Description:
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 FOR GREEN SYNTHESIS OF ANTIMICROBIAL MANGANESE FERRITE NANOPARTICLES USING PLANT EXTRACT
2. Applicant(s)
NAME: RK UNIVERSITY
NATIONALITY: INDIAN
ADDRESS: RK University, Bhavnagar Highway, Kasturbadham, Rajkot-360020, Gujarat, 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 FOR GREEN SYNTHESIS OF ANTIMICROBIAL MANGANESE FERRITE NANOPARTICLES USING PLANT EXTRACT

FIELD OF THE INVENTION
The present invention is relates to a process for green synthesis of antimicrobial manganese ferrite nanoparticles using plant extract. The present invention particularly relates to a process for green synthesis of antimicrobial manganese ferrite nanoparticles using either ginger root extract or cardamom seeds extract.

BACKGROUND OF THE INVENTION
Nanotechnology has received wide attention globally owing to the attractive surface properties of the nanoparticles that range between 1-100 nm in size. Nanotechnology plays a key role in the interdisciplinary research area. The physical property, sizes of nanoparticles may increase the performance of any material in the technological aspects. Nanotechnology holds enormous potential for healthcare, from delivering drugs more effectively, diagnosing diseases more rapidly and sensitively, and delivering vaccines via aerosols and patches.

Nanoparticles can be synthesized by various methods such as physical, chemical, and biological approaches. Generally, the physical and chemical methods are considered the best to get uniform-sized nanoparticles with long-term stability. However, these approaches are expensive and release toxic/hazardous materials into the environment. Because several nanoparticles have been widely utilized in medical products, disease diagnosis, and cosmetics, improving the biocompatibility of nanoparticles is highly important.

Green synthesis is an emerging area in the combined fields of biotechnology and nanotechnology and delivers loads of economic and environmental benefits. Green synthesis is a reliable, sustainable, and eco-friendly approach for synthesizing a wide range of materials/nanomaterials including metal/metal oxides nanomaterials, hybrid materials, and bio inspired materials.
In green synthesis, plant leaves, seeds, roots, extracts of the plant, biological wastage, agricultural wastes, etc. can be used. The plant extract is an irresistible preference for the conventional synthesis method. The substance obtained from plant extracts is helpful for the reduction of a metal compound into its corresponding nanoparticles. The biogenic routes are ecological, using harmless and biocompatible reagents, a comparatively consistent and important role to materials with supreme properties. In-plant extract various combinations of inorganic and organic reducing agents are present. The reaction of plant extract and the metal nitrate salt is so easy because of its uniform arrangement and high solubility.

Ferrite is a branch of ferrimagnetic materials. The group of Iron oxide is denoted as ferrites; having a general formula MOFe2O3, where M is divalent metal ion like Mn+2, Fe+2, Co+2, Ni+2, Cu+2, Zn+2, Mg+2 or Cd+2. The demand for soft magnetic materials is increasing daily in advanced technology. Manganese ferrites are soft ferrite materials that show high magnetic permeability. These materials can be used in many applications such as degradation of hazardous dye and antimicrobial activity. There is little research till now on the green synthesis of magnetic spinel ferrite nanoparticles using Aloe vera, Hibiscus rosa-sinensis leaf, sesame seed, lemon and turmeric curcumin, okra, coffee powder, Abrus precatorius extract. There is less to no study on the green synthesis of manganese ferrite (MnFe2O4) nanoparticles using ginger root extract or cardamom seeds extract.

Ginger (Zingiber officinale) and Cardamom (Elettaria cardamomum) are family members of Zingiberaceae. The main chemical components of ginger are phenyl propanoid, zingerone, gingerol, oxalic acid, ascorbic acid. Ginger has biological properties like antimicrobial, antioxidant, anticancer, and a stimulating effect on the immune system. Medicinal properties of ginger are due to the presence of gingerol and paradol, shogaols, etc.
Cardamom contains a wide variety of phenolic compounds including gallic acid, ferulic, isoferulic acid, chlorogenic acid, caffeic acid, luteolin acid, quercetin, rutin, etc. Cardamom seeds have a warm slightly pungent and highly aromatic flavour. Green cardamom has antioxidant, anti-inflammatory, and anti-carcinogenic properties. It would be interesting to explore the potential of ginger and cardamom for the synthesis of ferrite magnetic nanoparticles.

Therefore, the inventors of the present invention have developed green synthesis process for nanoparticle of manganese ferrite using cardamom extract and ginger extract. So, far it has been exploited to achieve the following objective.
OBJECTIVE OF THE INVENTION
The present invention is to provide a process for green synthesis of antimicrobial manganese ferrite nanoparticles using plant extract.

The main objective of the present invention is to provide a process for green synthesis of antimicrobial manganese ferrite nanoparticles using either cardamom extract or ginger extract.

Another objective of the present invention is to provide a process for green synthesis of antimicrobial manganese ferrite nanoparticles using plant extract which is eco-friendly.

Another objective of the present invention is to provide a process for green synthesis of antimicrobial manganese ferrite nanoparticles using plant extract which is spinel type.

Yet another objective of the present invention is to provide a process for green synthesis of antimicrobial manganese ferrite nanoparticles using plant extract which is inexpensive and easy to produce in large-scale synthesis.

One other object of the present invention is to provide a process for green synthesis of antimicrobial manganese ferrite nanoparticles using plant extract which is self-combustion method.

SUMMARY OF THE INVENTION
The main aspect of the present invention is to provide a process for green synthesis of antimicrobial manganese ferrite nanoparticles using plant extract comprises the steps of:
a) Preparing solution of nitrate salt of Mn (II) and Fe (III) in the molar ratio of 1:2;
b) Adding the plant extract dropwise into the solution of step (a) and mixing;
c) Measuring the pH of the solution of step (b);
d) Putting the solution of step (c) in the furnace at 80 °C for 1 hour to form a gel;
e) Putting the gel of step (d) in the furnace at 250 °C for 4 hour to get manganese ferrite powder;
f) Sintering the manganese ferrite powder of step (e) at 600 °C in the furnace for 2 hour to get the final manganese ferrite nanoparticles in air atmosphere.

BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1: Schematic Diagram for Preparation of Cardamom and Ginger extract
Figure 2: Schematic Diagram for Synthesis of Manganese Ferrite nanoparticles (MnC and MnG)
Figure 3: Spot EDAX spectrum of (a) MnG and (b) MnC
Figure 4: Scanning electron micrographs of (a) MnG and (b) MnC
Figure 5: XRD spectrum of (a) MnG and (b) MnC
Figure 6: FTIR spectrum of (a) MnG and (b) MnC
Figure 7: Dye degradation using (a) MnG and (b) MnC
Figure 8: Antimicrobial assay of MnG and MnC
DESCRIPTION OF THE INVENTION
The main embodiment of the present invention is to provide a process for green synthesis of antimicrobial manganese ferrite nanoparticles using cardamom seed extract or ginger root 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.

The term “manganese ferrite” used herein refers to soft magnetic materials with cubic spinel structure and high magnetic permeability which have been extensively used in various technological applications.

The term “spinel” used herein refers to any of a group of hard glassy minerals of variable colour consisting of oxides of aluminium, magnesium, chromium, iron, zinc, or manganese and occurring in the form of octahedral crystals.

The term “spinel ferrite” used herein refers to a complex oxide crystal structure with a face-centered cubic core and a unit formula of AFe2O4. This can be formed from the combination of a trivalent cation (Fe3+) and another divalent metallic cation, such as either a transition or post-transition metallic cation (A = Mn, Mg, Co, Ni, Zn).

The code MnC used herein refers to manganese ferrite nanoparticles obtained using cardamom seed extract.

The code MnG used herein refers to manganese ferrite nanoparticles obtained using ginger root extract.

As per main embodiment a process for green synthesis of antimicrobial manganese ferrite nanoparticles using plant extract comprises the steps of:
a) Preparing solution of nitrate salt of Mn (II) and Fe (III) in the molar ratio of 1:2;
b) Adding the plant extract dropwise into the solution of step (a) and mixing;
c) Measuring the pH of the solution of step (b);
d) Putting the solution of step (c) of pH ~4 in the furnace at 80 °C for 1 hour to form a gel;
e) Putting the gel of step (d) in the furnace at 250 °C for 4 hour to get manganese ferrite powder;
f) Sintering the manganese ferrite powder of step (e) at 600 °C in the furnace for 2 hour to get the final manganese ferrite nanoparticles in air atmosphere.

As per one embodiment, the plant extract used is either cardamom seed extract or ginger root extract. The ginger root and cardamom seeds were collected from local market of Rajkot.

As per one embodiment, use of cardamom seed extract in the process gives MnFe2O4 (Cardamom) nanoparticles (MnC).

As per one embodiment, use of ginger root extract in the process gives MnFe2O4 (ginger) nanoparticles (MnG).

As per one embodiment, the process for preparation of cardamom seeds extract comprises steps of:
a) Grinding cardamom seed in a mortar pestle to get yellow-brown powder;
b) Adding powder from step (a) in distilled water and mixing;
c) Keeping mixture of step (b) for constant stirring;
d) Boiling the mixture of step (c) for 4 hour at 90°C to get uniform cardamom solution;
e) Cooling the mixture of step (d) at room temperature and filtering;
f) Collecting and storing the brown extract of cardamom (pH ~ 5.4) for further use.

As per one embodiment, the process for preparation of ginger root extract comprises steps of:
a) Grinding ginger root in a mortar pestle to get yellow powder;
b) Adding powder from step (a) in distilled water and mixing;
c) Boiling the mixture of step (c) for 5 minutes at 90°C until the colour of the aqueous solution becomes yellow;
d) Cooling the mixture of step (d) at room temperature and filtering;
e) Collecting and storing the yellow extract of ginger (pH ~ 5.4) for further use.

As per one other embodiment, the present invention provides green synthesis process which utilizes plant extract for the preparation of manganese ferrite nanoparticles.

As per one embodiment, the MnFe2O4 nanoparticles (MnC/MnG) were obtained by the self-combustion method in which the chemical components from ginger/cardamom extracts act as heterocycles/reducing agents and as a fuel.

As per one other embodiment, the manganese ferrite nanoparticles obtained by green synthesis process of the present invention are useful for the photocatalytic dye degradation of hazardous dyes in visible light under the influence of various concentrations of H2O2.

As per one other embodiment, the manganese ferrite nanoparticles obtained by green synthesis process of the present invention showed selectivity for antimicrobial activity against two Gram-positive bacteria such as Bacillus subtilis, and Staphylococcus aureus.

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: PREPARATION OF MANGANESE FERRITE NANOPARTICLES USING PLANT EXTRACT
A process for green synthesis of antimicrobial manganese ferrite nanoparticles using plant extract comprises the steps of:
a) 50 ml nitrate salt solution of Mn (II) and Fe (III) in the molar ratio of 1:2 was prepared ;
b) The plant extract was added dropwise into the solution of step (a) and mixed;
c) pH of the solution of step (b) was measured;
d) The solution of step (c) of pH ~4 was kept in the furnace at 80 °C for 1 hour to form a gel;
e) The gel of step (d) was kept in the furnace at 250 °C for 4 hour to get manganese ferrite powder;
f) The manganese ferrite powder of step (e) was sintered at 600 °C in the furnace for 2 hour to get the final manganese ferrite nanoparticles in air atmosphere.

EXAMPLE 2: CHARACTERIZATION OF MANGANESE FERRITE NANOPARTICLES
2.1: ENERGY DISPERSIVE ANALYSIS OF X-RAY (EDAX) ANALYSIS
The Energy Dispersive Analysis of X-ray (EDAX) was used to check the proportion of Mn, Fe, and O in synthesized samples.

RESULT:
The stoichiometric proportion of MnFe2O4 (Ginger) / MnG and MnFe2O4 (cardamom) / MnC are confirmed by EDAX analysis which are shown in Figure 3 (a) and (b) respectively. EDAX analysis showed that the synthesized nanoparticles consist of highest Mn, Fe and O elements and few traces of Sulphur, Chlorine, and Potassium are found in MnG and MnC specimens due to presence of these elements in ginger and cardamom extract.

2.2: SCANNING ELECTRON MICROSCOPY (SEM) FOR MORPHOLOGY AND STRUCTURE ANALYSIS
The internal structure of samples was observed with the help of SEM images.

RESULT:
Figure. 4 (a) and (b) indicates the surface morphology of synthesized nanoparticles MnG and MnC respectively. Toxic gases escaped during the auto combustion process and created voids, pores, and fractured surface in the synthesized specimens.

2.3: X-RAY DIFFRACTION (XRD) ANALYSIS
The lattice parameters, particle size, and crystal structure were obtained by X-ray diffraction (XRD) characterization. The crystallite size was calculated by the Debye-Scherer formula using the highest intense peak (311) of XRD.

RESULT:
The formation of manganese ferrites (Ginger / Cardamom) is confirmed with cubic structure (JCPDS no: 74-2403, space group Fd3m) from the XRD pattern as shown in Figure 5. Few Bragg’s reflections related to unreacted phases of Fe2O3 (JCPDS no: 85-0599) are shown in the figures which is evolved during synthesizing of manganese ferrite via green synthesis route.

The synthesized nano ferrites are calcinated at 600 °C in a furnace for 2 hrs to improve the crystallinity of the same. The lattice parameter is 8.339 A° as well as the average crystallite size are 46.5 nm and 43.5 nm for MnC and MnG specimens, respectively, deduced from the XRD data.

2.4: FOURIER TRANSFORM INFRARED SPECTROSCOPY (FTIR)
The FTIR spectrums of MnG and MnC were recorded in the range of 400 – 800 cm-1 at room temperature. The force constants between metal-oxygen bonds were calculated with the help of FTIR spectroscopy. The octahedral force constants and tetrahedral forces constants of MnG and MnC were calculated using the standard formulae.

RESULT:
The three main metal-oxygen bonds were observed which are shown in Figure. 6. The values of octahedral force constant and tetrahedral forces constant for MnG specimen were 92.26 N/m and 121.79 N/m respectively while 93.05 N/m and 121.81 N/m respectively for MnC specimen.

EXAMPLE 3: DYE DEGRADATION OF METHYLENE BLUE USING FINAL MANGANESE FERRITE NANOPARTICLES (MnC/MnG)

Methylene blue to H2O2 ratio was fixed by 9:1 for the dye degradation study. The different combinations of 20 mg of MnG or MnC with methylene blue and H2O2 solutions were kept under sunlight to carry out the experiments of dye degradation. Also, the same combinations were used in UV-Visible spectrophotometer for the absorbance of light and dye degradation test.

RESULT:
Figure 7 show the plot of absorption of methylene blue dye with H2O2 molar proportion under the influence of UV/visible radiation for the nano ferrite specimens. The study showed that Spinel ferrite materials can be effectively used in photocatalysis in the presence of electromagnetic radiation to generate electron-hole pairs on the nano ferrite’s surface. Due to these electron-hole pairs, the reduction and oxidation process occurs in the solution and finally, it produces radicals OH and O2. These radicals can be utilized for the decomposition process of organic dyes like methylene blue. Furthermore, to enhance the formation of reactive oxygen species, H2O2 oxidants has been added to this reaction mixture.

EXAMPLE 4: ANTIMICROBIAL ACTIVITY OF FINAL MANGANESE FERRITE NANOPARTICLES (MnC/MnG)
The effects of synthesized nanoparticles were tested for antimicrobial activity against two Gram-positive bacteria such as Bacillus subtilis, and Staphylococcus aureus, and two Gram-negative bacteria such as, Escherichia coli, and Pseudomonas aeruginosa, on Mueller Hinton Agar (MHA) plates using well diffusion assay. Overnight grown cultures of the test organism (OD600 = 0.05) were spread plated on to MHA plates and 30 µL of nanoparticles suspension (100 µg/mL) was added in the wells followed by incubation at 37°C for 18 hrs, the zone of inhibition was measured.

RESULTS:
Bacteria Zone diameter (mm)
MnFe2O4 (C) MnFe2O4 (G)
E. coli Nil Nil
P. aeruginosa Nil Nil
B. subtilis 16 20
S. aureus 14 17
Table 1: Antimicrobial activity of MnFe2O4 (C) and MnFe2O4 (G) Nanoparticles
Nil: No zone of inhibition

Synthesized nanoparticles showed selectivity for antimicrobial activity against Gram-positive bacteria while they did not exhibit any zone of inhibition against Gram-negative bacteria. MnG was more potent with a larger zone diameter compared to MnC against both B. subtilis and S. aureus as shown in Figure 8.

, Claims:CLAIMS:
We claim;
1) A process for green synthesis of antimicrobial manganese ferrite nanoparticles using plant extract comprises steps of:
a) Preparing solution of nitrate salt of Mn (II) and Fe (III) in the molar ratio of 1:2;
b) Adding the plant extract dropwise into the solution of step (a) and mixing;
c) Measuring the pH of the solution of step (b);
d) Putting the solution of step (c) of pH ~4 in the furnace at 80 °C for 1 hour to form a gel;
e) Putting the gel of step (d) in the furnace at 250 °C for 4 hour to get manganese ferrite powder;
f) Sintering the manganese ferrite powder of step (e) at 600 °C in the furnace for 2 hour to get the final manganese ferrite nanoparticles, in air atmosphere.
2) The process for green synthesis of antimicrobial manganese ferrite nanoparticles using plant extract as claimed in claim 1, wherein said plant extract used is either cardamom seed extract or ginger root extract.
3) The process for green synthesis of antimicrobial manganese ferrite nanoparticles using plant extract as claimed in claim 1, wherein said cardamom seed extract gives manganese ferrite cardamom nanoparticles (MnC).
4) The process for green synthesis of antimicrobial manganese ferrite nanoparticles using plant extract as claimed in claim 1, wherein said ginger root extract gives manganese ferrite ginger nanoparticles (MnG).
5) The process for green synthesis of antimicrobial manganese ferrite nanoparticles using plant extract as claimed in claim 1, wherein said manganese ferrite cardamom nanoparticles (MnC) is having particle size 40-60 nm.
6) The process for green synthesis of antimicrobial manganese ferrite nanoparticles using plant extract as claimed in claim 1, wherein said manganese ferrite ginger nanoparticles (MnG) is having particle size 30-50 nm.

Dated this 23rd July, 2022