Description:Form 2

THE PATENT ACT, 1970
(39 of 1970)
&
THE PATENT RULES, 2003
COMPLETE SPECIFICATION
(See section 10 and rule 13)

“NOVEL COMPOSITION OF LOW COST WOUND DRESSING NATURAL NANOFORMULATION AND METHOD OF PREPARATION THEREOF”

in the name of PONDICHERRY UNIVERSITY an Indian national having address at CHINNA KALAPET, KALAPET, PONDICHERRY, PONDICHERRY - 605014, PONDICHERRY, INDIA.

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

SOURCE AND GEOGRAPHICAL ORIGIN OF THE BIOLOGICAL MATERIAL:

SL.
NO COMMON NAME SCIENTIFIC NAME PART OF BIOLOGICAL SOURCES SOURCE OF ACCESS DETAILS OF GEOGRAPHICAL LOCATION
1. Chicken Gallus gallus domesticus Keratin from chicken feathers Waste from Butcher shop after cleaning broiler chicken for meat Fathima Chicken Center M.G. Road, Opposite Devaraj Clinic,
Muthialpet, Puducherry,
605003
Banana Musa pradisca Pectin Waste from Banana Peel of Vegetable/Juice shops Almonds Café, East Coast Rd,
Chinna Kalapet, Auroville,
Kalapet,
Puducherry
605014.

FIELD OF THE INVENTION:
The present invention generally relates to a wound dressing nanoformulations viz. Ointment, cream, gel, lotion, transdermal patch, plasters etc. More particularly, the present invention includes composition and process of preparation of low cost wound dressing natural nanoformulation ointment exhibiting enhanced anti-oxidant, anti-microbial and wound healing activities and product thereof.

BACKGROUND OF THE INVENTION:

A wound is a puncture or damage of skin tissue which might be an open or closed wound. If the natural barrier, i.e., the epidermis, is broken due to environmental impact, a series of biochemical processes are initiated, known as a healing process. In general, the wound heals due to the natural healing mechanisms in which damaged tissues are replaced with new ones. This healing process occurs in four stages, i.e., hemostasis, inflammation, proliferation, and maturation. During the 1st phase, the clot is formed to halt bleeding. Platelet changes into an amorphous form which enables it to stick at the injured site and induces a signal to activate fibrinogen. In the 2nd stage, phagocytosis is initiated whereinthe debris and dead cells are engulfed and platelet-derived growth factors are released. During the 3rd stage, wound contraction occurs with a series of processes, viz. angiogenesis, collagen formation, and granulation. Finally, epithelization leads to tissue remodeling, the fourth stage of the wound-healing process. Wound healing becomes complicated when there is disruption due to environmental factors like comorbidity, physical attrition, microbial infection, etc., and the delay in the healing process leads to chronic wounds. Among these factors, microbial infection greatly influences the wound healing process, and prolonged elevation of proinflammatory cytokines such as interleukin- 1 and tumor necrosis factor–alpha is evidenced. This, in turn, causes disturbances in the production of matrix metalloproteinases and growth factors, thereby impairing the process of wound healing, especially in chronic and diabetic wounds.

Many wound dressing materials and dosage forms are available in the market. Moreover, several conventional wound management strategies and treatment methods are currently being studied for accelerated wound healing. However, they fail to be proven as a cost-effective and ideal option clinically due to poor reepithelialization of wounds, aggravated fluid loss, poor sensitivity, and durability. Out of the currently available wound healing therapies, nanotechnology-based approaches are considered an area of trustworthy research owing to their modified physicochemical properties. Consequently, innovative dressings should be based on biodegradable and biocompatible materials targeting specific events in the healing process to accelerate wound closure and restore the functionality of the healed tissue. Drug delivery approaches, particularly delivery using Nanoparticles (NPs), lead the way to new strategies to improve tissue regeneration therapies. The chemical nature of NPs may induce biological effects that can be combined with a loaded drug activity to enhance treatment efficacy. A superiority of drug delivery using NPs over current dressings results from their ability to carry, protect, and sustain active drug release. Side effects are decreased due to the greater efficacy of lowered drug doses directly released into the wound by NPs.

There are reports available in the state of art revealing the existence of nanoformulations for wound healing.

EP0468797A2 discloses a novel wound dressing material is produced by a chemical treatment of animal fibers, particularly, wool, to strip off the keratin layers of the surfaces of the fibers so as to expose the non-keratin protein in the under layers, which is hydrophilic. The wound dressing material provides healing and regenerating effects on the wounds, and can be produced at a low cost.

WO2008015475A1 discloses a gel which comprises a cross-linked matrix of albumin molecules and polysaccharide molecules having appended groups that are carboxyl groups 5 or activated derivatives or salts thereof. The albumin is preferably human serum albumin, most preferably in the form of a recombinant product. The polysaccharide molecules are preferably pectin molecules. The gels are useful for topical administration as wound dressings.
BR102019015537A2 discloses a nanoformulation developed based on copaíba oil for topical use in the healing process associated with laser in the present invention, copaíba oil (ocp) of the species copaifera reticulata ducke (ocpr), copaifera multijuga hayne (ocpm), and a commercial sample, were transmitted (preferential range 0.5% - 3.0%) in nanoemulsion SNEDDS (self-nanoemulsifying drug delivery system), for topical use associated with laser, in the treatment of cutaneous tissue repair. the sneddsocpr, ocpm or ocpc (snedds-ocp) formulations were prepared without co-surfactant, aiming to reduce carrier ingredients that can avoid adverse reactions. snedds-ocp contains copaiba oil mixed with sunflower oil (1: 1) and a surfactant for pharmacological use (preferential range 7% - 25%), dispersed in a neutral aqueous medium. The snedds-ocp formulations were evaluated in in vivo treatments (Rattus novergicus albinus) for wound healing. Histopathological and immunohistochemical analyzes were performed after 7 and 14 days which showed a reduction in neutrophils and lymphocytes, as well as increased collagenesis, proliferation of fibroblasts and greater epithelial thickening. The pharmacological results showed an anti-inflammatory, healing and antiseptic response, via topical use, as well as an antinociceptive effect, via oral administration. Comparatively, snedds-ocp formulations were more effective in the process associated with laser, in skin tissue repair.

US20160199447A1 discloses lipid nanoparticles comprising a growth factor and/or an antimicrobial peptide and to the method for their preparation. Moreover, it refers to pharmaceutical compositions comprising such lipid nanoparticles, and a pharmaceutical acceptable carrier. Finally, it also refers the said pharmaceutical composition for its use as medicament and for its use in promoting wound healing, particularly for topical administration.

WO2017122224A1 discloses solation of plant based CNCs from the leaves of S. cumini. For the formation of NCs, a novel greener approach using S. cumini LE as reducing agent for in situ impregnation of AgNPs as fillers into CNCs as matrix is reported. The silver nitrate solution in three different concentrations of 1 mM, 5 mM and 10 mM was used to form NCs where AgNPs have been incorporated into CNCs matrix. The CNCs and NCs were characterized using SEM, TEM, XRD, Zeta potential, FT-IR, and UV-Vis spectroscopy. NCs developed in the form of film and ointment showed strong antimicrobial activity against both gram negative and gram positive bacteria. NCs wound dressing is capable of regulating wound exudates and providing moisture to wound responsible for faster healing of acute wounds. The observations from histopathological and biochemical assays confirmed that NCs enhance healing because of lesser inflammation, rapid angiogenesis, early collagen formation and enhanced rate of reepithelization.

Siang-AnChen, Han-MinChen, Yeong-Der Yao, Chi-FengHung, Chi-ShunTu, Yao-JenLiang (2012) reported a Topical treatment with anti-oxidants and Au nanoparticles which promote healing of diabetic wound through receptors of advance glycation end-products.

Though many formulations with various ingredients and advantages have been disclosed in the prior arts, none of them discloses the characterized natural nanoformulation that exhibit enhanced anti-oxidant, anti-microbial and wound healing activities. Moreover, the process disclosed in the prior art is quite time-consuming and not economical.

Therefore, there lies a requirement for a novel composition which exhibits enhanced effects due to the individual and collective activities all the ingredients of the nanocomposite.
Hence, an attempt has been made to develop low-cost natural wound dressing nanoformulation with novel composition prepared using materials derived from biowaste and claimed to exhibit enhanced wound healing, anti-oxidant and anti-microbial activities.

OBJECT OF THE INVENTION:

The main object of the present invention is to develop a cost-effective composition and process for preparing wound dressing nanoformulations viz. Ointment, cream, gel, lotion, transdermal patch, plasters etc.

Another object of the present invention is to develop a low-cost natural wound dressing nanoformulation particularly ointment with appropriate combinations of biomolecules exhibiting enhanced anti-oxidant, anti-microbial and wound healing properties with effective re-epithelialization

Yet another object of the present invention is to develop a low-cost natural wound dressing nanocomposite comprising of low-cost biocompatible components which included protein-keratin, polysaccharide-pectin, phyto-molecule-Ferulic acid and inorganic metal-silver nanoparticles.

Further object of the present invention is to employ the developed nanoformulation ointment exhibiting enhanced wound healing, anti-oxidant and anti-microbial activities as wound dressing material to accelerate healing process.

BRIEF DESCRIPTION OF THE DRAWINGS:

Figure 1 depicts Nanoparticle characterization.
A –UV Vis spectra
B- FT-IR Spectra
C- CD Spectra
D- XRD Spectra
E-SEM images
F- DLS image

Figure 2 depicts the UV spectroscopy of
Pectin
Pectin Ferulic acid
C- Nanocomposite
Figure 3 depicts wound size before and after treatment of nanocomposite and nanoformulation of the present invention.

SUMMARY OF THE INVENTION:

The present invention discloses a composition and process of preparation of low-cost natural biocompatible wound dressing nanoformulations viz. Ointment, cream, gel, lotion, transdermal patch, plasters etc. particularly ointment exhibiting enhanced anti-oxidant, anti-microbial and wound healing activities with negligible toxicity. The process of the present invention comprises of
preparation of keratin/silver nanoparticles
dissolving predetermined amount of keratin powder in predetermined amount of 10% NaOH followed by sonication and filtration, in which filtrate is retained.;
adding predetermined amount of silver nitrate to the filtrate followed by addition of a predetermined amount of a solution comprising of sodium borohydride and distilled water in drop wise manner under continuous stirring at predetermined reaction conditions to form keratin capped nanosilver complex ;
preparation of pectin-Ferulic acid complex
mixing predetermined amount of pectin in water to form pectin dispersion;
dissolving predetermined amount of Ferulic acid in a solvent followed by mixing into the pectin dispersion slowly under magnetic stirring and evaporating at predetermined reaction conditions to obtain a suspension- pectin-Ferulic acid complex;
characterized in mixing the keratin/silver nanoparticles into the pectin-Ferulic acid complex under magnetic stirring followed by homogenization, centrifugation to obtain pellet and supernatant followed by drying the pellet at predetermined reaction conditions to obtain low-cost natural wound dressing nanocomposite.
The present invention also discloses low-cost natural biocompatible wound dressing nanocomposite exhibiting enhanced wound healing, anti-oxidant and anti-microbial activities with negligible toxicity prepared by the process as described above.

The present invention further discloses a process of preparation of low-cost, biocompatible natural wound dressing nanoformulations viz. Ointment, cream, gel, lotion, transdermal patch, plasters etc. particularly ointment exhibiting enhanced anti-oxidant anti-microbial and wound healing activities with negligible toxicity comprises of mixing the nanocomposite with an ointment base by trituration method.
The present invention further discloses a low-cost, biocompatible natural wound dressing nanoformulation ointment exhibiting enhanced wound healing, anti-oxidant and anti-microbial activities with negligible toxicity prepared by the process as described above.
DETAILED DESCRIPTION OF THE INVENTION:

The present invention discloses a composition and process of preparation of low-cost, biocompatible natural wound dressing nanoformulations viz. Ointment, cream, gel, lotion, transdermal patch, plasters etc. particularly ointment exhibiting enhanced anti-oxidant, anti-microbial and wound healing activities with negligible toxicity.

The process of the present invention comprises of
Preparation of keratin/silver nanoparticles.
Preparation of pectin-Ferulic acid complex.
Formation of low-cost wound dressing natural nanocomposite.
Preparation of nanoformulation ointment
Table 1: Composition of Nanocomposite of the present invention.

S.No. Ingredients Composition of 10gm of formulation of the present invention (ointment)
1. Ferulic acid 0.02%w/w
2. Keratin 0.02%w/w
3. Silver nitrate(for Nanosilver) 0.01% w/w ( for 0.01%w/w nano silver)
4. Pectin 0.02%w/w

Table 2 Composition of Nanoformulation ointment

S.No. Ingredients Composition of 10gm of formulation (ointment)
1. Nanocomposite 75 mg
2. Ointment base QS to 10gm

Preparation of keratin/silver nanoparticles:
Dissolving keratin powder in 10% NaOH followed by sonication and filtration, in which filtrate is retained and residue is discarded.
Addition of silver nitrate to the filtrate followed by addition of a solution comprising of sodium borohydride and distilled water in drop wise manner under continuous stirring to form keratin/silver nitrate nanoparticles.
The prepared keratin capped silver nanoparticles were then characterized using UV-Vis spectroscopy, DLS and FT-IR analysis (Figure 1).

UV-Vis spectroscopy

Two peaks at 280nm (keratin) and 390 nm (Silver nanoparticle) in the UV spectrum represent formation of keratin capped silver nanoparticle.

DLS

Formations of stable homogenized particles are confirmed by DLS in which nanosize of about 123.1nm±12, less PDI of about 0.289±0.12 are reported.

FT-IR

In the FT-IR spectra peak at 1454.5cm-1 shifted to 1384.6cm-1 which corresponds to -COO group. This confirms the possibility of bond formation of silver nanoparticle with negative carboxylic group of keratin through ionic interaction.
Thus the combined results of UV-Vis spectroscopy, DLS and FT-IR confirms the formation of Keratin capped silver nanoparticles which is further used for nanocomposite preparation.

Preparation of pectin-Ferulic acid complex:
mixing pectin in water to form pectin dispersion;
dissolving Ferulic acid in a solvent followed by mixing into the pectin dispersion slowly under magnetic stirring and evaporating to obtain suspension- pectin-Ferulic acid complex.

Formulation of low cost wound dressing natural nanocomposite.
mixing the keratin/silver nanoparticles into the pectin-Ferulic acid complex under magnetic stirring followed by homogenization, centrifugation to obtain pellet and supernatant followed by drying the pellet to obtain low cost wound dressing natural nano composite.
The nanocomposite was then subjected to spectral studies to determine the presence of pectin and Ferulic acid and its activities.

UV spectroscopy

The UV-Visible spectrum of nanocomposite, (Figure 2) the peaks of keratin and ferulic acid are masked by pectin coating and it is evidenced by spectra which resembles between pectin and silver nanoparticles. Stable peak shift of pectin at 2926cm-1(-OC-CH3) and surface charge of novel nanocomposite of the present invention is found to be -16.5mV which is similar to pectin surface charge and it is confirmed for presence of Pectin/silver nanoparticle complex at the outer layer.

The best activity is shown at a pH of around 5-6, which corresponds to the actual skin pH. It is easily miscible and soluble in the skin pH because of its hydrophilic and water absorptive qualities. Being at the outer coat, the activity of pectin is thought to be the primary cause of the commencement of antimicrobial action, after which the remaining components are exposed to continue in exerting their wound healing effect and it is also evidenced by preclinical evaluation of present invention.

Preparation of nanoformulation ointment:

The nanocomposite is then mixed with an ointment base by trituration method to form nanoformulation ointment.

The ointment base comprises of wool fat, hard paraffin, cetostearyl alcohol, and white soft paraffin.

The prepared nanoformulation was then subjected to experimental (in vitro and in vivo) studies to ascertain its efficacy.

As a result of the synergestic effect of all substances, the enhanced efficacy with a low dose is possible.

Nanosize of the composition proved by particle size, XRD and DSC-TGA also contribute to improved biological effects which is confirmed by antioxidant, antimicrobial and wound healing properties.

Free radical scavenging activity of the nanocomposite and the individual ingredients was analysed by DPPH scavenging assay. In the present invention maximum DPPH scavenging activity of the nanocomposite was at a concentration of 3.2mg/ml with percentage inhibition of 87.38 ± 0.233. It showed a maximum percentage inhibition in the same concentration compared to the individual ingredients. From the result it is evident that the nanocomposite showed better antioxidant activity, which is attributed to the synergistic effects of the API (ferulic acid), pectin and silver nanoparticle present in it.

Table 3 Anti-oxidant property of nanocomposite, nanoformulation and its components of the present invention

Conc. (µg/ml) % Inhibition
Vit C Keratin Silver nanoparticle Pectin Ferulic acid Nano composite Nanoformulation
Conc.
mg/ml % Inhibition
25 42.389 ± 3.588 -2.199 ± 0.417 8.528 ± 4.856 22.993 ± 2.058 15.552 ± 7.093 18.314 ± 4.100 1.25 30.78 ± 2.86
50 53.552 ± 10.198 -9.144 ± 2.279 9.010 ± 7.043 26.311 ± 3.441 22.600 ± 3.513 25.543 ± 7.404 2.5 44.22 ± 5.93
100 60.656 ± 0.203 2.508 ± 3.341 15.610 ± 4.466 27.970 ± 1.767 33.819 ± 3.821 39.513 ± 6.053 5 57.73 ± 1.12
200 92.155 ± 0.929 -2.623 ± 5.468 32.518 ± 6.510 39.969 ± 5.480 61.360 ± 5.555 58.576 ± 3.675 10 68.42 ± 3.17
400 92.896 ± 0.068 4.051 ± 4.209 25.102 ± 8.013 48.379 ± 2.018 76.508 ± 1.662 79.176 ± 1.444 19 77.96 ± 1.33
800 92.896 ± 0.068 -2.238 ± 4.775 43.048 ± 6.649 67.592 ± 2.217 89.955 ± 0.309 90.299 ± 0.324 38 84.54 ± 0.00
1600 93.130 ± 0.135 -8.025 ± 0.570 59.288 ± 8.979 75.887 ± 0.066 88.375 ± 0.260 88.277 ± 0.361 75 83.09 ± 0.00
3200 93.052 ±0.179 4.745 ± 0.347 66.852 ± 1.177 68.788 ± 0.467 86.998 ± 0.732 87.378 ± 0.233 150 75.43 ±
IC50 (µg) 42.04 - 962.5 744.83 166.36 138.12 IC50
(mg) 2.69
(equivalent to 213µg/ml of NC)

The antibacterial activities of the nanocomposite, nanoformulation and the individual components were evaluated compared to the positive drug control Gentamicin (10µg/disc) using the zone of inhibition method. As shown in Table 4, no inhibition zones were noted for pectin, keratin, Ferulic acid 15mg/ml, silver nanoparticles, nanocomposite, and nanoformulation showed a zone of inhibition. The antibacterial effect of the nanocomposite and nanoformulation is probably due to the synergistic effect of components present in it.

Table 4 Anti-microbial property of Nanocomposite, Nanoformulation and its Components

Compound
Concentration Zone of inhibition (mm)
Escherichia coli Acinetobacter baumannii Pseudomonas aeruginosa Staphylococcus aureus

Gentamicin (Control-C1) 10µg/disc 22±0.58
20±1.53 20±1.15 15±2.08
Silver Nanoparticle 150mg/disc 11±1.53 10±0.58 10±0.58 10±1.58
Ferulic acid 150mg/disc 14±1.73 14±1.73 13±1.53 12±0.58
Pectin 150mg/disc - - - -
Keratin 150mg/disc - - - -
Nanocomposite 150mg/disc 12.13±0.06 11.9±0.35 11.27±0.21 11.77±0.25
Nanoformulation 150mg/disc 12.30±0.20 12.27±0.21 11.27±0.21 11.13±0.06

Assessment of cell viability by MTT assay

Cell survival studies were carried out for ferulic acid,nanocomposite, and nanoformulation , and the viability of the L929 fibroblast cells against the tested concentrations were recorded. An IC50 value of 12.46µM,9.769µM and 5.638 µM was recorded for ferulicacid, nanocomposite, and nanoformulations respectively.

Wound size before and after treatment in Excision wounds in Animal model

Based on the results of the in-vitro studies, the nanocomposite and nanoformulation were tested on an Excision wound model in Albino rats. The percentage of wound closure was calculated, and the results are depicted in Figure 3. From the results, it is evident that the groups treated nanoformulation ointment (containing 75mg of nanocompsite) showed the highest percentage of wound contraction of about 78% within seven days of the study period, which is higher than the untreated control, standard and as well nanocomposite treated groups. It is also interesting to note that about 95% of the wound was sealed on the final day (21st day) of the study period for the both nanocomposite and nanoformulation. which is equivalent to the standard treated groups.

The nanocomposite and nanoformulation of the present invention was evaluated for its synergistic action, to study the effect of the formulation of the present invention viz-a-viz its components when individually applied.

Table 5: Synergistic effect of the nanoformulation
Sl. No. Name of the compound MTT Assay
Antioxidant activity (DPPH assay) Anti-microbial assay (Acinetobacter baumanni) (mm)
Conc (µM/mL)
%
inhibition Conc (µg/mL)
% inhibition Conc (mg/disc) Zone of inhibition (mm)
1 Silver nitrate (for Nanosilver) AgNP ---9.06------- ---50----- 962.5 50 150 10±0.58
2. Keratin ------- -------- Nil 50 150 Nil
3 Ferulic acid (FA) 12.46 50 166.36 50 150 14±1.73
4. Pectin -------- -------- 744.83 50 150 Nil
5. Nanocomposite (NC)
(Keratin capped Ag NP+ FA+ Pectin) 8.993 50 138.12 50 150 11.9±0.35
6 Nanoformulation ointment
(NC+ Ointment Base) 5.638 50 2.69 mg/ml (equivalent to 213µg/ml of NC) 50 150
12.27±0.21

From the above data, it is inferred that for treatment with the natural nanoformulation of the present invention only minimum dose amount is required when compared to individual constituents. This facilitates reduction in the quantity of constituents used. The formulation exhibits superior biocompatibility, anti-microbial and anti-oxidant activity compared to individual constituents. Eventhough ferulic acid exhibits comparable activity with that of the nanoformulation of the present invention, the nanoformulation of the present invention exhibits the said activity at lower concentration compared to Ferulic acid. Further the pure Ferulic acid dissolutes in 2 hours whereas the release of 98% of Ferulic acid from nanocomposites at the 12th hour demonstrates sustained release of Ferulic acid from the nanocomposite thereby making it suited for wound healing with low dose. The sustained release of ferulic acid is brought about by the pectin which is in the outer coat which swells in the wound exudate and also releases the silver ions from the keratin capped silver nanoparticles for antimicrobial action and reduces the toxicity too. Thus, the above discussion illustrates the synergism of the components present in the nanocomposite. Thus, the developed natural nanoformulation by the cost-effective process can be utilized as an effective and safe wound dressing agent.

Advantages:
The present invention has Ferulic acid which acts as API (active pharmaceutical ingredient) important in the development of therapeutic medicines for wound healing and keratinopathies by modulating β-catenin and Nrf2, with low adverse effects.
Ferulic acid shows free radical scavenging equivalent to Vitamin C. The release of 98% of Ferulic acid from nanocomposites at the 12th hour. But, the pure Ferulic acid dissolute in 2 hours and it demonstrates sustained release of Ferulic acid from nanocomposites thereby making it suited for wound healing with low dose.
Keratin protein, particularly type 2 alpha keratin isolated from chicken feather is been used in the nanoformulation which is found to be more beneficial in wound healing process, as the feather keratin infused with silver nanoparticles is responsible for antibacterial action.
Anti-bacterial activity: The smallest nanoparticles (approx. 90nm) with keratin capped at the lowest silver precursor concentrations and synthesis temperatures, binds to pectin after the cleavage of bond between keratin/silver nanoparticle. This results in remarkable antibacterial activity against Gram-negative E. coli and P. aeruginosa, A. baumannii as well as Gram-positive S. aureus.
The formulation has characteristic nanosize therefore has a greater surface area, leading to better cell–particle interaction.
The formulation has characteristic cubic shape therefore exhibits better antimicrobial activity
The formulation has Pectin, which form gel like structure with skin fluid to keep the wound in the moist form and aid in the release of active ingredients.
In one of the preferred embodiment, the present invention shall disclose a process of preparation of low-cost natural wound dressing nanocomposite exhibiting enhanced anti-oxidant anti-microbial and wound healing activities with negligible toxicity and faster wound contraction and reepithelization through tissue remodeling thereby attaining proper collagen deposition and neo-vessel. The process of the present invention comprises of;
preparation of keratin/silver nitrate nanoparticles comprises of
dissolving predetermined amount of keratin powder in predetermined amount of 10% NaOH followed by sonication and filtration, in which the filtrate is retained and residue is discarded;
adding predetermined amount of silver nitrate to the filtrate followed by addition of a predetermined amount of a solution comprising of sodium borohydride and distilled water in drop wise manner under continuous stirring at predetermined reaction conditions to form keratin/silver nitrate nanoparticles;
preparation of pectin-Ferulic acid complex comprises of
mixing predetermined amount of pectin in water to form pectin dispersion;
dissolving predetermined amount of Ferulic acid in a solvent followed by mixing into the pectin dispersion slowly under magnetic stirring and evaporating at predetermined reaction conditions to obtain suspension- pectin-Ferulic acid complex;
characterized in mixing the keratin/silver nitrate nanoparticles into the pectin-Ferulic acid complex under magnetic stirring followed by homogenization, centrifugation to obtain pellet and supernatant followed by drying the pellet at predetermined reaction conditions to obtain low cost natural wound dressing nanocomposite, in which the supernatant is discarded.
In another preferred embodiment, the present invention shall discloses a low-cost natural wound dressing nanocomposite exhibiting enhanced anti-oxidant anti-microbial and wound healing activities with negligible toxicity and faster wound contraction and reepithelization through tissue remodeling thereby attaining proper collagen deposition and neo-vessel prepared by the process described above.

In yet another preferred embodiment, the present invention shall disclose a process of preparation of low cost natural wound dressing nanocomposite exhibiting enhanced anti-oxidant anti-microbial and wound healing activities with negligible toxicity and faster wound contraction and reepithelization through tissue remodeling thereby attaining proper collagen deposition and neo-vessel. The process comprises of;
preparation of keratin/silver nitrate nanoparticles comprises of
dissolving 10g of keratin powder in 4ml of 10% NaOH followed by sonication and filtration, in which filtrate is retained and residue is discarded;
adding 5mg of silver nitrate to the filtrate followed by addition of 1ml of a solution comprising of 1mg of sodium borohydride and 3 ml distilled water in drop wise manner under continuous stirring for 10 minutes to form keratin/silver nitrate nanoparticles;
preparation of pectin-Ferulic acid complex comprises of
mixing 10mg of pectin in 20ml of water to form pectin dispersion;
dissolving 10mg of Ferulic acid in 2ml of acetone followed by mixing into the pectin dispersion slowly under magnetic stirring at 800 rpm for 1hour and evaporating the solvent to obtain suspension- pectin-Ferulic acid complex;
characterized in mixing the keratin/silver nitrate nanoparticles into the pectin-Ferulic acid complex under magnetic stirring 800-1000 rpm for 2 to 3 hours followed by homogenization at 10,000 rpm for 30 minutes and centrifugation at 12,000 rpm for 15 minutes to obtain pellet and supernatant followed by drying the pellet by lyophilization to obtain low cost natural wound dressing nanocomposite, in which the supernatant is discarded.
In yet another preferred embodiment, the present invention shall discloses a low-cost natural wound dressing nanocomposite exhibiting enhanced anti-oxidant anti-microbial and wound healing activities with negligible toxicity and faster wound contraction and reepithelization through tissue remodeling thereby attaining proper collagen deposition and neo-vessel prepared by the process described above.

In yet another preferred embodiment, the present invention shall disclose a process of preparation of low-cost natural wound dressing nanoformulation ointment exhibiting enhanced anti-oxidant, anti-microbial and wound healing activities with negligible toxicity and faster wound contraction and reepithelization through tissue remodeling thereby attaining proper collagen deposition and neo-vessel. The process comprises of
melting wool fat, hard paraffin, cetostearyl alcohol, and white soft paraffin in a steam bath to about 70-75 °C followed by allowing to cool slowly to form an ointment base;
preparation of keratin/silver nitrate nanoparticles comprises of
dissolving predetermined amount of keratin powder in predetermined amount of 10% NaOH followed by sonication and filtration, in which filtrate is retained and residue is discarded;
adding predetermined amount of silver nitrate to the filtrate followed by addition of a predetermined amount of a solution comprising of sodium borohydride and distilled water in drop wise manner under continuous stirring at predetermined reaction conditions to form keratin/silver nitrate nanoparticles;
preparation of pectin-Ferulic acid complex comprises of
mixing predetermined amount of pectin in water to form pectin dispersion;
dissolving predetermined amount of Ferulic acid in a solvent followed by mixing into the pectin dispersion slowly under magnetic stirring and evaporating at predetermined reaction conditions to obtain suspension- pectin-Ferulic acid complex;
characterized in mixing the keratin/silver nitrate nanoparticles into the pectin-Ferulic acid complex under magnetic stirring followed by homogenization, centrifugation to obtain pellet and supernatant followed by drying the pellet at predetermined reaction conditions to obtain low cost natural wound dressing nanocomposite, in which the supernatant is discarded.
adding predetermined amount of the nano composite of step(d) with continuous trituration to predetermined amount of the ointment base of step (a) to form an homogenous mixture of nanoformulation ointment
In yet another preferred embodiment, the present invention shall discloses a low-cost natural wound dressing nanoformulation ointment exhibiting enhanced anti-oxidant, anti-microbial and wound healing activities with negligible toxicity and faster wound contraction and reepithelization through tissue remodeling thereby attaining proper collagen deposition and neo-vessel prepared by the process described above.

In yet another preferred embodiment, the present invention shall discloses a process of preparation of low-cost natural wound dressing nanoformulation ointment exhibiting enhanced anti-oxidant, anti-microbial and wound healing activities with negligible toxicity and faster wound contraction and reepithelization through tissue remodeling thereby attaining proper collagen deposition and neo-vessel. The process comprises of
melting 0.5gm of wool fat, 0.5 gm of hard paraffin, 0.5 gm of cetostearyl alcohol, and 8.5 gm of white soft paraffin in a steam bath to about 70-75 °C followed by allowing to cool slowly to form an ointment base;
preparation of keratin/silver nitrate nanoparticles comprises of
dissolving 10g of keratin powder in 4ml of 10% NaOH followed by sonication and filtration, in which filtrate is retained and residue is discarded;
adding 5mg of silver nitrate to the filtrate followed by addition of 1ml of a solution comprising of 1mg of sodium borohydride and 3 ml distilled water in drop wise manner under continuous stirring for 10 minutesto form keratin/silver nitrate nanoparticles;
preparation of pectin-Ferulic acid complex comprises of
mixing 10mg of pectin in 20ml of water to form pectin dispersion;
dissolving 10mg of Ferulic acid in 2ml of acetone followed by mixing into the said pectin dispersion slowly under magnetic stirringat 800 rpm for 1hourand evaporating the solvent to obtain suspension- pectin-Ferulic acid complex;
characterized in mixing the keratin/silver nitrate nanoparticles into the pectin-Ferulic acid complex under magnetic stirring 800-1000 rpm for 2 to 3 hours followed by homogenization at 10,000 rpm for 30 minutes and centrifugation at 12,000 rpm for 15 minutes to obtain pellet and supernatant followed by drying the pellet by lyophilization to obtain low cost natural wound dressing nanocomposite, in which the supernatant is discarded.
adding the 75mg of nano composite of step(d) with continuous trituration to 10gm of the ointment base of step (a) to form an homogenous mixture of nanoformulation ointment
In further preferred embodiment, the present invention shall disclose a low-cost natural wound dressing nanoformulation ointment exhibiting enhanced anti-oxidant, anti-microbial and wound healing activities with negligible toxicity and faster wound contraction and reepithelization through tissue remodeling thereby attaining proper collagen deposition and neo-vessel prepared by the process described above.
Working example:

A process of preparation of low-cost natural wound dressing nano formulation exhibiting enhanced wound healing, anti-oxidant and anti-microbial activities with negligible toxicity.
Preparation of keratin/silver nanoparticles.
Preparation of pectin-Ferulic acid complex.
Formation of low cost wound dressing natural nanocomposite.
Preparation of nanoformulation ointment.

Preparation of keratin/silver nanoparticles

10 mg of Keratin powder was dissolved in 4 ml 10% NaOH. The solution was then sonicated and filtered to obtain filtrate and residue. The residue was discarded. To the filtrate, 5 mg of silver nitrate was added and stirred rapidly for approximately 5 min. on addition of silver nitrate; the pH was changed to 6.7. In a separate vial a solution was prepared by adding 1mg of sodium borohydride to 3mL of DI water. Exactly 1 mL of the solution was added in drop wise manner to the rapidly stirring keratin/silver nitrate solution over the course of 10 min.

The solution darkened to a dark orange color and the final pH of the solution is typically measured to be 7.7.

Preparation of pectin-Ferulic acid complex.

10mg of pectin was mixed in 20ml of water. 10mg Ferulic acid is dissolved in 2ml acetone (solvent) and poured into the pectin dispersion slowly under magnetic stirring at 800 rpm for 1h.

Formation of low cost wound dressing natural nanocomposite
The prepared Keratin/silver nanoparticles was mixed with the pectin-Ferulic acid complex under magnetic stirring 800-1000 rpm for 2 to 3 hours. It was then homogenized at 10,000 rpm for 30 minutes and then centrifuged at 12,000rpm for 15minutes to obtain pellet and supernatant. The pellet was lyophilized to obtain low cost wound dressing natural nanocomposite.

Preparation of nanoformulation ointment
Melting 0.5gm of wool fat, 0.5 gm of hard paraffin, 0.5 gm of cetostearyl alcohol, and 8.5 gm of white soft paraffin in a steam bath to about 70-75 °C followed by allowing to cool slowly to form an ointment base followed by adding 75mg of nanocomposite with continuous trituration to the 10gm of ointment base to form an homogenous mixture of natural nanoformulation ointment

Although preferred embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be made without departing from the scope of the invention as defined in the claims which follow.
, Claims:WE CLAIM:

1. A process of preparation of low-cost natural wound dressing nanocomposite exhibiting enhanced anti-oxidant anti-microbial and wound healing activities with negligible toxicity and faster wound contraction and reepithelization through tissue remodeling thereby attaining proper collagen deposition and neo-vessel, the claimed process comprises of;
• preparation of keratin/silver nitrate nanoparticles comprises of
 dissolving predetermined amount of keratin powder in predetermined amount of 10% NaOH followed by sonication and filtration, wherein filtrate is retained and residue is discarded;
 adding predetermined amount of silver nitrate to the said filtrate followed by addition of a predetermined amount of a solution comprising of sodium borohydride and distilled water in drop wise manner under continuous stirring at predetermined reaction conditionsto form keratin/silver nitrate nanoparticles;
• preparation of pectin-Ferulic acid complex comprises of
 mixing predetermined amount of pectin in water to form pectin dispersion;
 dissolving predetermined amount of Ferulic acid in a solvent followed by mixing into the said pectin dispersion slowly under magnetic stirring and evaporating at predetermined reaction conditions to obtain suspension- pectin-Ferulic acid complex;
• characterized in mixing the said keratin/silver nitrate nanoparticles into the said pectin-Ferulic acid complex under magnetic stirring followed by homogenization, centrifugation to obtain pellet and supernatant followed by drying the pellet at predetermined reaction conditions to obtain low cost natural wound dressing nanocomposite, wherein the supernatant is discarded.

2. A low-cost natural wound dressing nanocomposite exhibiting enhanced anti-oxidant anti-microbial and wound healing activities with negligible toxicity and faster wound contraction and reepithelization through tissue remodeling thereby attaining proper collagen deposition and neo-vessel prepared by the process as claimed in claim 1.

3. A process of preparation of low cost natural wound dressing nanocomposite exhibiting enhanced anti-oxidant anti-microbial and wound healing activities with negligible toxicity and faster wound contraction and reepithelization through tissue remodeling thereby attaining proper collagen deposition and neo-vessel as claimed in claim 1 comprises of;
• preparation of keratin/silver nitrate nanoparticles comprises of
 dissolving 10g of keratin powder in 4ml of 10% NaOH followed by sonication and filtration, wherein filtrate is retained and residue is discarded;
 adding 5mg of silver nitrate to the said filtrate followed by addition of 1ml of a solution comprising of 1mg of sodium borohydride and 3 ml distilled water in drop wise manner under continuous stirring for 10 minutesto form keratin/silver nitrate nanoparticles;
• preparation of pectin-Ferulic acid complex comprises of
 mixing 10mg of pectin in 20ml of water to form pectin dispersion;
 dissolving 10 mg of Ferulic acid in 2ml of acetone followed by mixing into the said pectin dispersion slowly under magnetic stirringat 800 rpm for 1hourand evaporating the solvent to obtain suspension- pectin-Ferulic acid complex;
• characterized in mixing the said keratin/silver nitrate nanoparticles into the said pectin-Ferulic acid complex under magnetic stirring 800-1000 rpm for 2 to 3 hours followed by homogenization at 10,000 rpm for 30 minutes and centrifugation at 12,000 rpm for 15 minutes to obtain pellet and supernatant followed by drying the pellet by lyophilization to obtain low cost natural wound dressing nanocomposite, wherein the supernatant is discarded.

4. A low-cost natural wound dressing nanocomposite exhibiting enhanced anti-oxidant anti-microbial and wound healing activities with negligible toxicity and faster wound contraction and reepithelization through tissue remodeling thereby attaining proper collagen deposition and neo-vessel prepared by the process as claimed in claim 3.

5. A process of preparation of low-cost natural wound dressing nanoformulation ointment exhibiting enhanced anti-oxidant, anti-microbial and wound healing activities with negligible toxicity and faster wound contraction and reepithelization through tissue remodeling thereby attaining proper collagen deposition and neo-vessel comprises of

a. melting wool fat, hard paraffin, cetostearyl alcohol, and white soft paraffin in a steam bath to about 70-75 °C followed by allowing to cool slowly to form an ointment base;
b. preparation of keratin/silver nitrate nanoparticles comprises of
 dissolving predetermined amount of keratin powder in predetermined amount of 10% NaOH followed by sonication and filtration, wherein filtrate is retained and residue is discarded;
 adding predetermined amount of silver nitrate to the said filtrate followed by addition of a predetermined amount of a solution comprising of sodium borohydride and distilled water in drop wise manner under continuous stirring at predetermined reaction conditionsto form keratin/silver nitrate nanoparticles;
c. preparation of pectin-Ferulic acid complex comprises of
 mixing predetermined amount of pectin in water to form pectin dispersion;
 dissolving predetermined amount of Ferulic acid in a solvent followed by mixing into the said pectin dispersion slowly under magnetic stirring and evaporating at predetermined reaction conditions to obtain suspension- pectin-Ferulic acid complex;
d. characterized in mixing the said keratin/silver nitrate nanoparticles into the said pectin-Ferulic acid complex under magnetic stirring followed by homogenization, centrifugation to obtain pellet and supernatant followed by drying the pellet at predetermined reaction conditions to obtain low cost natural wound dressing nanocomposite, wherein the supernatant is discarded.
e. adding predetermined amount of the said nano composite of step(d) with continuous trituration to predetermined amount of the said ointment base of step (a) to form an homogenous mixture of nanoformulation ointment
6. A low-cost natural wound dressing nanoformulation ointment exhibiting enhanced anti-oxidant, anti-microbial and wound healing activities with negligible toxicity and faster wound contraction and reepithelization through tissue remodeling thereby attaining proper collagen deposition and neo-vessel prepared by the process as claimed in claim 5.

7. A process of preparation of low-cost natural wound dressing nanoformulation ointment exhibiting enhanced anti-oxidant, anti-microbial and wound healing activities with negligible toxicity and faster wound contraction and reepithelization through tissue remodeling thereby attaining proper collagen deposition and neo-vessel as claimed in claim 5 comprises of
a. melting 0.5gm of wool fat, 0.5 gm of hard paraffin, 0.5 gm of cetostearyl alcohol, and 8.5 gm of white soft paraffin in a steam bath to about 70-75 °C followed by allowing to cool slowly to form an ointment base;
b. preparation of keratin/silver nitrate nanoparticles comprises of
 dissolving 10g of keratin powder in 4ml of 10% NaOH followed by sonication and filtration, wherein filtrate is retained and residue is discarded;
 adding 5mg of silver nitrate to the said filtrate followed by addition of 1ml of a solution comprising of 1mg of sodium borohydride and 3 ml distilled water in drop wise manner under continuous stirring for 10 minutes to form keratin/silver nitrate nanoparticles;
c. preparation of pectin-Ferulic acid complex comprises of
 mixing 10mg of pectin in 20ml of water to form pectin dispersion;
 dissolving 10 mg of Ferulic acid in 2ml of acetone followed by mixing into the said pectin dispersion slowly under magnetic stirringat 800 rpm for 1hourand evaporating the solvent to obtain suspension- pectin-Ferulic acid complex;
d. characterized in mixing the said keratin/silver nitrate nanoparticles into the said pectin-Ferulic acid complex under magnetic stirring 800-1000 rpm for 2 to 3 hours followed by homogenizationat 10,000 rpm for 30 minutes and centrifugation at 12,000 rpm for 15 minutes to obtain pellet and supernatant followed by drying the pellet by lyophilization to obtain low cost natural wound dressing nanocomposite, wherein the supernatant is discarded.
e. adding the 75mg of said nano composite of step(d) with continuous trituration to 10gm of the said ointment base of step (a) to form an homogenous mixture of nanoformulation ointment

8. A low-cost natural wound dressing nanoformulation ointment exhibiting enhanced anti-oxidant, anti-microbial and wound healing activities with negligible toxicity and faster wound contraction and reepithelization through tissue remodeling thereby attaining proper collagen deposition and neo-vessel prepared by the process as claimed in claim 7.

Dated this 05th day of FEB 2023

For PONDICHERRY UNIVERSITY
By its Patent Agent

Dr.B.Deepa
IN/PA 1477