DESC:

FORM 2
THE PATENTS ACT, 1970
(39 OF 1970)
&
The Patents Rules, 2003
COMPLETE SPECIFICATION
(See section 10; rule 13)

1. Title of the invention – A PROCESS OF PREPARING SILVER DOPED ZINC OXIDE NANOPARTICLES USING PLUMBAGO AURICULATA LEAF EXTRACT
2. Applicant(s)
NAME: RK University
NATIONALITY: INDIAN
ADDRESS: RK University, School of Science, Bhavnagar Highway, Tramba, Rajkot-360020, Gujrat, India.

3. PREAMBLE TO THE DESCRIPTION

The following specification particularly describes the invention and the manner in which it is to be performed.

A PROCESS OF PREPARING SILVER DOPED ZINC OXIDE NANOPARTICLES USING PLUMBAGO AURICULATA LEAF EXTRACT
FIELD OF THE INVENTION
The present invention is relates to a synthesis of silver doped zinc oxide nanoparticles using Plumbago auriculata leaf extract. The present invention particularly relates to process for preparation of silver doped zinc oxide nanoparticles using Plumbago auriculata leaf extract which shows significant antimicrobial effect.

BACKGROUND OF THE INVENTION
Nanotechnology plays an important role in modern material science. Nanotechnology is a rapid growing field with its application in science and technology for the purpose of manufacturing new materials at nano scale level. Nanoparticles are small sized particles with large surface area which help the drug molecules in faster absorption to cure diseases.

Synthesis of nanomaterials includes various physical, chemical and biological methods. Nanoparticles synthesized using chemical and physical methods have several disadvantages as compared to biological synthesis. Since, they employ toxic and corrosive chemicals for reduction and stabilization. Microorganisms mediated synthesis of nanoparticles are slower and gives only a limited number of morphological feature.

Most of the nanoparticles are synthesized by ball milling, sol-gel process, chemical vapor deposition, sputtering, hydrothermal method etc. But, these chemical methods are complex, expensive, hazardous and leading to generation of unwanted by-products.

Use of medicinal plants for nanoparticles synthesis has acquire a great attention due to the ease in scaling up for larger production, apart from being cost effective and environmental friendly. Several nanoparticles composed of elemental gold, silver, copper, platinum and palladium are synthesized using plants which show antimicrobial, antifungal, antibiofilm, cytotoxic and photocatalytic properties. Green synthesis using plant extracts offers a simple and effective approach to synthesis nanoparticles at large scale.

Metal oxide nanoparticles has wide range of industrial application due to their unique properties. Doping is a widely used method for the modification of nanoparticles to enhance their electrical, optical and biological activities. Metal oxide nanoparticles are chemically stable and they are used in a variety of different applications such as adsorption, photocatalytic activities, antibacterial and antifungal activities.

The synthesis method plays a vital role in determining the properties of metal oxide nanoparticles. The existing synthesis methods have their drawbacks that they do not comply with the need of an appropriate method which gives enhanced properties of nanoparticles.

Therefore the inventors of the present invention have developed a green route for synthesis of silver doped zinc oxide nanoparticles (ZnOAgNPs) using P. auriculata leaf extract (PALE) to check the effect against various bacteria. The prepared silver doped zinc oxide nanoparticles using P. auriculata leaf extract exhibits notable antimicrobial effect. So, far it has been exploited to achieve the following objective.

OBJECTIVE OF THE INVENTION
The main object of the present invention is to provide a process of preparing silver doped zinc oxide nanoparticles using Plumbago auriculata leaf extract.

Another object of the present invention is to provide a process of preparing silver doped zinc oxide nanoparticles using Plumbago auriculata leaf extract which is green synthesis process.

Another object of the present invention is to provide a process of preparing silver doped zinc oxide nanoparticles using Plumbago auriculata leaf extract which is easy to manufacture.

Yet another object of the present invention is to provide a process of preparing silver doped zinc oxide nanoparticles using Plumbago auriculata leaf extract which is simple and cost effective.

One other object of the present invention is to provide a process of preparing silver doped zinc oxide nanoparticles using Plumbago auriculata leaf extract which is having significant anti-microbial effect.

SUMMARY OF THE INVENTION
The main aspect of the present invention is to provide a process of preparing silver doped zinc oxide nanoparticles using Plumbago auriculata leaf extract.

Another aspect of the present invention is to provide a process of preparing silver doped zinc oxide nanoparticles using Plumbago auriculata leaf extract comprises the steps of,
a) mixing of 10 mM zinc acetate and 1 mM of silver nitrate;
b) adding 5 ml of Plumbago auriculata leaf extract in 95 ml of step (a) reaction mixture with constant stirring at 150 rpm for 24 hr;
c) recovering synthesized nanoparticles from step (b) mixture by centrifugation at 10,000 rpm for 20 min followed by washing twice with distilled water.
d) collecting pellets from step (c) mixture and further calcinating at 400° C for 4 h in a muffle furnace to gel final silver doped zinc oxide nanoparticles.

BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1: UV-Vis absorption spectra of the (A) ZnONPs; (B) ZnOAg1; (C) ZnOAg10; (D) ZnO10Ag1nanoparticles synthesized by using PALE at room temperature.
Figure 2:(A) Photoluminescence emission spectra of ZnONPs, ZnOAg1, ZnOAg10, ZnO10Ag1; (B)-(E) the Gaussian fitted PL emission spectra of ZnONPs, ZnOAg1, ZnOAg10, ZnO10Ag1, respectively.
Figure 3: HRTEM images of nanoparticles synthesized using PALE. (A) ZnONPs with hexagonal shape; (B) spherical shaped ZnONPs and inset showed the bar diagram of DLS data; (C) Rod shaped ZnOAg1NPs; (D) Spherical ZnOAg1NPs and inset showed the bar diagram of DLS data; (E) Rod shaped ZnOAg10NPs and inset showed the bar diagram of DLS data; (F) spherical shaped ZnOAg10NPs; (G) Rod shaped ZnO10Ag1NPs, (H) spherical shaped ZnO10Ag1NPs, and inset showed the bar diagram of DLS data.
Figure 4: Representative spot EDS spectra of (A) ZnONPs; (B) ZnOAg1NPs; (C) ZnOAg10NPs; (D) ZnO10Ag1NPs.
Figure 5: FTIR spectra of nanoparticles synthesized by PALE.(A) PALE and ZnONPs; (B) ZnOAg1NPs, ZnOAg10NPs, and ZnOAg10NPs.

DESCRIPTION OF THE INVENTION
The main embodiment of the present invention is to process of preparing silver doped zinc oxide nanoparticles using Plumbago auriculata leaf extract.

The detailed description set forth below is intended as a description of exemplary embodiments and is not intended to represent the only forms in which the exemplary embodiments may be constructed and/or utilized. The description sets forth the functions and the sequence of steps for constructing and/or operating the exemplary embodiments. However, it is to be understood that the same or equivalent functions and sequences which may be accomplished by different exemplary methods are also intended to be encompassed within the spirit and scope of the invention.

As defined herein, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs.

Although any process and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described.

As stated in the present invention herein, the singular forms “a,” “an” and “the” specifically also encompass the plural forms of the terms to which they refer, unless the content clearly dictates otherwise. The term “about” is used herein to means approximately, in the region of, roughly, or around.

As stated herein, that it follows in a transitional phrase or in the body of a claim, the terms “comprise(s)” and “comprising” are to be interpreted as having an open ended meaning. That is, the terms are to be interpreted synonymously with the phrases “having at least” or “including at least”. When used in the context of a process, the term “comprising” means that the process includes at least the recited steps, but may include additional steps. When used in the context of a composition, the term “comprising” means that the composition includes at least the recited features or components, but may also include additional features or components.

As used herein “nanoparticles” are defined as means a small particles that ranges between 1 to 100 nanometers in size. Undetectable by the human eye, nanoparticles can exhibit significantly different physical and chemical properties to their larger material.

As used herein “metal oxide nanoparticle” are defined as compounds obtained by reaction of metal ions at particular reaction conditions.

As used herein “identical concentration” is defined as equivalent molar concentration of salt solutions which are used in the proposed invention.

As per main embodiment of the present invention is to process of preparing silver doped zinc oxide nanoparticles using Plumbago auriculata leaf extract.

Plumbago auriculata leaf extract:
The plant specimen of P. auriculata along with flower was collected from the campus of RK. University, Rajkot, India. On the basis of ethno botanical knowledge, Plumbago auriculata is widely used medicinal plant that possesses many secondary metabolites in its root and aerial parts. Phytochemicals such as phenols, flavonoids, alkaloids, carbohydrates and saponins present in P. auriculata may help in synthesis of nanoparticles and may act as stabilizing and capping agent. The leaf and stem are rich in plumbagin, a napthoquinone that possesses antimicrobial, antibiofilm, anticancer and antifungal activities.

As per one embodiment, Plumbago auriculata used herein is an extract form.

As per one embodiment of the present invention, process for preparation of Plumbago auriculata leaf extract comprises the steps of,
a) collecting Plumbago auriculata leaves, washing and drying at room temperature for 10 days;
b) pulverizing dried leaves from step (a) into fine powder using electrical blender;
c) mixing of 5 gm of Plumbago auriculata leaf powder from step (b) with distilled water followed by heating at 60°C for 20 min;
d) filtering step (c) mixture through Whatman no 1 filter paper to get Plumbago auriculata leaf extract;
e) cooling Plumbago auriculata leaf extract from step (d) at room temperature and storing at 4°C until further use.
As per main embodiment a process of preparing silver doped zinc oxide nanoparticles using Plumbago auriculata leaf extract comprises the steps of,
a) mixing of 10 mM zinc acetate and 1 mM of silver nitrate;
b) adding 5 ml of Plumbago auriculata leaf extract in 95 ml of step (a) reaction mixture with constant stirring at 150 rpm for 24 hr;
c) recovering synthesized nanoparticles from step (b) mixture by centrifugation at 10,000 rpm for 20 min followed by washing twice with distilled water;
d) collecting pellets from step (c) mixture and further calcinating at 400° C for 4 h in a muffle furnace to gel final silver doped zinc oxide nanoparticles.

As per one embodiment of the present invention, silver doped zinc oxide nanoparticles were synthesized via green synthesis method using Plumbago auriculata leaf extract.

As per one embodiment of the present invention, Silver (Ag) doped ZnONPs were fabricated by varying the concentration of AgNO3 and ZnC4H6O4.The salt solutions of AgNO3 and ZnC4H6O4 were mixed in identical concentration of 1 mM for the synthesis of ZnOAg1NPs. The salt solutions of AgNO3 and ZnC4H6O4 were mixed in identical concentration of 10 mM for the synthesis of ZnOAg10NPs. For the synthesis of ZnO10Ag1NPs, 10 mM of ZnC4H6O4 was mixed with 1 mM of AgNO3.

As per one embodiment, the present invention provides a process for preparation of silver doped zinc oxide nanoparticles from Plumbago auriculata leaf extract which provides mostly rod shaped nanoparticles.

As per one embodiment, the silver doped zinc oxide nanoparticles from Plumbago auriculata leaf extract are having particle size in the range from 1-200 nm, more preferably 1-150 nm and most preferably 10-100 nm.

As per one embodiment of the present invention, silver doped zinc oxide nanoparticles using Plumbago auriculata leaf extract which are having very high potential for the antimicrobial effect. Plumbago auriculata leaf extract (PALE) mediated biofabrication is a green route for synthesis of ZnOAgNPs.

The invention is further illustrated by the following examples which are provided to be exemplary of the invention and do not limit the scope of the invention. While the present invention has been described in terms of its specific embodiments, certain modifications and equivalents will be apparent to those skilled in the art and are intended to be included within the scope of the present invention.

EXAMPLE 1: PROCESS OF PREPARING SILVER DOPED ZINC OXIDE NANOPARTICLES USING PLUMBAGO AURICULATA LEAF EXTRACT
A process of preparing silver doped zinc oxide nanoparticles using Plumbago auriculata leaf extract comprises the steps of,
a) mixing of 10 mm zinc acetate and 1 mm of silver nitrate;
b) adding 5 ml of Plumbago auriculata leaf extract in 95 ml of step (a) reaction mixture with constant stirring at 150 rpm for 24 hr;
c) recovering synthesized nanoparticles from step (b) mixture by centrifugation at 10,000 rpm for 20 min followed by washing twice with distilled water.
d) collecting pellets from step (c) mixture and further calcinating at 400° C for 4 h in a muffle furnace to gel final silver doped zinc oxide nanoparticles.

EXAMPLE 2: PHYTOCHEMICAL ANALYSIS
The plants belonging to family of Plumbaginaceae are known to be a potential source of various phytochemicals such as phenols, flavonoids, napthoquinone, alkaloids, and carbohydrates that might play an important role in formation of nanoparticles by redox reaction. Table 1 shows high flavonoid content (960±2.88 µg/mL) and phenolic content (314.3±0.33 µg/mL) in Plumbago auriculata leaf extract (PALE). The Plumbago is known to contain a napthoquinone called plumbagin which showed its presence even in PALE at a concentration of 260 µg/mL. Other phytochemicals such as citric acid, ascorbic acid and starch were also noted to be present in PALE. Hence, the presence of these phytochemicals may help convert reduce zinc acetate to zinc oxide nanoparticles (ZnONPs).
The results are shown below:
Phenolic (µg/mL) Flavonoid (µg/mL)
Reducing sugar (µg/mL) Starch (µg/mL) Citric acid (µg/mL) Ascorbic acid (µg/mL) Plumbagin (µg/mL)
314.3±0.33 960 ± 2.88 121.3± 4.6 150.3± 3.17 109.4±2.36 1.97 ± 1.55 260.4 ± 8.9

EXAMPLE 3: UV-VISIBLE ABSORPTION AND PHOTOLUMINESCENCE SPECTROSCOPY ANALYSES
The UV-Vis absorption spectra of the synthesized nanoparticles were recording as shown in Figure 1A-D. Brown color precipitates were formed during reaction which converted to greyish white after calcination indicating synthesis of ZnONPs and ZnOAgNPs. Figure 1A indicates the UV-Vis absorption spectrum of ZnONPs which showed a broad absorption in the range of 300-490 nm. The main characteristics peak of ZnONPs was observed at 352 nm. The slightly blue shift was observed implying lower particle size of ZnONPs.

On silver doping, the UV-Vis absorption spectrum of the ZnOAg1 revealed two characteristics peaks at 370 nm and 635 nm, respectively. Further, with the increase of the concentration of salts in ZnOAg10, the absorbance peak became prominent at ~560 nm and blue shifted in comparison to that of ZnOAg1 sample. This blue shift may be arise due to the decrease of optical scattering caused by grain growth and the reduction of grain boundary density. The UV-Vis absorbance spectrum of ZnO10Ag1 sample showed only the main characteristics peak of ZnONPs at 371 nm while there is no sharp peak observed for silver except some enhancement of visible absorbance. Here, the peak of ZnONPs is predominant over the peak of silver because during the preparation of the sample, the concentration of the zinc acetate was much higher in compare to that of silver nitrate. It is observed that the relative visible absorbance in the UV-Vis spectra of ZnOAg1, ZnOAg10, and ZnO10Ag1 increased significantly compared to that of pure ZnONPs.

Further, the movement, separation, and recombination of photo-generated electron and hole (e--h+) pairs in the synthesized nanoparticles were measured by recording the room temperature PL emission spectra at 300 nm excitation which are shown in the Figure 2A. The PL intensity of the ZnOAgNPs decreased as compared to that of the ZnONPs which indicated an effective interfacial charge transfer from ZnO to Ag, performing as an electron sink that hinders the recombination of photo-induced carriers. Thus, the ZnOAg nanocomposites are estimated to show improved photocatalytic dye degradation performance than that of the pristine ZnONPs. The Gaussian fitted PL emission spectra of the synthesized samples are shown in Figure 2B-E, respectively. It was observed that ZnONPs show a strong near band edge emission band centred at ~340 nm in all samples. The Gaussian fitted deconvoluting PL emission spectra of the synthesized nanoparticles showed some additional bands at ~405 nm, ~420 nm, ~456 nm and ~470 nm wavelengths, which are characteristic to the singly and doubly charged oxygen vacancy states (Vo+ and Vo++) and defects in ZnONPs.

EXAMPLE 4: HRTEM IMAGE AND DLS ANALYSES
The morphology of the synthesized nanoparticles were determined using HRTEM images, as shown in Figures 3. The HRTEM image of ZnONPs clearly showed two anisotropic nanostructures of hexagonal and spherical (Figure 3A-B) nanoparticles. The size and diameter of the hexagonal and spherical nanoparticles are observed to be 350 nm and 500 nm, respectively. The particle size of ZnONPs is also found by DLS analysis and the corresponding bar diagram is shown in the inset of Figure 3B. The size of ZnONPs is ranging from 289-1635 nm with maximum number of particles of size 407 nm. A nanorod of thickness ~40 nm and length ~350 nm was observed in ZnOAg1NPs sample (Figure 3C). There were some polydispersed spherical nanoparticles of size ~50-200 nm as observed in ZnOAg1NPs (Figure 3D). The particle size distribution of ZnOAg1NPs were in a range from 85 to 409 nm with majority of size ~98.3 nm as seen in the inset of Figure 3D. Similar nanorods were also spotted for ZnOAg10NPs that were ~50 nm × ~10 nm in dimension as evident from Figure 3E. Apart from the nanorods, there were some spherical particles of size ~30 nm as well. The size of the ZnOAg10NPs was ~171 to 1635 nm with majority being around 231 nm as shown in inset of Figure 3F. A similar trend was also observed for ZnO10Ag1NPs where the rod shaped nanoparticles were larger in size (~758 nm) as seen in Figure 3G and 3H. The size of the particles ranged from 72 to 687 nm most of which were around 90 nm.

EXAMPLE 5: Energy Dispersive X-Ray Analysis (EDAX)
The presence of Zn, O, and Ag in the synthesized sample was confirmed by EDAX spectra as shown in Figure 4. The purity of ZnONPs was confirmed by the presence of Zn and O in Figure 4A. Figure 4b indicated the presence of Zn and Ag up to 16.84% and 16.61%, respectively in ZnOAg1NPs. Figure 4c showed 25.32% Zn and 24.68% Ag in ZnOAg10 whereas ZnO10Ag1 exhibited 57.28% Zn and 8.52% Ag as evident from Figure 4d.

EXAMPLE 6: FTIR SPECTROSCOPY ANALYSIS
The PALE comprises different phytochemicals such as phenolic, flavonoid, reducing sugar, starch, citric acid, ascorbic acid, plumbagin, etc. which played an key role in the reduction of Ag+ and Zn2+ ions and stabilized the nanoparticles. The PALE shows several characteristic bands in the FTIR spectra given in Figure 5A-B. The peak at 3550 cm-1 is due to the N–H stretch vibrations of the peptide linkages, and the peak at 2994 cm-1 corresponds to the stretching vibration of methyl groups. The peak at 1395 cm-1 is attributed to germinal methyl group. The peak at 1067 cm-1 is responsible for the bending vibration of C–OH groups and the ant symmetric stretching band of C–O–C groups of polysaccharides and/or chlorophyll. These peaks indicated the presence of different phytochemicals in the PALE extract. The FTIR spectra of the synthesized ZnOAg nanocomposites by PALE are shown in Figure 5b. The peak at 3550 cm-1 is due to the N–H stretch vibrations of the peptide linkages is slightly blue shifted for ZnAg1NPs and ZnAg10NPs while it remains at the same position for Zn10Ag1NPs. The main characteristic peaks of Zn-O vibration at 584 cm-1 was observed for all three samples. Additionally, there are some other peaks observed at 2166, 2042, 1573, 1383, 1203, 1075, and 640 cm-1 which may be originated from the different vibrational bands of phenolic, flavonoid, sugar, starch, citric acid, ascorbic acid, and plumbagin. The FTIR spectra confirmed the involvement of the active functional groups of the phytochemicals present in PALE for the synthesis and stabilization of the nanoparticles.

EXAMPLE 7: IN-VITRO ANTIOXIDANT ACTIVITY
Example 7.1: DPPH radical scavenging activity
The biogenic nanoparticles (600µL) with a concentration of 1 mg/mL were allowed to react with 2400 µL of (100 mM) 2, 2- diphenyl-1-picrylhydrazyl (DPPH) prepared in methanol. The reaction was incubated in the dark for 30 min at room temperature. The absorbance was measured at 517 nm. The scavenging activity was calculated using the given formula:

% DPPH radical scavenging = (A517control -A517test)/A517control * 100

The phytogenically synthesized ZnONPs did not show any scavenging activity, while Ag-doped ZnONPs showed a significant DPPH radical scavenging activity of 51.50 ± 0.16 (ZnOAg10NPs), 49.21 ± 0.17 (ZnO10Ag1NPs), and 32.07 ± 0.24 (ZnOAg1NPs).

Nanoparticles % scavenging
ZnONPs ND
ZnOAg10NPs 51.50 ± 0.16
ZnO10Ag1NPs 49.21 ± 0.17
ZnOAg1NPs 32.07 ± 0.24
Table 1: DPPH radical scavenging activity of biogenic nanoparticles
Note: ND = Not determined, the data is represented as the mean ± SEM; (n=3)

It is reported that many chronic diseases such as diabetes and cancer are caused due to generation of reactive oxygen species (ROS) in the body. The oxidative stress plays an important role in the damage of DNA and proteins of the cell. The oxidative stress can be prevented by various free radical scavengers. The nanoparticles synthesized via biogenic process showed good antioxidant activity which can further contribute to the prevention of oxidative stress. These nanoparticles can play a crucial role in curing inflammation and wound healing caused by diabetes.
,CLAIMS:Claims
We claim;
1. A process of preparing silver doped zinc oxide nanoparticles using Plumbago auriculata leaf extract comprises the steps of,
a) mixing zinc acetate and silver nitrate by varying the concentration;
b) adding 5 ml of Plumbago auriculata leaf extract in 95 ml of step (a) reaction mixture with constant stirring at 150 rpm for 24 hr;
c) recovering synthesized nanoparticles from step (b) mixture by centrifugation at 10,000 rpm for 20 min followed by washing twice with distilled water.
d) collecting pellets from step (c) mixture and further calcinating at 400° C for 4 h in a muffle furnace to gel final silver doped zinc oxide nanoparticles.
2. The process of preparing silver doped zinc oxide nanoparticles using Plumbago auriculata leaf extract as claimed in claim 1, wherein the zinc acetate and silver nitrate were mixed in identical concentration of 1 mM for the synthesis of ZnOAg1NPs.
3. The process of preparing silver doped zinc oxide nanoparticles using Plumbago auriculata leaf extract as claimed in claim 1, wherein the zinc acetate and silver nitrate were mixed in identical concentration of 10 mM for the synthesis of ZnOAg10NPs.
4. The process of preparing silver doped zinc oxide nanoparticles using Plumbago auriculata leaf extract as claimed in claim 1, wherein the 10 mm zinc acetate and 1 mm silver nitrate was mixed for the synthesis of ZnO10Ag1NPs.
5. The process of preparing silver doped zinc oxide nanoparticles using Plumbago auriculata leaf extract as claimed in claim 1, wherein said silver doped zinc oxide nanoparticles is having particle size 10-100 nm.

Dated this 28th Jul, 2023