected from a stagnant water body in Pimpri, Pune, Maharashtra, India. Further, the method includes separating the swollen petioles from the water hyacinth plant and washing the swollen petiole with sterile distilled
water.
Furthermore, the method includes grinding the swollen petioles in a grinder to get the extract from the petioles of water hyacinth and filtering the extract. The extract is filtered and is used immediately for synthesis of gold nanoparticles.
Thereafter, the method includes adding the prepared extract of water hyacinth petioles to equal volumes of 10 mM gold tetrachloride (AuCI4) to form mixture. The reaction mixture thus formed is incubated for 4 hours at 37°C until the color of the mixture is changed. The rate of the reaction increases after elevating the temperature to 100°C and the color change is observed in 1 hour. The effect of pH change on the reaction is also studied; the optimum pH for the reaction is 10.5.
The synthesis of gold nanoparticles is performed using swollen petiole extract of water hyacinth as a reducing agent. The reduction of gold tetrachloride to nanogold resulted in color change as shown in figure 1. The typical dark purple

color was observed when equal volume of leaf extract was added to gold tetrachloride solution.
CHARACTERIZATION OF NANOPARTICLES
The reduction of pure Au+ ions was studied by observation of surface plasmon resonance using a double beam spectrophotometer (Shimadzu, UV-2450). The topography of the nanoparticles was studied using Field Emission Gun Scanning Electron Microscope (FEG-SEM) (JSM-76Q0F) and the elemental analysis was performed by Energy Dispersive spectroscopy (EDS). FTIR (Fourier Transform Infrared Spectroscopy) was also performed to ascertain the presence of plant peptides that may have coated the particles during synthesis procedure. FTIR spectra were measured on Jasco FT/IR-16100.
The gold nanoparticles were characterized by observation of surface plasmon resonance maxima of gold nanoparticles. This peak is attributed to nanoparticles with sizes ranging to 120 nm to 240 nm as shown in Figure 2.
An absorption maximum was observed at 520-570 nm which is characteristic of gold nanoparticles. The gold nanoparticles were further chracterized using SEM and FTIR analysis. FTIR analysis reveals a set of unique peaks that prove the presence of amino groups indicating involvement of plant peptides in biosynthesis of nanoparticles. The FTIR spectra of biostabilised gold nanoparticles are presented in figure 3.

As seen in figure 3, the bonds observed in the FTIR spectra indicate the presence of -C=0- and -C=C- stretching suggesting the presence of phytopeptides and the possible role of plant bioactive molecules in synthesis and stabilization of gold nanoparticles. The FEG-SEM image revealed that the particles are spherical in shape and their EDS confirmed the presence of gold ions in the peaks as shown in figure 4b.
ANTIMICROBIAL ACTIVITY OF GOLD NANOPARTICLES
The antimicrobial activity of gold nanoparticles synthesized using water hyacinth was studied against Escherichia coli, Staphylococcus aureus and Bacillus subtilis using the agar well diffusion method. 0.1 ml of 16 h old log phase test bacterial culture (Absorbance - 0.5 at 600 nm) was spread on Mueller Hinton agar plates and 6 mm wells were prepared on the agar plates using cork borer. 20 pi of nanoparticles solution (40mg/ml) was added aseptically to the wells and the plates were incubated at 37°C overnight. After 24 h the zone of inhibition was measured.
The antibacterial activity of gold nanoparticles was studied against Escherichia coli, Bacillus subtilis, Staphlococcus aureus and Klebsiella pneumoniae. The gold nanoparticles demonstrated a zone of inhibition against all the test organisms with maximum inhibition against gram negative E.coli (16 mm) followed by Klebsiella sp. (9 mm) while antimicrobial activity against gram positive Bacillus sp. (7mm) and 5. aureus ( 8mm) was lesser.
The biostabilized gold nanoparticles of the present invention synthesized using water hyacinth extract were spherical, stable and were characterized using UV- Vis

spectrophotometry, SEM and FTIR analysis. The gold nanoparticles demonstrated potent antimicrobial activity against bacteria. Thus a rapid, eco friendly and economical method for synthesis of biostabilized gold nanoparticles was developed using an invasive aquatic plant.
The foregoing object the invention is accomplished and the problems and shortcomings associated with prior art techniques and approaches are overcome by the present invention described in the present embodiment. Detailed descriptions of the preferred embodiment are provided herein; however, it is to be understood that the present invention may be embodied in various forms. Therefore, specific details disclosed herein are not to be interpreted as limiting, but rather as a basis for the claims and as a representative basis for teaching one skilled in the art to employ the present invention in virtually any appropriately detailed system, structure, or matter. The embodiments of the invention as described above and the methods disclosed herein will suggest further modification and alterations to those skilled in the art. Such further modifications and alterations may be made without departing from the spirit and scope of the invention.

We Claim:
1. A method for synthesis of bio stabilized gold nanoparticles, the method comprising:
collecting water hyacinth plants;
separating swollen petioles from the water hyacinth plant;
washing the swollen petioles with sterile distilled water;
crushing the swollen petioles to get the extract from the petioles of water
hyacinth;
filtering the extract;
adding the prepared extract from water hyacinth petioles to equal volumes
of 10 mM gold tetrachloride(AuCl4) to form mixture; and
incubating the mixture for 4 hours at 37°C until the color of the mixture is
changed.