Description:1
FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENTS RULES, 2003 5
[See Section 10, Rule 13]
COMPLETE SPECIFICATION
GREEN BIO-SYNTHESIS OF ANTIBACTERIAL NANOPARTICLES (NPs)-10 BASED EPITHELIUM PROPITIOUS DISINFECTANT
APPLICANT
Griftech Innovation Private Limited
House no-276/2/3, Jail Road, Tehshil Sadar Mandi (T), Mandi, (H. P)
Nationality: India 15
Indian Institute of Technology Mandi,
Kamand - 175005, Himachal Pradesh,
Nationality: India
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The following specification particularly describes the invention and the manner in which it is to be performed.
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FIELD OF INVENTION
The present invention relates to creating an epithelium propitious green disinfectant that effectively combats harmful microorganisms and viruses. More specifically, the present invention relates to formulations that include green bio-synthesized nanoparticles enriched with one or more natural phenol compounds. These 5 formulations are well-suited for disinfecting and cleansing extensive surfaces often found in agricultural, residential, and commercial environments and ecosystems.
BACKGROUND OF THE INVENTION:
Despite contemporary hygiene and infection prevention advancements, health has become a prominent issue. This is partly due to the increase in viral and fungal diseases 10 stemming from infectious diseases that may be spread from person to person, between humans and animals, and across the globe due to increased interconnectedness.
Numerous infectious diseases and ailments that impact both; humans and animals, as well as the contamination of various substances like food, biological specimens, and environmental samples, are caused by various pathogens, including bacteria, fungi, 15 viruses (both living and non-living), and bacterial spores. Moreover, environmental factors play a crucial role in determining whether a host will encounter any of these pathogens. The subsequent interactions between the pathogen and the host will dictate the outcome of this exposure. These interactions occur in a series of stages, including infection, the development of the disease, and either recovery or mortality. In cases of 20 microbial infections in humans or animals, the initial stage typically involves attachment to or colonization of their skin or mucous membranes, followed by the invasion and spread of the infectious bacteria. Pathogenic bacteria commonly enter the body through the skin and mucous membranes.
Over time, the advancement and widespread use of vaccines have greatly contributed 25 to the prevention of a wide range of diseases. However, even vaccinated individuals
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and animals may still face severe threats to their health. Furthermore, since vaccines are not available for all infectious diseases, there has been a growing recognition of the importance of implementing well-designed, safe, and effective disinfection protocols to maintain the ecosystem of overall health and well-being of society.
The concept of developing disinfectants emerged in the 1970s and initially involved 5 the selection of a basic chemical disinfectant, which was then enhanced by adding other chemicals. An approach frequently employed to augment the efficacy of simple chemical-based disinfection is the inclusion of proactive nanoparticles (NPs) containing substances like calcium, zinc, silver, or copper salts, either individually or in conjunction with other nanoparticles such as TiO2 NPs. This strategy is utilized to 10 expand the spectrum of disinfection activity.1,2
Currently, in the market, antimicrobial compositions often contain metal-oxide NPs that are typically produced through chemically driven synthesis methods, which are not naturally occurring. Many of these "synthetic disinfectants" adversely affect the environment and human health. Moreover, issues related to metal-oxide-based NP 15 synthesis, such as agglomeration, stability, and toxicity, have also raised concerns.
To address, the current challenges and mitigate the negative impacts associated with synthetic disinfectants, there is a growing need for the green synthesis technique of metal-oxide NPs. In this context, the biosynthesis of NPs using biological components like plant leaves, plant roots, flowers, bacteria, fungi, yeast, etc. is the ideal method for 20 the development of disinfectants. Among the aforementioned biological components, plants are abundant on the earth’s surface and easily extractable.
This method eliminates the need for expensive equipment and demanding operating conditions like high pressure, high temperature, and hazardous chemicals exposure, which adversely affects human health. Plants contain various phytochemicals and 25 polyphenolic compounds, such as phenol carboxylic acids, suited for green synthesis of various nanoparticles from plant extracts safe for humans and other organisms.
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Nature serves as a vast source of medicinal and other plants. Early humans, driven by instinct, taste, and experience, treated their illnesses using plants. With an abundance of nutrients like minerals, amino acids, vitamins, and water, rhododendron (brass flowers), for instance, finds applications in various medicines. Here, the studies have demonstrated rhododendron flowers' antibacterial, antifungal, and antiviral properties. 5 Green synthesizing approach for biogenic nanoparticles from plant extracts is a straightforward and environmentally friendly technique suitable for large-scale production. Phytochemicals present in plant leafs/flowers and extracts act as capping and reducing agents during NP synthesis. Heterocyclic and water-soluble compounds can effectively stabilize and reduce metal ions. 10
It is worth noting that the Globally Harmonized System (GHS) for classifying and labeling chemicals does not classify TiO2 as a hazardous material. TiO2 is known for its non-toxicity, strong oxidizing power, thermal stability, and enhanced photocatalytic properties. These attributes make TiO2-based materials valuable for disinfection and the purification of air and water etc. TiO2 exists in three different crystalline forms, 15 namely anatase, rutile, and brookite, at different temperatures. Its robust photocatalytic activity and chemical stability endorse TiO2 NPs as highly practical for various disinfection and air and water purification applications.
So far, it has been established that the use of chemically synthesized disinfectants poses risks to human health, well-being, and the environment. To address this issue, the 20 adoption of a green synthesis approach appears to be a promising solution. Recognizing the potential advantages of bio-reduction-based synthesis of NPs, the present invention focuses on producing a novel series of TiO2 NPs using plant-based extract, aiming to provide versatile disinfectants.
Here, the green synthesis approach for TiO2, NP is followed by using flowers extract 25 from rhododendron. This innovative approach to green NPs represents a new platform
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for developing disinfectants that are safe for the environment and gentle on the skin while maintaining high efficacy.
To better elucidate the technological distinctions between the present invention and the closest cited prior art, the following defining characteristics of the present invention are described below: 5
ADVANTAGES:
The present invention encompasses a set of nanoparticles, including TiO2, SnO2, ZnO, Au, Ag, Pd, Cu, and others, which are prepared using natural extract derived from natural herbs likewise rhododendron flowers. These nanoparticles offer significant 10 advantages in the field of medicine due to their harmless and non-toxic compositions. The developed nanoparticles, ranging in size from 6 to 15nm, exhibit a uniform size distribution and are well-dispersed in alcohol- and compatible-based solvents with low viscosity. This characteristic facilitates easy and extensive coverage of large surfaces, ensuring efficient surface sterilization. 15
Furthermore, the green antimicrobial nanoparticles, including TiO2, SnO2, ZnO, Au, Ag, Pd, Cu, and more, presented in this invention can also serve as the foundational material for creating dry nanopowders, such as TiO2, SnO2, ZnO, Au, Ag, Pd, Cu, etc., suited for applications in metallurgy, material science and beauty products. To produce high-quality nanopowder from such mixtures, whether they are aqueous or not, 20 atomization at a high-temperature setting can be employed.
Since the compositions of this invention can be produced in substantial quantities and are derived from relatively affordable and non-hazardous metal salts and flowers extract, a commercial process for their production may be both practical and competitive when compared to traditional dry methods for manufacturing nanopowders 25 of TiO2, SnO2, ZnO, Au, Ag, Pd, Cu, and more. The average particle size of the nanoparticles in the compositions of this invention, intended for producing dry
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nanopowders, typically falls within the range of 6 - 15 nm. This small size and generally monodisperse size distribution provide them with a competitive advantage over nanopowders of TiO2, SnO2, ZnO, Au, Ag, Pd, Cu, and others with larger average sizes and broader size distributions typically produced through dry processes.
OBJECTIVES: 5
To develop a green synthesis route for metal oxide NP-based disinfectant with versatile applicability, suitable for use across a broad spectrum of industrial, clinical, and environmental settings/applications.
To furnish a durable and enduring antimicrobial solution capable of effectively sanitizing contaminated hands and surfaces, thereby providing long-lasting protection. 10
SUMMARY OF INVENTION:
The present invention involves methodologies for producing and utilizing antimicrobial compositions containing stabilized NPs formulation, as hereby TiO2 synthesized using extract of rhododendron flowers. The size distribution of the NPs typically varies from approximately 6 to 15 nm, and this size may vary corresponding 15 on selection of the specific plant extract used during the formulation process.
The present invention primarily focuses on NP-based disinfection formulations incorporating one or more naturally occurring phenolic compounds. Broadly speaking, the present invention revolves around the green bio-reduction-based synthesis of TiO2 NPs, (but not limited hereto, similar line-optimized protocols are also applicable for 20 other above stated NPs formulations) by harnessing extract from rhododendron flowers. Furthermore, it pertains to developing disinfectant formulations based on bio-synthesized TiO2 NPs, enriched with one or more natural-origin phenolic compounds, designed for effectively cleaning and disinfection large area households and commercial surfaces/platforms. 25
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The structural, morphological, phase, particle size, and antibacterial properties of the TiO2 NPs (but not limited hereto, similar line-optimized protocols are also applicable for other above-stated NPs formulations) are influenced by the selection of the specific type of plant extract utilized for the green synthesis of these NPs.
In one embodiment, the present invention encompasses a disinfectant formulation 5 comprising the as following:
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Herein, TiO2 NPs (but not limited hereto, similar line-optimized protocols are applicable for other above-stated NPs formulations also) that are synthesized using extract of rhododendron flowers and incorporate one or more natural-origin phenolic compounds. 10
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A solvent that is gentle on both the skin and the environment, designed for mixing with TiO2 NPs (but not limited hereto, similar line-optimized protocols are also applicable for other above-stated NPs formulations) to create a disinfectant that can be easily spread over large area households and commercials surfaces. 15
In an embodiment, the present invention pertains to disinfectant formulations that possess:
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Antibacterial properties, effectively combating bacterial contamination.
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Antimicrobial properties, provide defense against a wide range of microorganisms. 20
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Antiviral properties, capable of combating viral infections.
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Antifungal properties, targeting and preventing fungal growth and infections.
Furthermore, in a specific embodiment, the present invention involves a disinfectant formulation comprising well-dispersed TiO2 NPs (but not limited hereto, similar line-optimized protocols are also applicable for other above-stated NPs formulations) in 25 isopropanol alcohol.
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As outlined in the present invention, these formulations can be manufactured in large quantities through relatively straightforward processes. The constituents of these compositions are non-hazardous and can be easily removed by washing the surfaces treated with antibacterial TiO2 NPs here (but not limited hereto, similar line-optimized protocols are also applicable for other above-stated NPs formulations). These 5 formulations may also exhibit characteristics such as optical clarity, low viscosity, and the ability to be stored at room temperature for extended periods without requiring special storage conditions.
The NPs described in this invention can find applications in other formulations where the creation of an antimicrobial environment is desired or reducing microbial growth 10 or eliminating odors would be beneficial.
BRIEF DESCRIPTION OF THE FIGURES
For the purposes of the following detailed description, it is to be understood that the invention may assume various alternative variations and step sequences, except where expressly specified to the contrary. Moreover, other than in any operating examples, or 15 where otherwise indicated, all numbers expressing, for example, quantities of ingredients used in the specification are to be understood as being modified in all instances by the term "about". It is noted that, unless otherwise stated, all percentages given in this specification and appended claims refer to percentages by weight of the total composition. 20
Thus, before describing the present invention in detail, it is to be understood that this invention is not limited to particularly exemplified systems or method parameters that may of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to limit the scope of the invention in any manner. 25
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The use of examples anywhere in this specification including examples of any terms discussed herein is illustrative only, and in no way limits the scope and meaning of the invention or of any exemplified term. Likewise, the invention is not limited to various embodiments given in this specification.
Unless otherwise defined, all technical and scientific terms used herein have the same 5 meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the case of conflict, the present document, including definitions will control.
It must be noted that, as used in this specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the content clearly dictates 10 otherwise. Thus, for example, reference to a “polymer” may include two or more such polymers.
The terms “preferred” and “preferably” refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation 15 of one or more preferred embodiments does not imply that other embodiments are not useful and is not intended to exclude other embodiments from the scope of the invention.
As used herein, the terms “comprising” “including,” “having,” “containing,” “involving,” and the like are to be understood to be open-ended, i.e., to mean including 20 but not limited to.
The current invention is best comprehended through the detailed explanation provided in the appended drawings. These drawings illustrate particular elements or embodiments of the invention (but not limited hereto, similar line-optimized protocols are also applicable for other above-stated NPs formulations). It is crucial to emphasize 25 that while the summary of the present disclosure has been presented here, the drawings
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are intended to showcase specific aspects and should not be construed as imposing limitations on the scope of the invention.
The detailed description of drawings to understand the complete process is as follows:
Figure 1: Schematic diagram illustrating the green synthesis process of TiO2 NPs using extract of Rhododendron flowers. Step-1: Preparation of Rhododendron flowers 5 extract; Step-2: Titanium isopropoxide solution preparation; Step-3: formation of TiO2 NPs-based precursor solution; Step-4: calcination; Step-5: TiO2 NPs and extract of Rhododendron flowers-based disinfectant (final product). (but not limited hereto, similar line-optimized protocols are also applicable for other above-stated NPs formulations). 10
Figure 2: (a) Field emission electron microscopy (FESEM) images and (b) X-ray differaction (XRD) patterns patterns of TiO2 NPs synthesized using extract of Rhododendron flowers.
Figure 3: Absorbance for the different concentration of TiO2 NPs in Isopropanol Alcohol (IPA). 15
Figure 4: (a-b) Antibacterial inhibition effect of green synthesized TiO2 NPs on bacterial species, and provide in-depth analysis of the inhibition of bacterial growth concerning Escherichia coli, Staphylococcus aureus, and Shigella with and without green synthesized TiO2 NPs (but not limited hereto, similar line-optimized protocols are also applicable for other above-stated NPs formulations). 20
Figure 5. Camera images of the bottles under ultraviolet light exposure containing (a) IPA and TiO2 nanoparticles immersed in IPA (25 µgm/ml). (b) FESEM images of the TiO2 nanoparticles filter with 0.2µm filter. FESEM images for Staphylococcus aureus (c) untreated and (d) treated with synthesized TiO2 NPs.
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Figure 6: (a) Agar diffusion test against non-commensal bacteria “Staphylococcus aureus” showing the antimicrobial effect of samples alone or in combination with TiO2. Staphylococcus aureus was incubated with different concentration of TiO2 particles and Rhododendron flowers Extract. Toxicity behavior of (b) TiO2 alone at different concentrations 2.5mg, 5mg, and 10mg, (c) Rhododendron flowers extract 5 alone (d) TiO2 with Rhododendron flowers extract and growth in control sample for an optimum concentration against Staphylococcus aureus (but not limited hereto, similar line-optimized protocols are also applicable for other above-stated NPs formulations).
Figure 7: (a) Agar diffusion test against commensal bacteria “Staphylococcus epidermidis” showing the antimicrobial effect of sample alone or in combination with 10 TiO2. Staphylococcus epidermidis was incubated with different concentrations of TiO2 particles and Rhododendron flowers Extract. (b) Toxicity behavior of TiO2 at different concentrations from 2.5mg, 5mg, and 10mg (c) only Rhododendron flowers extract (d) TiO2 with Rhododendron flowers extract and growth in the control sample for an optimum concentration TiO2 against Staphylococcus Epidermidis. (but not limited 15 hereto, similar line-optimized protocols are also applicable for other above-stated NPs formulations).
Figure 8: HaCaT cells (cells of Skin keratinocytes) viability with TiO2 and different concentrations of TiO2 with Rhododendron flowers extract. 20
BRIEF DESCRIPTION OF THE INVENTION:
The present invention encompasses green formulation of TiO2 NPs using natural extract from Rhododendron flowers (but not limited hereto, similar line-optimized protocols are also applicable for other above-stated NPs formulations) and outlines procedures for their preparation and use. These NPs are typically uniform in size and 25 exhibit most probably a spherical shape. The formulations of the current invention are created using Rhododendron flowers extract that are not hazardous to environment or
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human skin/organs. The compositions containing TiO2 NPs (but not limited hereto, similar line-optimized protocols are also applicable for other above-stated NPs formulations) in this invention are water and alcohol-based and are produced through a wet technique. This method ensures that the TiO2 NPs (but not limited hereto, similar line-optimized protocols are also applicable for other above-stated NPs formulations) 5 remain well-dispersed in the solution, which is in contrast to dry nanopowders produced through methods like thermal evaporation and other vacuum-based processes. Unlike dry powders, TiO2 NPs (but not limited hereto, similar line-optimized protocols are also applicable for other above-stated NPs formulations) in these compositions do not present a dust hazard, even in waste materials. Dry powders 10 can potentially pose health risks, and the extent of exposure to them is not well understood.
A composition according to the present invention includes TiO2 NPs with average size of 6nm to 15nm, which are synthesized from extract of Rhododendron flowers. These NPs typically exhibit relatively narrow particle size distributions, as depicted in Figure 15 2a. It is noteworthy that X-ray differaction (XRD) patterns patterns also confirm the successful synthesis of TiO2 NPs, as shown in Figure 2b. (but not limited hereto, similar line-optimized protocols are also applicable for other above-stated NPs formulations).
The primary embodiment of the current invention is further elucidated below, with a focus on the incorporation of antimicrobial TiO2 (but not limited hereto, similar line-20 optimized protocols are also applicable for other above-stated NPs formulations) in the disinfectant formulation, aiming to provide a solution to the issues associated with chemically driven NPs synthesis.
In an embodiment of the present invention, the present invention discloses a process for the green synthesis of TiO2 NPs using extract of Rhododendron flowers (available 25 in Himalayan region). The process includes providing flowers from plant sources; cleaning the flowers to remove contaminants and dust; placing the cleaned flowers in
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separate containers; adding deionized water to the containers at a weight/volume ratio of 1:5; heating the mixtures to a temperature of 90°C and maintaining this temperature for a duration of two hours; allowing the solutions to cool, followed by filtration to obtain clear and clean plant extract; combining the obtained plant extract with a solution of titanium isopropoxide in isopropyl alcohol; monitoring and adjusting the 5 pH of the mixture to 7 using a microprocessor-based pH meter; formation of the TiO2 nanoparticles by stirring the solution for a duration of four hours; drying the solution at 100°C and performing a calcination process in an open-air oven at 500°C; preparing a final disinfectant formulation by mixing the synthesized TiO2 nanoparticles with isopropanol alcohol and Rhododendron flowers extract; stirring the mixture overnight 10 to obtain a stable and effective disinfectant.
In another embodiment of the present invention, the size of the nanoparticles ranging from 6-15nm.
In another embodiment of the present invention, the extract is selected from Rhododendron flowers. 15
In an embodiment of the present invention, the present invention discloses successful synthesis of well-dispersed and polydispersed NPs (e.g., TiO2, SnO2, ZnO, Au, Ag, Pd, Cu, etc.) using an eco-friendly method that utilizes medicinal herb extract from Rhododendron flowers as reducing and capping agents.
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In another embodiment of the present invention, the characterization of the synthesized nanoparticles (e.g., TiO2, SnO2, ZnO, Au, Ag, Pd, Cu, etc.) with particle sizes ranging from 6 to 15 nm, providing insights into sustainable synthesis using herb extract.
In another embodiment of the present invention, development of an effective 25 disinfectant by combining dispersed TiO2 NPs and herbal Rhododendron flowers
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extract, demonstrating high efficacy against both commensal and non-commensal bacteria.
In another embodiment of the present invention, analysis of HaCaT cell viability using the MTT assay, confirming that the disinfectant is safe for epithelial cells and does not 5 harm Keratinocytes skin cells.
In another embodiment of the present invention, metal oxide NPs-based disinfectant exhibits potent antibacterial properties against non-commensal bacteria (Staphylococcus aureus) while remaining non-toxic to commensal bacteria 10 (Staphylococcus epidermidis).
In another embodiment of the present invention, nanoparticles (TiO2, SnO2, ZnO, Au, Ag, Pd, Cu, etc.) synthesized through an eco-friendly method demonstrate effectiveness against a wide range of bacterial strains, including gram-positive and 15 gram-negative species such as Escherichia coli, Staphylococcus aureus, and Shigella.
In an embodiment of the present invention, the present invention discloses a process for the green synthesis of titanium dioxide (TiO2) nanoparticles. The present invention comsprises: 20
Preparation of Plant Extract
In the synthesis process, flowers from Rhododendron were carefully cleaned to eliminate any dust or contaminants. These clean flowers were then placed in separate containers. Deionized water was added to the containers at a ratio of 1:5 (weight/volume), and the mixtures were heated to a temperature of 90 °C. This heating 25 was maintained for a duration of two hours.
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Subsequently, after allowing the solution to cool, it was filtered to remove any particulate matter, ensuring a clear and clean solution. This solution was then stored for future use in the synthesis process, particularly in the creation of the desired TiO2 nanoparticles (but not limited hereto, similar line-optimized protocols are also applicable for other above-stated NPs formulations). 5
Green Synthesis of TiO2 Nanoparticles
Green synthesis of TiO2 nanoparticles was performed by taking titanium isopropoxide and Rhododendron flowers extract solutions (Figure 1). The 20 ml of flowers extract solution was added dropwise into 0.1 M Titanium Isopropoxide ethanol solution of 100ml. The pH of the mixture was carefully monitored and adjusted to 7 using a 10 microprocessor-based pH meter. The flowers extract and titanium isopropoxide solution were allowed to stir for a duration of 4 hours. During this time, the formation of nanoparticles was observed. (but not limited hereto, similar line-optimized protocols are also applicable for other above-stated NPs formulations).
Subsequently, the solution was dried at 100°C, followed by a calcination process 15 carried out in an open-air oven at 500°C. The final disinfectant formulation was prepared by mixing the TiO2 nanoparticles (but not limited hereto, similar line-optimized protocols are also applicable for other above-stated NPs formulations) with commonly used isopropanol alcohol and stirring the mixture overnight.
The resulting TiO2 NPs exhibited different average particle size approximately 20 approximately 6-15 nm for nanoparticles synthesized with Rhododendron flowers extract. (but not limited hereto, similar line-optimized protocols are also applicable for other above-stated NPs formulations).
In the XRD (X-ray diffraction) patterns displayed in Figure 2 b a prominent peak associated with TiO2 was more pronounced for nanoparticles synthesized using 25 rhododendron flowers extract.
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In the present invention, the presence of TiO2 NPs (but not limited hereto, similar line-optimized protocols are also applicable for other above-stated NPs formulations) was verified through ultraviolet (UV) spectroscopy at a wavelength of 365 nm, as shown in Figure 3. This spectroscopic analysis provided conclusive evidence regarding the successful synthesis of the nanoparticles, as the specific absorption pattern at this 5 wavelength is indicative of TiO2 nanoparticles. This step in the analysis serves as confirmation of the formation of the nanoparticles, further validating the outcomes of the synthesis process.
In another embodiment of the present invention, the invention discloses a process for a disinfectant formulation comprising titanium dioxide (TiO2) nanoparticles, the 10 process includes providing one or more flowers from one or more plant sources. Cleaning the one or more flowers to re ove contaminants and dust. Placing the cleaned one or more flowers in separate containers. Adding deionized water to the separate containers at a weight/volume ratio of 1:5. Heating mixture of the one or more flowers and deionized water to a temperature 15 of 90°C and maintaining the temperature for a duration of two hours. Allowing the mixture of the one or more flowers and deionized water to cool, followed by filtration to obtain clear and clean flower extract. Preparing isopropoxide ethanol solution by mixing titatnium isopropoxide in isopropyl alcohol. Adding the 20 ml of the flower extract dropwise into 0.1 M Titanium Isopropoxide ethanol solution of 100ml. 20 Monitoring and adjusting the pH of the mixture of the flower extract and the Titanium Isopropoxide ethanol solution to 7 using a microprocessor-based pH meter. Formation of the TiO2 nanoparticles by stirring the mixture of the flower extract and the Titanium Isopropoxide ethanol solution for a duration of four hours. Drying the mixture of the flower extract and the Titanium Isopropoxide ethanol solution at 100°C and performing 25 a calcination process in an open-air oven at 500°C. Stirring mixture of the synthesized titanium dioxide TiO2 nanoparticles with isopropanol alcohol and extracts overnight to obtain a stable and effective disinfectant. The process is green process. The size of the
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nanoparticles ranging from 6-15nm. The flower extract influences the properties of the nanoparticles, including size, crystallinity, and antimicrobial efficacy, thereby tailoring the nanoparticles for specific applications. The flower is selected from Aloe barbadensis, Rhododendron, and Murraya koenigii. The disinfectant formulation of the present invention being non-hazardous and easily removable by washing, exhibiting 5 characteristics such as optical clarity, low viscosity, and the ability to be stored at room temperature for extended periods without special storage conditions. The nanoparticles are selected but not limited to tin dioxide (SnO2), zinc oxide ZnO, Au, Ag, Pd, and Cu.
In another embodiment of the present invention, the process for a disinfectant formulation comprising titanium dioxide (TiO2) nanoparticles is made but not limited 10 to plant leaves extract and plant roots extract.
In another embodiment of the present invention, the flower extract comprises phytochemicals such as phenol carboxylic acids, for green synthesis of nanoparticles.
MATERIALS AND METHOD
General Antimicrobial Analysis 15
In accordance with yet another desirable embodiment, the assessment of the antimicrobial properties of the present invention was conducted as follows:
The Minimum Inhibitory Concentration (MIC) values for TiO2 synthesized using Rhododendron was determined through the conventional broth microdilution test. During this evaluation, three different concentrations - 10 mg, 5 mg, and 2.5 mg were 20 tested, as outlined in Figure 4. Throughout the entire testing process, continuous observation was rigorously maintained, and close attention was paid to discern the presence or absence of an antibacterial zone. The establishment of an antibacterial zone was indicated by the emergence of a clear zone around the designated pinhole, indicating an absence of bacterial growth. The diameter of this clear zone directly 25 reflected the antimicrobial efficacy of the tested samples. It is important to highlight
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that all tested concentrations effectively demonstrated inhibition of microbial activity, a significant deviation from the growth observed in the control group. These outcomes are visually depicted in Figure 4a & 4b, showcasing the distinct ability of the tested sample to curtail microbial growth in comparison to the control. (but not limited hereto, similar line-optimized protocols are also applicable for other above-stated NPs 5 formulations).
In the context of assessing in vitro antimicrobial activity, the recorded inhibitory diameters resulting from the use of TiO2 nanoparticles (but not limited hereto, similar line-optimized protocols are also applicable for other above-stated NPs formulations) in combination with Rhododendron flowers extract or individually were evaluated 10 against the bacterial strains listed in Table 1. It is noteworthy that the combined application of TiO2 (but not limited hereto, similar line-optimized protocols are also applicable for other above-stated NPs formulations) and Rhododendron flowers extract demonstrated effective inhibitory effects, with the exception of Staphylococcus Epidermidis, where this inhibitory effect was not observed. Among the tested bacterial 15 strains, the highest degree of inhibition was observed in the case of E. Coli, followed by Staphylococcus Aureus, and Shigella. These findings underscore the substantial inhibitory potential of the combination of TiO2 and extract, particularly in suppressing the growth of these specific bacterial strains. This indicates a promising avenue for the development of antimicrobial agents and highlights the potential utility of these 20 materials in combating bacterial infections.
Table 1. The inhibitory zone diameter resulting on the use of the TiO2 and Rhododendron flower extract alone or in combination against the bacterial strains.
Samples
Shigella
S. Aureus
E. Coli
S. Epi
TiO2
129.03 ± 0.85
106.23 ± 0.11
111.23 ± 0.98
10.14 ± 0.12
Rhododendron Flower Extract
101.24 ± 0.14
110.87 ± 0.89
92.16 ± 1.65
11.89 ± 0.33
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TiO2 + Rhododendron Flower Extract
109.25 ± 0.99
112.22 ± 0.14
125.11 ± 0.14
9.15 ± 0.63
The distinction in microbial surface morphology between untreated microbes and those exposed to green-synthesized TiO2 NPs (but not limited hereto, similar line-optimized protocols are also applicable for other above-stated NPs formulations) is vividly depicted in the FESEM images, as illustrated in Figure 5 (c, d). Untreated microbes 5 exhibited their normal, local morphology. However, after their treatment with green-synthesized TiO2 nanomaterials, we observed damage to the cell membrane, which could lead to the leakage of intracellular content, cell death, or lysis. In this case, the behavior of the microorganisms exhibited signs of stress, suggesting potential susceptibility to degradation due to the impact of the nanoparticles. These visual 10 insights captured by FESEM images provide valuable evidence of the antimicrobial effects of green-synthesized TiO2 nanoparticles (but not limited hereto, similar line-optimized protocols are also applicable for other above-stated NPs formulations) on bacterial species.
When analyzing the antimicrobial activity of the present invention against non-15 commensal bacteria, the dose-dependent assay revealed toxic effects at a higher concentration of 10 mg/ml compared to the concentrations of 5 mg/ml and 2.5 mg/ml. The agar diffusion test conducted to demonstrate the antimicrobial efficacy of the TiO2 sample, (but not limited hereto, similar line-optimized protocols are also applicable for other above-stated NPs formulations) either individually or in combination with 20 extract, is illustrated in Figure 6a.
For TiO2 alone, a reduction in bacterial growth was noticeable at the 10 mg/ml concentration after 4 hours of incubation, and this trend persisted until the 24-hour mark (Figure 6b). However, no such effect was evident at the 5 mg/ml and 2.5 mg/ml concentrations. Rhododendron extract demonstrated toxicity at the 10 mg/ml 25
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concentration after 4 hours of incubation (Figure 6c). (but not limited hereto, similar line-optimized protocols are also applicable for other above-stated NPs formulations).
The combination of TiO2 with Rhododendrone extract did not exhibit the same level of toxicity as TiO2 alone (Figure 6d). These observations highlight the complex interactions between different compounds and bacterial strains, contributing to varying 5 degrees of toxicity and antimicrobial effects. (but not limited hereto, similar line-optimized protocols are also applicable for other above-stated NPs formulations).
The comprehensive results indicated that the combination of extract with TiO2 demonstrated toxicity against non-commensal bacteria. Notably, the combination of TiO2 with Rhododendron flowers extract exhibited the substantial effect. Upon 10 comparing all it was evident that combination displayed a good level of toxicity towards Staphylococcus aureus (Figure 6d). (but not limited hereto, similar line-optimized protocols are also applicable for other above-stated NPs formulations).
Testing of TiO2 alone and in combination against Staphylococcus epidermidis was carried out using similar concentrations and combination as those used for 15 Staphylococcus aureus, which is a commensal microbe. In this case, no toxic effects were observed in any of the tests. However, both TiO2 and the extract exhibited the ability to hinder the growth of this bacteria. The antimicrobial effects of samples tested individually or in combination are illustrated in Figure 7(b-d). The most significant inhibitory effect was noted with concentrations of 10mg/ml and 5mg/ml of TiO2 and 20 Rhododendron extract when tested alone (Figure 7b & 7c). The combination exhibited an equally potent ability as TiO2 in inhibiting bacterial growth, which is evident in Figure 7d. (but not limited hereto, similar line-optimized protocols are also applicable for other above-stated NPs formulations).
The overall data revealed that extract in combination with TiO2 are not toxic towards 25 Staphylococcus epidermidis (a commensal microbe). However, the effective growth of Staphylococcus epidermidis is not comparable to the control microbe alone (bacteria
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only without any nanoparticles or extract), showing inhibition of its growth but not killing. Upon comparing these tested combination against Staphylococcus epidermidis, no significant differences were observed in their effectiveness in inhibiting bacterial growth (Figure 7d). (but not limited hereto, similar line-optimized protocols are also applicable for other above-stated NPs formulations). 5
General Toxicity Analysis
In addition to the antimicrobial analysis, the present invention conducted tests for toxicity against human skin cells, specifically the HaCaT cells, as follows:
HaCaT cell viability analysis was performed using the MTT assay. None of the tested TiO2 nanoparticles and Sample showed any toxicity toward HaCaT cells. The 10 combination of TiO2 (but not limited hereto, similar line-optimized protocols are also applicable for other above-stated NPs formulations) and extract also exhibited no toxicity. No toxic effect was observed at any concentration and with any of the tested combination (Figure 8). This result demonstrates that the tested combination are not harmful to skin cells, specifically Keratinocytes. 15
These findings provide valuable assurance regarding the safety of the tested compositions when it comes to their potential use on human skin, indicating that they do not pose any toxicity risks to skin cells. , C , C , Claims:22
I/We Claims:
1. A process for disinfectant formulation comprising titanium dioxide (TiO2) nanoparticles, the process comprising the steps of:
providing one or more flowers from one or more plant sources;
cleaning the one or more flowers to remove contaminants and dust; 5
placing the cleaned one or more flowers in separate containers;
adding deionized water to the separate containers at a weight/volume ratio of 1:5;
heating mixture of the one or more flowers and deionized water to a temperature of 90°C and maintaining the temperature for a duration of two hours; 10
allowing the mixture of the one or more flowers and deionized water to cool, followed by filtration to obtain clear and clean flower extract;
preparing isopropoxide ethanol solution by mixing titatnium isopropoxide in isopropyl alcohol;
adding the 20 ml of the flower extract dropwise into 0.1 M Titanium 15 Isopropoxide ethanol solution of 100ml;
monitoring and adjusting the pH of the mixture of the flower extract and the Titanium Isopropoxide ethanol solution to 7 using a microprocessor-based pH meter;
formation of the TiO2 nanoparticles by stirring the mixture of the flower extract 20 and the Titanium Isopropoxide ethanol solution for a duration of four hours;
drying the mixture of the flower extract and the Titanium Isopropoxide solution at 100°C and performing a calcination process in an open-air oven at 500°C; and
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stirring mixture of the synthesized titanium dioxide TiO2 nanoparticles with isopropanol alcohol and extracts overnight to obtain a stable and effective disinfectant,
wherein, the process is green process.
2. The process as claimed in claim 1, wherein the size of the nanoparticles ranging from 5 6 - 15nm.
3. The process as claimed in claim 1, wherein the flower extract influences the properties of the nanoparticles, including size, crystallinity, and antimicrobial efficacy, thereby tailoring the nanoparticles for specific applications.
4. The process as claimed in claim 1, wherein the flower/leave can be selected from 10 Aloe barbadensis, Rhododendron, and Murraya koenigii.
5. The process as claimed in claim 1, wherein, the disinfectant formulation of the present invention being non-hazardous and easily removable by washing, exhibiting characteristics such as optical clarity, low viscosity, and the ability to be stored at room temperature for extended periods without special storage conditions. 15
6. The process as claimed in claim 1, wherein the nanoparticles are selected but not limited to tin dioxide (SnO2), zinc oxide (ZnO), Au, Ag, Pd, and Cu.
7. The process as claimed in claim 1, wherein process for a disinfectant formulation comprising titanium dioxide (TiO2) nanoparticles is comprising but not limited to plant leaves extract and plant roots extract. 20
8. The process as claimed in claim 1, wherein flower extract comprises phytochemicals such as phenol carboxylic acids, for green synthesis of nanoparticles.
Dated this 27th day of November 2023
Signature
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-Digitally Signed-
Santosh Vikram Singh
IN/PA: 414
Agent For the Applicant