Description:FIELD OF INVENTION:
[001] The present invention relates to curcumin loaded c-lobe nanoparticles. Particularly, the present invention relates to a process for the preparation of curcumin loaded c-lobe nanoparticles.

BACKGROUND OF THE INVENTION:
[002] In Asia, Curcuma longa has been identified as a significant medicinal herb due to the presence of the hydrophobic bioactive component curcumin (diferuloylmethane) in its rhizome. This polyphenolic compound has been chosen to combine with LF C-lobe (cLF) since it has received a great deal of interest off lately due to the variety of biological and pharmacological actions like antitumor, antidiabetic, antimicrobial, wound healing and antioxidant nature along with additional activities like antirheumatic and anti-Alzheimer, which may add to synergism. However, despite its alleged advantages, curcumin's useful applications are restricted by its poor bioavailability, poor pharmacokinetics, and low target efficacy, which are currently being tackled by generating its nano-formulations. Even in low quantities, curcumin is sufficient for its biological activity and can be used as a supplement in multiple target therapy in addition to other drugs to increase the effectiveness of the therapy. Nanocarriers are also intended to improve the encapsulated medications' bioavailability and pharmacokinetic characteristics as they can precisely control the release of drugs by encasing them. Although several synthetic polymers are used to create nanocarriers, they could have harmful side effects. As an alternative, LF being biocompatible and non-immunogenic can act as a good nanocarrier. This red protein-based nanoparticles have been reported to be potentially anti-inflammatory. Numerous curcumin carriers, like, liposomes, nanoparticles (NPs), ultrasound microbubbles, etc have been created as a drug-delivery systems to date. There have been recent reports of noteworthy antibacterial activity of curcumin nanocarriers in combination with other antibiotics, against multidrug resistant ESKAPE pathogens. On similar leads, we have prepared novel curcumin based bLF’s C-lobe nanoparticles which have inhibitory potential towards ESKAPE pathogens specifically, A. baumannii.
[003] World is moving toward safer options to combat various illness. These curcumin loaded c LF NPs makes a good candidate for a safer drug option for various illness, though much more studies are yet to be done so its full potential can be documented. We are presenting its antibiotic effectiveness against ESKAPE pathogen. The idea of combining a smaller functional part of natural immune modulator and a herbal medicine with the new day technology of nano-formulation can act as double edge sword in combating serious illness. WHO has determined 3mg/kg of body weight as an acceptable daily intake of curcumin. But due to its low absorption in gut, rapid metabolism and elimination its effect is not ideal. Nano-formulations combined with c-LF can increase curcumin’s duration and availability in body so that its full effect can be observed.

OBJECTIVE OF THE INVENTION:
[004] An objective of the present invention is to provide curcumin loaded c-lobe nanoparticle from bovine lactoferrin.
[005] An objective of the present invention is to provide a process for curcumin loaded c-lobe nanoparticle from bovine lactoferrin.

SUMMARY OF THE INVENTION:
[006] Accordingly, the present invention provides a process for the synthesis of curcumin loaded c-lobe nano-particles, wherein the process comprises the steps of hydrolysing bovine Lactoferrin with proteinase K and purifying c-lobe of the bLF; suspending c-lobe and curcumin in sodium acetate buffer and mixing; resuspending in olive oil, stabilising and capping agent, and sonicating for 15 cycles of 30 seconds sonication and 1-minute pause; and extracting the c-lobe nano-particles formed and washing.
[007] In an embodiment, the present invention provides that the sonication is performed using centrifugation at 6000 rpm for 10 minutes.
[008] In an embodiment, the present invention provides that the washing of the pellet is performed with 700 µl diethyl ether and sodium acetate buffer thrice individually.
[009] In an embodiment, the present invention provides that the purifying c-lobe of the bLF using ion-exchange chromatography CMC column with 50mM Tris pH 8.
[0010] In an embodiment, the present invention provides that the sonicating is performed for 15 cycles of 30 seconds sonication and 1-minute pause.
[0011] In an embodiment, the present invention provides that the suspending c-lobe and curcumin are mixed together and incubated for 30 minutes on ice.
[0012] In an embodiment, the present invention provides a c-lobe nanoparticle obtained from bovine lactoferrin according the process disclosed above.

BRIEF DESCRIPTION OF DRAWINGS:
[0013] These and other features, aspects, and advantages of the present invention will become better understood when the following detailed description is read concerning the accompanying drawings in which like characters represent like parts throughout the drawings, wherein:
Fig. 1: SDS-PAGE gel: Image showing bands of fraction of bLF C-lobe at 40KD molecular weight marker;
Fig. 2: TEM Image: Image showing c-lobe nanoparticle of size (a) 7.82 and 7.25 nm (b) 7.59 and 7.65 nm; and
Fig. 3: Antibiotic sensitivity assay.
[0014] Further, skilled artisans will appreciate that elements in the drawings are illustrated for simplicity and may not have been necessarily been drawn to scale. Furthermore, the drawings may show only those specific details that are pertinent to understanding the embodiments of the present invention so as not to obscure the drawings with details that will be readily apparent to those of ordinary skill in the art having the benefit of the description herein.

DETAILED DESCRIPTION:
[0015] To promote an understanding of the principles of the invention, reference will now be made to the embodiment illustrated in the drawings and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended, such alterations and further modifications in the illustrated system, and such further applications of the principles of the invention as illustrated therein being contemplated as would normally occur to one skilled in the art to which the invention relates.

[0016] It will be understood by those skilled in the art that the foregoing general description and the following detailed description are exemplary and explanatory of the invention and are not intended to be restrictive thereof.

[0017] Reference throughout this specification to “an aspect”, “another aspect” or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrase “in an embodiment”, “in another embodiment”, and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment.

[0018] The terms "comprises", "comprising", or any other variations thereof, are intended to cover a non-exclusive inclusion, such that a process or method that comprises a list of steps does not include only those steps but may include other steps not expressly listed or inherent to such process or method. Similarly, one or more devices or sub-systems or elements or structures or components proceeded by "comprises...a" does not, without more constraints, preclude the existence of other devices or other sub-systems or other elements or other structures or other components or additional devices or additional sub-systems or additional elements or additional structures or additional components.

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The system, methods, and examples provided herein are illustrative only and not intended to be limiting.
[0020] Embodiments of the present invention will be described below in detail concerning the accompanying drawings.
The present invention discloses a curcumin loaded c-lobe nanoparticle from bovine lactoferrin and a process for the preparation thereof.
Synthesis procedure:
[0021] Bovine Lactoferrin is hydrolysed using Proteinase K and c-lobe is purified using ion-exchange chromatography CMC column. Protein is eluted using NaCl linear gradient and peak is obtained between 0.2-0.3M fraction. The fraction showing peak is collected and concentrated as c-lobe of bLF, which is confirmed using SDS-PAGE showing a band at 40 KD.
[0022] Nanoparticles of curcumin-cLF are produced using the sol-oil method, 10mg of c-lobe is suspended in 500 µl and 3mg curcumin (Sigma Aldrich, USA) in 100 µl of sodium acetate buffer pH4. Both are mixed together and incubated for 30 minutes on ice. It is resuspended in 20ml of edible olive oil, a stabilising and capping agent, and sonicated for 15 cycles of 30 seconds sonication and 1-minute pause. After which, it is flash frozen and stored at -20 ℃ until further processing.
[0023] The nanoparticles formed after sonication is extracted using centrifugation at 6000 rpm for 10 minutes and washing the pellet with 700 µl diethyl ether and sodium acetate buffer (pH 4) thrice individually. The obtained pellet is resuspended in 100µl buffer and stored at -20 ℃.
[0024] 40 KD molecular weight reddish-brown coloured c-lobe of lactoferrin is isolated and determined using SDS-PAGE. Nanoparticle formation of curcumin loaded cLF is ensured using transmission electron microscopy, this showed nanoparticles of size ranging 7 to 15 nm. The cLF NPs are studied for its antimicrobial activity against 4 bacterial strain, namely E. coli, E. faecalis, S. aureus and A. baumannii. Minimum inhibitory concentration against these bacteria’s are obtained via broth microdilution method. For E. coli, S. aureus MIC of 0.5mg is obtained, whereas for E .faecalis and A. baumannii MIC is seen at the 1 mg. Further, AST done using Kirby-Bauer disk diffusion method against E. coli and A. baumannii showed the zone of inhibition at 19mm and 10 mm respectively.
[0025] The following examples define the invention by way of illustration which does not limit the scope of the invention.
Example 1:
1. Validation of nanoparticle using TEM:
[0026] Preparation of grid using negative staining procedure: add 1-2 drop of nanoparticle solution on the carbon grid and incubate at room temperature for 2-3 minutes and stain the sample with an electron dense stain and let it stand for 10-40 seconds. Dry the grid and visualised using transmission electron microscope Morgagni 268D (Fei Electron Optics).

[0027] Determination of antimicrobial activity: Bacterial cultures (ATCC, Manassas, VA, United States) of E. coli, E .faecalis, S. aureus and A. baumannii were grown in MH broth overnight. Minimum inhibitory concentration of c-lobe nanoparticle is determined on above mentioned bacterial spp. according to CLSI. Procedure for Optimizing Disk Contents (Potencies) for Disk Diffusion Testing of Antimicrobial Agents Using Harmonized CLSI and EUCAST Criteria, 1st Edition CLSI guideline M23S. Wayne, PA: Clinical and Laboratory Standards Institute; 2020. In 96 well plate overnight grown cultures of 0.5 macfarlands OD were incubated in the presence of c-lobe nanoparticle. 30 µl of resazurin dye is added in each well for determination of bacterial viability.

[0028] Antibiotic sensitivity test: AST is done using disk diffusion method, CLSI, M23S 1st edition, Wayne 2020. Antibacterial disk is prepared by making 1mm diameter disc of Whatman paper no. 1 which is soaked with 10 µl of curcumin c-LF at its MIC concentration specific to bacterial sp. determined above. The disk is air dried for 15 minutes in aseptic condition and placed on bacterial lawn. After incubation at 37℃ overnight the diameter of inhibition zone is measured.
, Claims:We Claim:
1. A process for the synthesis of curcumin loaded c-lobe nano-particles, wherein the process comprises the steps of:
a) hydrolysing bovine Lactoferrin with proteinase K and purifying c-lobe of the bLF;
b) suspending c-lobe and curcumin in sodium acetate buffer and mixing;
c) resuspending in olive oil, stabilising and capping agent, and sonicating for 15 cycles of 30 seconds sonication and 1-minute pause; and
d) extracting the c-lobe nano-particles formed and washing.
2. The process as claimed in claim 1, wherein the sonication is performed using centrifugation at 6000 rpm for 10 minutes.
3. The process as claimed in claim 1, wherein washing the pellet is performed with 700 µl diethyl ether and sodium acetate buffer thrice individually.
4. The process as claimed in claim 1, wherein the purifying c-lobe of the bLF using ion-exchange chromatography CMC column with 50mM Tris pH 8.
5. The process as claimed in claim 1, wherein the sonicating is performed for 15 cycles of 30 seconds sonication and 1-minute pause.
6. The process as claimed in claim 1, wherein suspending c-lobe and curcumin are mixed together and incubated for 30 minutes on ice.
7. A curcumin loaded c-lobe nanoparticle obtained from bovine lactoferrin according the process as claimed in claim 1.
Dated this 11th day of February, 2023

POOJA
Agent for the Applicant
IN/PA/1838