Method for synthesizing multifunctional biopolymer from coconut shell and / or coconut shell fiber wood pith powder and application of multifunctional biopolymer

Abstract

The invention relates to a multifunctional biopolymer synthesized from coconut shell and / or coconut shell fiber wood pith powder and application thereof. The present invention relates to the synthesis and production of biopolymers from natural waste-coconut shell and / or coconut shell fiber wood pith powder. More specifically, it relates to a novel multifunctional biopolymer obtained from a copolymer, cross-linking agent and plasticizer combination with coconut shell and / or coconut shell fiber wood pith powder (coconut fruit) for use in the pharmaceutical, nutraceutical, cosmetic and leather industries. Lignin components are extracted and purified from coconut shells, physicochemical characteristics of the lignin components are studied, and the lignin components are processed into various biopolymers to be used as burn wound dressings and leather substitutes.

Description

TECHNICAL FIELD

[0001] The present invention relates to the extraction of lignin from coconut shell and / or coconut coir pith powder. The extracted lignin is then purified and the physico-chemical properties of the purified lignin are studied. The lignin is further processed along with copolymer, cross-linking agent and plasticizer to make bio-polymer sheets / films. These sheets / films exhibit multi-functional properties and have been found to be very effective for wound dressing in case of severe burn accidents. These sheets / films are also used for the prototype production of leather substitute materials. The properties of the developed bio-polymer sheets / films have been characterized and have been found promising for various applications. BACKGROUND

[0002] Coconut shell and / or coconut coir pith powder is a versatile, environment friendly material which is popular in various applications. These natural lignocellulosic fibers are obtained from the fruit of coconut (Cocos nucifera), a tropical plant belonging to the family Arecaceae (Palmae). Their unique properties make them suitable for a wide range of applications, while their sustainable source makes them an attractive option for those who prioritize environmental responsibility. Coconut shell and / or coconut coir pith powder is produced globally by Indonesia, Philippines, India and Sri Lanka among the major producing countries. Coconut fiber is the most important fibrous by-product of the coconut plantation process, with a global annual production of at least 30 million tons. Coconut is mostly used in the food, fiber and other industries, but parts of the shell are mainly used in crafts and decoration, most of which remains unutilized, and its plant components remain in an area to be explored in the pharmaceutical sector. According to the study by Panyakaew and Fotios (2011), the composition of coconut shell contains 30% fiber and 70% pith, which is known for its high lignin and phenolic content. Van Dam et al. (2006) concluded that coconut shell contains 35% cellulose, 35% lignin and 17% hemicellulose (refer Figure 1 ). Due to the high lignin content, coconut fiber exhibits excellent elasticity, durability and resistance to rot.

[0003] Lignin is a very beneficial complex organic polymer that plays a key role in the cell wall structure, particularly in wood and bark. It provides important rigidity and rot resistance, making it an excellent building material. In fact, lignin is the second most abundant biopolymer in lignocellulosic biomass next to cellulose. In addition, it forms a strong and durable complex with cellulose and hemicellulose, resulting in excellent strength, flexibility, and high wear resistance, which is extremely useful in various industries. Lignin is composed of phenolic monomers (mainly coniferyl, sinapyl, and p-coumaryl alcohol) that are interconnected by various carbon-carbon and carbon-oxygen bonds, forming a highly branched irregular structure. This complex network of aromatic rings and hydrocarbon chains enhances the strength and flexibility of lignin, which is essential for plant growth and a valuable resource for many industry applications.

[0004] Lignin is a highly complex organic polymer present in plant cell walls that plays a key role in numerous industries due to its exceptional properties. It is an impressive biopolymer that plays a key role in enhancing the structural support, hardness, and durability of plant tissues. Although cellulose and hemicellulose have traditionally received more attention in the utilization of biomass, the unique and complex properties of lignin have recently sparked interest in its potential applications in sustainable materials, bioenergy, and various industry sectors. In the pulp and paper sector, lignin, which was previously considered a waste byproduct, is now being used to produce valuable chemicals such as vanillin. It is also used as a sustainable adhesive in wood products, reducing dependence on synthetic resins. In the field of bioplastics and composites, lignin enhances mechanical properties and aids in biodegradation, particularly in the production of vehicle components. Furthermore, lignin derivatives are utilized in agriculture as soil conditioners and controlled-release fertilizers, improving practicality and minimizing environmental impact. In the pharmaceutical industry, the antioxidant properties and antimicrobial properties of lignin help to enhance drug stability and delivery. In addition, the cosmetics industry also values lignin for its UV protection in sunscreens and anti-aging products. Moreover, lignin can be converted into biofuels, supporting renewable energy initiatives. Our previous patent application PCT / KR2022 / 019441 has expanded their application range to include nutraceuticals and cosmetics. Finally, its unique chemical structure and properties present more significant potential for development in various new fields, particularly in the development of biopolymers.

[0005] The demand for new biopolymers based on natural resources is increasing in the leather, pharmaceutical, and cosmetic industries, as these materials have sustainable development, regulatory compliance, and enhanced functional properties. Consumers and governments are increasingly inclined to replace conventional synthetic polymeric materials with eco-friendly, biodegradable alternatives to mitigate environmental impact and health risks. Biopolymers from natural sources such as lignin have unique advantages, such as biocompatibility, antimicrobial properties, and customization, making them highly suitable for various applications, including sustainable leather alternatives, advanced drug delivery systems, and natural skincare products. This shift towards natural biopolymers promotes market diversification, encourages innovation, and aligns with global trends towards more sustainable industrial practices.

[0006] Lignin-based biopolymers offer innovative and sustainable solutions across various industries. In the leather industry, they can serve as environmentally friendly alternatives while maintaining high quality and durability. The pharmaceutical industry can benefit from the biocompatibility and functional properties of lignin, making it ideal for drug delivery systems, wound dressings, and other medical applications. In the cosmetics industry, integrating lignin-derived biopolymers into skincare products can provide antioxidant and UV protection benefits for natural ingredients, enhancing product efficacy and sustainability.

[0007] In the leather industry, animal leather presents several disadvantages, including the slaughter process for obtaining leather. Additionally, the environmental pollution caused by the discharge of waste and carbon dioxide from livestock farming during leather production is increasingly being addressed. The use of toxic materials such as chromium in the manufacturing process of leather not only poses health risks to manufacturers but also causes environmental pollution when discharged with wastewater. On the other hand, lignin can improve leather treatment and finishing by improving texture, appearance, and durability, providing natural antioxidant protection. Lignin can also serve as a natural dye and color stabilizer, as well as a biodegradable adhesive, contributing to a more eco-friendly manufacturing process. In summary, the application of lignin in leather production can promote the sustainability and high quality of leather products.

[0008] Burns are one of the most common types of injuries, second only to traffic accidents, falls, and physical violence. In the early 20th century, treatment options for burn patients were extremely limited, often resulting in death from hypovolemic shock within the first day of injury. However, significant progress has been made in regenerative medicine, burn treatment, and pharmacotherapy in the late 20th century. Despite these advances, effectively managing burn wounds remains challenging. The recovery of a burn wound is a complex and lengthy process that relies on multiple immune system-mediated repair mechanisms. It involves the restoration of tissue continuity disrupted by unforeseen events such as fires. To achieve complete restoration, a burn wound goes through three successive stages: inflammation, formation of granulation tissue (proliferation), and remodeling, which can lead to scar formation. The patient's immune system plays a key role in coordinating the entire wound healing process. During the initial stages of healing, the injury is most susceptible to bacterial infection, which can exponentially decrease the healing process and be fatal if not treated promptly. To overcome this issue, although various types of wound dressings have been attempted, such as surface dressings, hydrogels, hydrocolloids, foams, fibers, and membranes, they all have corresponding issues, such as biocompatibility issues, properties of the dressing material, ease of use, dressing removal, shape and size, activity of pathogenic organisms, drug loading, etc. To address these issues, the development of innovative biocompatible polymers is needed.

[0009] Lignin-based biopolymer sheets and films can offer significant advantages for burn and wound dressings compared to conventional materials. Their natural antimicrobial and antioxidant properties can help prevent infection and reduce oxidative stress, promoting faster healing. These biopolymers can form hydrogels, creating a moist healing environment while controlling excess exudate, promoting cell migration, tissue regeneration, and removal of necrotic tissue. These processes are crucial for optimal wound healing outcomes. Their biocompatibility also reduces the risk of allergic reactions and minimizes possible irritation. Furthermore, they can be used to manufacture soft and durable dressings that not only protect the wound from contamination but also control drug delivery. In other words, lignin-based biopolymers have the potential to be designed to deliver therapeutic agents, such as antimicrobials or growth factors, directly to the wound site in a controlled manner, ensuring sustained treatment. This targeted drug delivery approach aims to enhance therapeutic efficacy while minimizing systemic side effects. Additionally, they are biodegradable and more cost-effective, making them consistent with environmentally friendly, sustainable healthcare practices. In summary, lignin-based biopolymer dressings provide a multifunctional and effective solution for advanced wound care, contributing to improved patient treatment outcomes and reduced environmental impact.

[0010] The present invention discloses the development of various lignin based biopolymer blends using lignin extracted from coconut shell and / or coconut shell fiber pith powder for the preparation of biopolymer sheets and / or films for burn healing / wound dressing and as leather substitute material. Lignin polymers are produced by three major oxidative radical polymerization reactions of p-hydroxycinnamyl alcohols known as monolignols, namely p-coumaryl alcohol, coniferyl alcohol and sinapyl alcohol. These alcohols are responsible for the formation of p-hydroxyphenyl (H), guaiacyl (G) and syringyl (S) units which are incorporated into the lignin polymer.

[0011] Hardwood consists of syringyl (S) and guaiacyl (G) units, whereas softwood consists mainly of guaiacyl (G) units and a small amount of p-hydroxyphenyl (H) units. Figure 2 SUMMARY

[0012] TECHNICAL PROBLEM

[0013] The main object of the present invention is to extract lignin from the coconut shell and / or coconut shell fiber powder obtained from the fruit of the tropical plant coconut (Cocos nucifera) belonging to the family Arecaceae, to develop a new natural biocompatible multifunctional biopolymer which can be used for pharmaceutical, nutraceutical, cosmeceutical, cosmetic and / or leather industry applications.

[0014] Another object of the present invention is to extract lignin components from coconut shell and / or coconut shell fiber pith powder, purify, study its physicochemical properties, process it into various biopolymer sheets / films / gels using other additional copolymers, crosslinking agents and plasticizers.

[0015] Another object of the present invention is to develop biopolymer sheets / films / gels for severe burn accident victims, the protective wound dressing layer is made from a combination of the following ingredients: 1) lignin extracted from coconut shell and / or coconut shell fiber pith powder, and 2) alpha-lactalbumin extracted from whey protein isolate, and 3) additional other copolymers, crosslinking agents and plasticizers. Since lignin polymers have antimicrobial, antioxidant and moisturizing properties, they have the potential to be used for wound dressing in severe burn accidents to restore new growth which replaces the damaged tissue at the site of the burn wound, especially when combined with API class recombinant human growth factors. It can also be further used for healing and restoration of damaged tissue through a variety of immune system-mediated repair mechanisms, as well as regeneration and replacement of damaged burn tissue with new growth.

[0016] Another object of the present invention is to provide leather substitute material which can be converted into leather (non-animal source) using leather enhancing material obtained from the fibers of coconut shell and / or coconut shell pith powder, thereby exhibiting excellent wear resistance and flexibility, especially as a leather substitute. ​

[0017] Technical Solution

[0018] As an agricultural waste, coconut shell and / or coconut shell fiber pith powder has been proven to be a rich source of lignin as well as cellulose. The improvement in solubility of lignin depends on the method of extraction and drying as well as the adjustment of pH and temperature. Lignin was successfully converted into multifunctional biopolymer sheets / films / gels using various additional copolymers, crosslinkers, and plasticizers, although the effect of each additive was studied at a basic level. The present invention developed multifunctional biopolymer sheets / films / gels and studied their application effects in the manufacturing process and final quality of wound dressings and leather replacement materials, respectively.

[0019] Lignin is an excellent organic polymer with multiple chemical components, which makes it very suitable for use in burn and wound healing dressings. Its phenolic compounds have natural antimicrobial and antioxidant properties, effectively preventing infection and reducing oxidative stress, thereby promoting healing. The main monolignols - coniferyl alcohol, sinapyl alcohol, and p-coumaryl alcohol - play a key role in maintaining the structural integrity of lignin, antimicrobial activity, and supporting tissue repair. Methoxyl groups increase chemical reactivity, allowing the development of customized derivatives to improve moisture retention and controlled drug release. In addition, carboxylic acid groups increase hydrophilicity, forming hydrogels that maintain a moist healing environment and enhance the binding capacity of therapeutic agents. Additionally, quinones introduce redox properties, which help control infection and inflammation, while lignin-carbohydrate complexes promote biocompatibility, making lignin a multifunctional and efficient material for advanced wound care solutions.

[0020] On the other hand, alpha-lactalbumin has been reported to play an excellent role in promoting new skin regeneration in burn wounds by stimulating fibroblast proliferation and adhesion, as well as by increasing local serotonin concentration, thereby accelerating the healing of burn wounds. It supports the supply of essential amino acids, especially the highest levels of tryptophan, which are required for protein synthesis and tissue repair. In addition, it helps regulate the immune response, minimizing excessive inflammatory reactions, and controls the production of cytokines, promoting collagen synthesis for structural integrity. When alpha-lactalbumin is added to burn wound dressings incorporating lignin, alpha-lactalbumin also helps create a better moist wound environment that is essential for cell migration and tissue formation, while the antioxidant properties of lignin reduce oxidative stress, further promoting effective skin regeneration.

[0021] In addition to the above uses for burn wound dressings, alpha-lactalbumin can be incorporated into a multi-functional lignin biopolymer that constitutes a prototype leather substitute material derived from coconut shell and / or coconut shell pith fiber powder, with or without additional leather reinforcing materials. If the ideal composition is provided, the resulting leather substitute can provide excellent wear resistance from lignin and soft, enveloping flexibility from alpha-lactalbumin.

[0022] More particularly, the present invention relates to a natural biocompatible multi-functional biopolymer comprising: a) lignin extracted from coconut shell and / or coconut shell fiber pith powder obtained from the fruit of the tropical plant coconut (Cocos nucifera) belonging to the family Arecaceae; b) a copolymer; c) a crosslinking agent; d) a plasticizer; and e) at least one active agent.

[0023] In another embodiment, the present disclosure relates to a novel natural biocompatible multi-functional biopolymer, wherein the at least one copolymer is selected from natural polymers or water-soluble synthetic polymers. Natural polymers are, for example, hyaluronic acid and its derivatives, starch, modified starch, alginate, chitosan, chitin, natural gums; proteins, such as gelatin, collagen, casein, zein, gluten, soy protein isolate, and whey protein isolate; land plant extracts, such as pectin; and combinations thereof. Water-soluble synthetic copolymers include cross-linked polyacrylic acid chains, polyvinylpyrrolidone, polyvinyl alcohol; modified cellulose ethers, such as hydroxypropyl methylcellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, carboxymethyl cellulose; ion exchange resins, and combinations thereof. The effective amount of the copolymer is about 10% to 90% by dry weight, preferably 30% to 70% by dry weight, depending on the application of the developed biocompatible multi-functional polymer.

[0024] In another embodiment, the present disclosure relates to a novel natural biocompatible multi-functional biopolymer, wherein the at least one crosslinking agent is selected from glutaraldehyde, formaldehyde, polyethylene glycol (PEG) and its derivatives, genipin, dextran, polyethyleneimine (PEI), ethylenediamine (EDA), and sugar alcohols / polyols, such as erythritol, xylitol, maltitol, mannitol, lactitol, polyglycitol, isomaltitol, sorbitol, glycerol, and combinations thereof, to facilitate the crosslinking chemical reaction. The effective amount of the crosslinking agent is about 1% to 10% by dry weight, preferably 2% to 5% by dry weight, depending on the application of the developed biocompatible multi-functional polymer.

[0025] In another embodiment, the present disclosure relates to a novel natural biocompatible multifunctional biopolymer, wherein at least one plasticizer is selected from sucrose ester derivatives, glycerol, propylene glycol, polyethylene glycol, magnesium stearate, bovine tallow, neatsfoot oil, lanolin, mineral oil, silicone oil, jojoba oil, castor oil, coconut oil, whale oil, squalene, grape seed oil, safflower oil, canola oil, and almond oil, and the like, and combinations thereof. The effective amount of the plasticizer is about 0.5% to 10% by dry weight, preferably 1% to 3% by dry weight, depending on the application of the developed biocompatible multifunctional polymer.

[0026] In another embodiment, the present disclosure relates to a novel natural biocompatible multifunctional biopolymer, wherein the optional emulsifier is selected from xanthan gum, lanolin, sodium alginate, lecithin, casein, whey protein, pectin, ammonium phosphatides, gum arabic, and oleylamine. The effective amount ranges from 0.5% (w / w) to 5% (w / w), depending on the application of the developed biocompatible multifunctional polymer.

[0027] In another embodiment, the present disclosure relates to a novel natural biocompatible multifunctional biopolymer, wherein the optional emulsifier is selected from xanthan gum, lanolin, sodium alginate, lecithin, casein, whey protein, pectin, ammonium phosphatides, gum arabic, and oleylamine.

[0028] In another embodiment, the present disclosure relates to a novel natural biocompatible multifunctional biopolymer, wherein it is used as a wound dressing or a part of a wound dressing on severe burn wounds of a patient to restore new tissue growth and replace damaged tissue on burn wounds, wherein at least one active agent can be loaded from turmeric extract (curcumin), honey, green tea extract, gota kola extract (Centella asiatica), alpha-lactalbumin, whey protein isolate, comfrey extract, tea tree oil, lavender oil, chamomile extract, aloe vera gel, silver sulfadiazine, mupirocin, hyaluronic acid, collagen, vitamin E, allantoin, sphingosine / phytosphingosine-1 -phosphate, dexpanthenol (vitamin B5), and combinations thereof. The effective amount of the at least one active agent is about 0.01% to 50% by dry weight, preferably 1% to 20% by dry weight, depending on the application and severity of the burn wound.

[0029] In another embodiment, the present disclosure relates to a novel natural biocompatible multifunctional biopolymer, wherein it is used as a wound dressing or a part of a wound dressing on severe burn wounds of a patient to restore new tissue growth and replace damaged tissue on burn wounds, wherein at least one active agent can be loaded from human growth factors and / or cytokines such as epidermal growth factor (EGF), growth hormone / somatotropin (hGH / STH), vasoactive intestinal peptide (VIP), fibroblast growth factor family proteins, i.e. fibroblast growth factor-1 (FGF1), fibroblast growth factor-2 (FGF2), fibroblast growth factor-7 / kertinocyte growth factor (FGF7; KGF), fibroblast growth factor-10 / kertinocyte growth factor-2 (FGF10;KGF2), Fibroblast growth factor-19 (FGF19), Fibroblast growth factor-21 (FGF21), Fibroblast growth factor-23 (FGF23), etc., Vascular endothelial growth factor (VEGF), Thymosin beta-4 (TMS), Stem cell factor / Steel factor (SCF), Platelet-derived growth factor (PDGF), Prolactin (PRL), Placental lactogen (hPL), Nerve growth factor (NGF), Brain-derived neurotrophic factor (BDNF), Glial cell-derived neurotrophic factor (GDNF), Ciliary neurotrophic factor (CNTF), Interleukin-1 receptor antagonist (IL1Ra), Interleukin family proteins, i.e., Interleukin-2 (IL-2), Interleukin-4 (IL-4), Interleukin-13 (IL-13), Interleukin-17 (IL-17), Interleukin-18 (IL-18), etc., Adiponectin (ApN), TSLP (thymic stromal lymphopoietin), Bone morphogenetic protein family proteins, i.e., BMP-2, BMP-4, BMP-7, etc., Cluster of differentiation-34 (CD34), Desmoplakin (Dsp), Erythropoietin (EPO), Erythroid differentiation regulatory-1 (ERDR1), Fibronectin, Follicle-stimulating hormone (FSH), Luteinizing hormone (LH), Human chorionic gonadotropin (hCG), Follistatin (FST), Growth differentiation factor family proteins, i.e., Growth differentiation factor 11 / Bone morphogenetic protein-11 (GDF11 / BMP-11), Growth differentiation factor 15 (GDF15), etc., Irisin, Kisspeptins, Klotho, Matrikines (extracellular matrix-derived peptides), Scube3 (signal peptide, CUB and EGF-like domain protein 3), Osteonectin, Oxytocin, Pregnancy-associated plasma protein-A (PAPP-A), Platelet factor 4 (PF4), Reelin, Stem cell antigen-1 (Sca-1), Stanniocalcin-1 (STC1), Stanniocalcin-2 (STC2), Transforming growth factor (TGF) family proteins, i.e., TGF-alpha, TGF beta-1, TGF beta-2, TGF beta-3, Thyrotropin-releasing hormone (TRH), Thyroid-stimulating hormone (TSH), Heat shock protein family proteins, i.e., HSP90A and its derivatives, etc., and combinations thereof. The effective amount of at least one or more active agents is about 0.000001% to 1% dry weight, preferably 0.00001% to 0.1%, and more preferably 0.00005% to 0.005%, depending on the application and severity of the burn wound.

[0030] In another embodiment, the present disclosure relates to a novel natural biocompatible multifunctional biopolymer wherein the combination of lignin and alpha-lactalbumin has been proven to be useful in the preparation of an effective wound dressing for severe burn wounds, wound healing, reducing scar formation and accelerating epithelial regeneration, as well as restoring new tissue growth and replacing damaged burn wound tissue.

[0031] In another embodiment, the present disclosure relates to a novel natural biocompatible multifunctional biopolymer wherein it is used for developing leather substitute or a part of leather substitute, wherein leather substitute prototypes with or without alpha-lactalbumin or leather enhancing material obtained from fibers of coconut shell and / or coconut shell fiber pith powder both exhibit excellent abrasion resistance and flexibility of natural leather. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 : Depicts a) percentage distribution of components in coconut shell; and b) percentage distribution of lignin in different plant materials.

[0033] Figure 2 : Depicts the structure of lignin component.

[0034] Figure 3a and Figure 3b : Depicts Figure 3a : Extraction of lignin from coconut shell; Figure 3b : Isolation of alpha-lactalbumin from whey protein.

[0035] Figure 4 : Depicts zeta potential and particle size of lignin.

[0036] Figure 5 : Depicts ATR-IR graph of extracted lignin.

[0037] Figure 6 : Depicts X-ray diffraction graph (XRD) of extracted lignin.

[0038] Figure 7 : Depicts differential scanning calorimetry graph (DSC) of extracted lignin.

[0039] Figure 8 : Depicts polarized light microscope photograph of ferric chloride test.

[0040] Figure 9 : Depicts lignin-based biopolymer films - HLG (F1), XGPG (F2) and PXLG (F3).

[0041] Figure 10 : Depicts thickness of films.

[0042] Figure 11 : Depicts folding endurance of lignin-based biopolymer films.

[0043] Figure 12 Texture and elastic modulus diagrams of lignin-based biopolymer membranes HLG (F1), XGPG (F2), and PXLG (F3) are depicted.

[0044] Figure 13 The tensile strength and elastic modulus of the lignin-based biopolymer membrane were analyzed.

[0045] Figure 14 The swelling degree of the lignin-based biopolymer membrane was described.

[0046] Figure 15 The water retention rate of the lignin-based biopolymer membrane was described.

[0047] Figure 16 The water vapor permeability of the lignin-based biopolymer membrane is described.

[0048] Figures 17a-17d : Depicted Figure 17a At 2360cm- 1 The membrane showed excessive photodegradation of glutaraldehyde into CO2. Figure 17b Infrared spectrum of HLG film; Figure 17c Infrared spectrum of PXLG film; and Figure 17d Infrared spectrum of XGPG film.

[0049] Figure 18 XRD analysis of lignin-based biopolymer membranes is depicted.

[0050] Figure 19 : A DSC overlay of an XGPG membrane loaded with α-lactalbumin is depicted.

[0051] Figure 20 SEM image depicting the lignin-based biopolymer XGPG(F2) membrane.

[0052] Figure 21 The histopathology of tissue samples is described as follows: (Normal group - no damage, as a reference; negative control group - damaged, but no treatment; blank membrane group - received a blank lignin-based biopolymer membrane without α-lactalbumin; standard group - also damaged, but treated with commercially available silver sulfadiazine cream; treatment group - damaged, and treated with a membrane containing α-lactalbumin).

[0053] Figure 22 : Depicts a prototype of an animal leather alternative derived from lignin-based biopolymers. Detailed Implementation

[0054] Those skilled in the art will recognize, given the benefit of this disclosure, that the present disclosure has additional features and modifications which are within the scope of the disclosure. It is understood that the present disclosure encompasses all such variations and modifications. The present disclosure also encompasses all of the steps, features, compositions and compounds individually or collectively referred to or indicated in the specification and any combination of any and all of the steps or features.

[0055] For convenience, before further description of the present disclosure, some of the terms used in the specification and examples are collected here. The terms used herein have the meanings recognized and known by those skilled in the art, however, for the sake of convenience and completeness, specific terms and their meanings are listed below.

[0056] The articles "a", "an" and "the" are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.

[0057] The terms "comprising" and "including" are used in the inclusive, open- ended sense and mean that other elements can also be included. It is not intended to be construed as "only comprising" or "only including".

[0058] Throughout this specification, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated element or step or group of elements or steps but not the exclusion of any other element or step or group of elements or steps.

[0059] The term "comprising" is used to mean "including but not limited to". "Comprising" and "including but not limited to" can be used interchangeably.

[0060] Proportions, concentrations, amounts, and other numerical data can be expressed herein in a range format. It is to be understood that such range format is used merely for convenience and brevity and should be construed as having been followed to following the principle that more restrictive ranges are within the scope of the ranges given, as if each narrower range is explicitly written herein.

[0061] In one embodiment of the present disclosure, a method for extraction and purification of lignin from coconut shell and / or coconut shell fiber pith powder is provided, which comprises: 1) extracting lignin from coconut shell using Kraft pulping process; 2) making a solution by adding 10 g to 80 g of coconut shell powder, more preferably 20 g to 40 g of coconut shell powder, in 200 ml of 12.5% w / v aqueous NaOH solution, keeping in an autoclave (Lequitron) at 130°C for 3 hours; 3) then filtering the NaOH solution containing dissolved lignin through muslin cloth using a vacuum filter, separating and keeping the residue / filter cake containing delignified material, collecting the filtrate (black liquor) and keeping in a beaker; 4) treating the collected black liquor with concentrated sulfuric acid until the pH reaches 2, at which time the precipitated lignin is clearly visible, then letting the solution stand for 4 hours (lignin precipitate at the bottom, while the acidic liquid forms a separate layer at the top); 5) carefully discarding the liquid portion, then centrifuging the remaining solution at 10,000 RPM for 10 minutes, discarding the supernatant, and obtaining the residue in the form of lignin; 6) further washing the lignin with distilled water, then passing it through a vacuum filter using filter paper (4.5 um pore size, nylon 66); 7) drying the collected lignin in a hot air oven at 60°C overnight, then pulverizing it into fine powder using a mortar and pestle, and storing in a plastic bag Figure 3a

[0062] In one embodiment of the present disclosure, the development of a new natural biocompatible multifunctional biopolymer is set forth herein, which comprises a lignin component extracted from coconut shell and / or coconut shell fiber pith powder, a copolymer, a crosslinking agent, and a plasticizer.

[0063] In one embodiment of the present disclosure, the development of a new natural biocompatible multifunctional biopolymer is set forth herein, which comprises a lignin component extracted from coconut shell and / or coconut shell fiber pith powder, a copolymer, a crosslinking agent, and a plasticizer, wherein the polymer can be natural and / or synthetic. Some examples of natural polymers are hyaluronic acid and its derivatives, starch, modified starch, alginate, chitosan, chitin, natural gums, proteins such as gelatin, collagen, casein, zein, gluten, soy protein isolate, and whey protein isolate; land plant extracts such as pectin; and combinations thereof. Water-soluble synthetic copolymers include crosslinked polyacrylic acid chains, polyvinylpyrrolidone, polyvinyl alcohol; modified cellulose ethers such as hydroxypropyl methylcellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, carboxymethyl cellulose; and ion exchange resins. The effective amount of the copolymer is about 10% to 90% by dry weight, preferably 30% to 70%, depending on the application of the developed biocompatible multifunctional polymer.

[0064] ​The term "copolymer" is a material formed by combining two or more polymers to form a network structure, wherein at least one polymer is synthesized or cross-linked in the direct presence of the other polymer. These different polymers and / or copolymers are physically intertwined or internally intertwined with each other, forming a single intertwined network, thereby enhancing the mechanical and physical properties, and thus are ideally used for various applications such as biomedical adhesives and impact-resistant materials. Although the polymers and copolymers are not necessarily covalently bonded to each other (i.e., covalently bonded, for example, by a cross-linking agent), they form a cohesive structure that cannot be separated without breaking some of the chemical bonds that connect them.

[0065] In one embodiment of the present application, the development of a new natural biocompatible multifunctional biopolymer is set forth herein, which comprises a lignin component extracted from coconut shell and / or coconut shell fiber pith powder, a copolymer, a cross-linking agent, and a plasticizer, wherein the cross-linking agent can be selected from glutaraldehyde, formaldehyde, polyethylene glycol (PEG) and its derivatives, genipin, dextran, polyethyleneimine (PEI), ethylenediamine (EDA), and sugar alcohols / polyols such as erythritol, xylitol, maltitol, mannitol, lactitol, polyglycitol, isomaltitol, sorbitol, glycerol, and combinations thereof, to facilitate the cross-linking chemical reaction. The effective amount of the cross-linking agent is about 1% to 10% by dry weight, preferably 2% to 5%, depending on the application of the developed biocompatible multifunctional polymer.

[0066] In one embodiment of the present application, the development of a new natural biocompatible multifunctional biopolymer is set forth, which comprises a lignin component extracted from coconut shell and / or coconut shell fiber pith powder, a copolymer, a cross-linking agent, and a plasticizer, wherein the plasticizer can be selected from sucrose ester derivatives, glycerol, propylene glycol, polyethylene glycol, magnesium stearate, tallow, neatsfoot oil, lanolin, mineral oil, silicone oil, jojoba oil, castor oil, coconut oil, whale oil, squalene, grape seed oil, safflower oil, canola oil, almond oil, and combinations thereof. The effective amount of the plasticizer is about 0.5% to 10% by dry weight, preferably 1% to 3%, depending on the application of the developed biocompatible multifunctional polymer.

[0067] In one embodiment of the present application, the development of a new natural biocompatible multifunctional biopolymer is set forth, which comprises a lignin component extracted from coconut shell and / or coconut shell fiber pith powder, a copolymer, a cross-linking agent, and a plasticizer, wherein the optional emulsifier is selected from xanthan gum, lanolin, sodium alginate, lecithin, casein, whey protein, pectin, phosphatidyl, gum arabic, and oleylamine. The effective amount ranges from 0.5% (w / w) to 5% (w / w), depending on the application of the developed biocompatible multifunctional polymer.

[0068] In one embodiment of the present application, there is provided the development of a new natural biocompatible multifunctional biopolymer which has been successfully researched for wound dressing in severe burn accidents to restore new growth in place of damaged tissue at the burn wound, wherein at least one or more active agents can be loaded from turmeric extract (curcumin), honey, green tea extract, gota kola extract (gotu kola), alpha-lactalbumin, whey protein isolate, comfrey extract, tea tree oil, lavender oil, chamomile extract, aloe vera gel, silver sulfadiazine, mupirocin, hyaluronic acid, collagen, vitamin E, allantoin, sphingosine / phytosphingosine-1 -phosphate and dexpanthenol (vitamin B5). The effective amount of at least one or more active agents is about 0.01% to 50% by dry weight, preferably 1% to 20%, depending on the application and severity of the burn wound.

[0069] In one embodiment of the present application, there is provided the development of a new natural biocompatible multifunctional biopolymer that has been successfully researched for use in wound dressing for severe burn accidents to restore new growth to replace the damaged tissue at the site of burn wounds, wherein at least one or more active agents can be loaded from human growth factors and / or cytokines such as epidermal growth factor (EGF), growth hormone / somatotropin (hGH / STH), vasoactive intestinal peptide (VIP), fibroblast growth factor family proteins, namely fibroblast growth factor-1 (FGF1), fibroblast growth factor-2 (FGF2), fibroblast growth factor-7 / kertinocyte growth factor (FGF7; KGF), fibroblast growth factor-10 / kertinocyte growth factor-2 (FGF10;KGF2), Fibroblast growth factor-19 (FGF19), Fibroblast growth factor-21 (FGF21), Fibroblast growth factor-23 (FGF23), etc., Vascular endothelial growth factor (VEGF), Thymosin beta-4 (TMS), Stem cell factor / Steel factor (SCF), Platelet-derived growth factor (PDGF), Prolactin (PRL), Placental lactogen (hPL), Nerve growth factor (NGF), Brain-derived neurotrophic factor (BDNF), Glial cell-derived neurotrophic factor (GDNF), Ciliary neurotrophic factor (CNTF), Interleukin-1 receptor antagonist (IL1Ra), Interleukin family proteins, i.e., Interleukin-2 (IL-2), Interleukin-4 (IL-4), Interleukin-13 (IL-13), Interleukin-17 (IL-17), Interleukin-18 (IL-18), etc., Adiponectin (ApN), TSLP (thymic stromal lymphopoietin), Bone morphogenetic protein family proteins, i.e., BMP-2, BMP-4, BMP-7, etc., Cluster of differentiation-34 (CD34), Desmoplakin (Dsp), Erythropoietin (EPO), Erythroid differentiation regulatory-1 (ERDR1), Fibronectin, Follicle-stimulating hormone (FSH), Luteinizing hormone (LH), Human chorionic gonadotropin (hCG), Follistatin (FST), Growth differentiation factor family proteins, i.e., Growth differentiation factor 11 / Bone morphogenetic protein-11 (GDF11 / BMP-11), Growth differentiation factor 15 (GDF15), etc., Irisin, Kisspeptins, Klotho, Matrikines (extracellular matrix-derived peptides), Scube3 (signal peptide, CUB and EGF-like domain protein 3), Osteonectin, Oxytocin, Pregnancy-associated plasma protein-A (PAPP-A), Platelet factor 4 (PF4), Reelin, Stem cell antigen-1 (Sca-1), Stanniocalcin-1 (STC1), Stanniocalcin-2 (STC2), Transforming growth factor (TGF) family proteins, i.e., TGF-alpha, TGF beta-1, TGF beta-2, TGF beta-3, Thyrotropin-releasing hormone (TRH), Thyroid-stimulating hormone (TSH), Heat shock protein family proteins, i.e., HSP90A and its derivatives, etc., and combinations thereof. The effective amount of at least one or more active agents is about 0.000001% to 1% dry weight, preferably 0.00001% to 0.1%, and more preferably 0.00005% to 0.005%, depending on the application and severity of the burn wound.

[0070] In one embodiment of the present application, there is provided a process for isolation of alpha-lactalbumin from whey protein (purchased from Suzhou Greenway Biotech Co. Ltd, China), the process comprising extracting alpha-lactalbumin from whey protein isolate (90%) and removing casein, which makes milk white, by centrifugation. Subsequently, NaCI is used as a detergent and a penetrating agent. Ca++ bound to the protein is released from alpha-lactalbumin by lowering the pH to 4.2 with citric acid monohydrate (40 mM / L). This results in the conversion of Ca++ bound alpha-lactalbumin to a Ca++ free moltenglobule apo-form, which can be collected by a vacuum filter. Restoration of the pH equilibrium results in the apo-form reverting to its native form in the presence of Ca++. The method is efficient and extracts alpha-lactalbumin (90%) with high efficiency. Figure 3b

[0071] In one embodiment of the present application, there is provided development of a new natural biocompatible multifunctional biopolymer, which has been successfully researched for wound dressing in severe burn accidents to restore new growth in place of damaged tissue at the site of burn wounds, wherein alpha-lactalbumin, a tryptophan-rich protein, has been proven to have healing properties in various studies, however, the use of whey-extracted alpha-lactalbumin as a wound dressing has not been explored. In this study, it was demonstrated that the combination of lignin and alpha-lactalbumin effectively promoted wound healing in Swiss albino mice. Further, the combination of lignin and alpha-lactalbumin was found to reduce scar formation at a faster rate and accelerate epithelial regeneration as compared to the commercially available silver sulfadiazine cream. Notably, animals treated with the membrane loaded with alpha-lactalbumin exhibited greater stimulation in hair follicles and the time of hair regeneration was earlier as compared to their control group. These findings suggest that the combination of lignin and alpha-lactalbumin holds promise as an effective wound dressing to promote wound healing and reduce scar formation.

[0072] In one embodiment of the present application, there is provided development of a new natural biocompatible multifunctional biopolymer for further development of a leather substitute prototype, wherein the production of the prototype involves complete adhesion and sealing of the leather substitute material and leather enhancing material. This is achieved by placing a weight on top of the material for 48 hours at 25°C, followed by completion of the coating of the wax emulsion, softener, and wetting agent. The result is a completely sealed prototype, which can be cut to specifications, wherein the leather enhancing material can be obtained from fibers of coconut shell and / or coconut shell fiber pith powder, foam fillers using EVA (ethylene-vinyl acetate), mesh fabrics such as polyester or nylon, glass fibers, polyethylene or polycarbonate, rubber, and combinations thereof, which are uniformly attached to the cellulose water-based adhesive present in the leather substitute material. DETAILED DESCRIPTION​

[0074] Examples

[0075] Working examples of the present disclosure will now be described. These examples are neither limiting nor intended to limit the scope of the application. 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 application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, exemplary methods, devices, and materials are described herein. It is to be understood that this application is not limited to the particular methodology, protocols, and reagents described, as these can vary.

[0076] Example 1

[0077] Extraction and characterization of lignin from coconut shell and / or coconut shell pith powder

[0078] Materials

[0079] Coconut and / or coconut shell pith powder was procured from various vendors of local market of Shirpur, Maharashtra, India; sodium hydroxide, sulfuric acid, glutaraldehyde, other organic solvents and starch were procured from Rankem, Mumbai, India; xanthan gum, sorbitol, glycerol were obtained from Loba Chemie Pvt. Ltd, Mumbai; HPC (hydroxypropyl cellulose) and PVA Hot (polyvinyl alcohol) were procured from Research Lab Pvt. Ltd, Mumbai. Coconut fibers were manually removed, shells were ground in a kitchen grinder and the powder was dried in an oven overnight to remove excess moisture, and stored in sealed plastic bags at room temperature (25 °C) for further use.

[0080] Method for extraction of lignin from coconut shell powder

[0081] Lignin was extracted from coconut shell powder using Kraft pulping method. For extraction of lignin, two solutions of coconut shell powder samples were prepared, each in 200 ml of 12.5% w / v aqueous NaOH solution containing 10 g to 80 g (more preferably 20 g to 40 g) of dry coconut shell powder, one solution was then kept in an autoclave (Lequitron) at 130 °C for 3 hours and the other solution was kept in a microwave oven at 140 °C for 1 hour (with 10 minutes interval) for further processing. Figure 3a

[0082] Characterization of lignin

[0083] Yield of extract:

[0084] ​The weight of extracted lignin / weight of coconut shell before lignin extraction was used to determine the percentage yield of lignin. The coconut shell powder was dried in a hot air oven at 60 °C for 12 hours to remove excess moisture. After the alkaline treatment of the pre-weighed dried coconut shell powder (W2) and extraction of lignin through a muslin filter, the lignin in the filtered black liquor was precipitated by drop-wise addition of concentrated sulphuric acid to pH 2. The precipitated lignin was separated and collected by centrifugation or filtration on Whatman paper (grade 42, 2.5 pm pore size), dried in a hot air oven at 60 °C for 8 hours, washed twice with water to remove impurities and then dried again in a hot air oven at 60 °C for 12 hours. Subsequently, the dried lignin was pulverized using a mortar and pestle and weighed again (W1).

[0085]

[0086] W1 is the weight of the dried lignin powder obtained.

[0087] W2 is the initial weight of the coconut shell powder taken.

[0088] For a coconut shell powder of initial weight 30 g (W1), about 8.4 g of lignin was obtained (W2), thus the yield of lignin from the coconut shell powder was 28%. The theoretical yield for various studies is about 34%, thus the percentage yield of Kraft extraction is about 82.35%. The yield can also be improved in future implementations by improving the heating method and optimizing the pH in the extraction. Further micronization of the coconut shell powder should also improve the yield.

[0089] Particle size and zeta potential

[0090] The particle size and surface charge of the lignin thus prepared was determined to assess the effect of pH on the lignin particles obtained. For this experiment, 10 mg of lignin was dissolved in 100 ml of aqueous buffer solution at neutral (pH 7) and alkaline pH (pH 12) and then using a syringe, the solution was transferred to a Zeta Sizer (Malvern) cuvette to ¾ of its volume. The particle size and zeta potential were determined.

[0091] The objective of this study was to examine the effect of pH on the change in particle size of lignin depending on the different pH ranges of the solvent. It was found that the particle size of lignin decreased significantly as the pH increased from 5 to 13. This can be attributed to the solubility of lignin in alkaline solutions (> pH 12) with an isoelectric point estimated to be pH 2. At neutral pH, the particle size of lignin was observed to be 323 nm, while when the pH level exceeded 9, the particle size decreased to 205 nm. At neutral pH, lignin exhibited a value of -13.5 mV (anionic) which falls within the stability range of -30 mV to +30 mV (Figure 4 ).

[0092] ATR-IR (attenuated total reflectance infrared) of lignin

[0093] Lignin powder was dried in a hot air oven for 8 hours prior to analysis to remove moisture, thus reducing noise in the following results. The lignin powder was crushed using a mortar and pestle, then pressed under an ATR-IR sampler. The chemical composition of the lignin was analysed using a Brookfield ATR-IR spectrophotometer in the range of 4000 cm -1 to 400 cm -1 .

[0094] The FTIR (Fourier Transform Infrared) spectrum provided shows the transmittance (%) of a lignin sample over a range of wave numbers (cm -1 ). The spectrum shows distinct peaks which indicate the presence of functional groups in the lignin. Specifically, there is a broad peak near 3400 cm 1 corresponding to O-H stretching, a sharp peak near 2930 cm -1 indicating C-H stretching. Additionally, there are characteristic peaks near 1600 cm -1 and 1510 cm -1 associated with aromatic C=C stretching, a peak near 1260 cm -1 corresponding to C-O stretching (aryl ether), and peaks near 1120 cm -1 and 1030 cm -1 associated with guaiacyl units in alcohols and ethers, respectively. Figure 5 .

[0095] X-ray Diffraction (XRD)

[0096] In order to determine the properties of the lignin powder and its purity with respect to amorphous to crystalline state, an X-ray diffractometer was used. The sample was crushed in a mortar and pestle, transferred to a cuvette, and then analysed using a Bruker D2 Phaser X-ray diffractometer. The voltage and current used were 40 kV and 30 mA, respectively, and the XRD pattern was obtained in the range of 5° to 50° 2-theta with a step size of 0.02° and a scan rate of 5° / min.

[0097] The extracted lignin has 86% amorphousness and 14% crystallinity without any sharp peaks. All the peaks are kept in a relatively short range of 200 to 500 counts. Lignin generally exhibits a broad diffuse scattering pattern. The amorphous nature of lignin is due to its complex and irregular polymeric structure. The phenolic monolignols form a three-dimensional network, lacking regular repeating units and long-range order as a feature of crystalline structure. This lesser crystallinity is due to the lignin precipitation process transfer and the presence of sulfur impurities remaining during the washing step Figure 6 ).

[0098] Differential scanning calorimetry

[0099] To evaluate the thermal properties of lignin, a differential scanning calorimeter (TA Instruments, DSC Discovery 250) was used to monitor the thermal events during the curing process. 2.4 mg of lignin powder was placed in an aluminum open pan and subjected to thermal analysis in the temperature range of 30 °C to 325 °C. The DSC curve was recorded under N2 atmosphere (50 mL / min) with a heating rate of 10 °C / min.

[0100] The initial slope at 40 °C is due to the instrument calibration. The presence of moisture in lignin due to the initial resolution of moisture, lignin exhibits a sharp endothermic peak. Drying the lignin powder at higher temperatures can have a reduced moisture content, but it can affect the appearance of lignin and its solubility. The absence of any secondary peaks indicates that lignin has been successfully extracted and isolated from cellulose, hemicellulose, and xylan, as they degrade well in this range. There is no appearance of any phase transition of lignin in the range of 300 °C Figure 7 ).

[0101] UV-visible spectrophotometry

[0102] The wavelength and absorption spectrum were used to identify and determine the linearity of lignin. Using a Shimadzu 1900 UV spectrophotometer, 10 mg of lignin was dissolved in 10 ml of deionized water using ultrasonication to obtain a stock solution of 1000 ppm, which was then diluted to 100 ppm. The λ max values and linearity range were determined.

[0103] In the UV-visible spectrum, lignin appears to have a peak at 272 nm (λ max ), which is very common for kraft lignin, which originates from the aromatic ring / non-conjugated phenolic group. Lignin has typical absorption peaks at 230 nm and 275 nm due to the electronic transition of non-conjugated phenolic structures in aromatic compounds. The linearity was determined in distilled water in the concentration range of 20 μg / ml to 100 μg / ml (R 2 = 0.999).

[0104] Other tests

[0105] The phenol ferric chloride test was performed on 1% w / v aqueous solution of lignin by drop wise addition of freshly prepared ferric chloride solution and observed whether the resulting solution showed visible color change. Polarized light microscopy (PLM) was used to understand the particle shape, uniformity and size. The lignin liquid solution in deionized water was observed under PLM at 40X and 100X magnification.

[0106] At neutral pH, the range of undissolved particles appeared was 10 pm to 37 pm with few particles of around 70 pm. Most of the dissolved lignin particles were in the range of 200 nm to 300 nm at neutral pH and 200 nm at pH 8 as shown in Zeta Sizer. The particles appeared uniform in round to rhombus shape. The extracted lignin was dissolved in distilled water and the resulting solution showed positive ferric chloride test by turning dark green in color. Figure 8 ).

[0107] Example 2

[0108] Pharmaceutical, nutraceutical and cosmetic applications of bio-polymer formulations of lignin extracted from coconut shell and / or coconut coir pith powder

[0109] In the present invention, three different bio-polymers were manufactured using different ways by following the methods.

[0110] A free flowing solution was obtained by mixing HPC and lignin in the ratio of 3:2 using 2.5% concentration of glutaraldehyde as cross-linking agent. Sulfuric acid was added drop wise during mixing. The resulting solution was named as HLG bio-polymer (F1). A thick viscous solution coded as XGPG bio-polymer (F2) was obtained by mixing polyethylene glycol of concentration 10% with xanthan gum and lignin in the ratio of 1:1. The mixture was stirred at 2000 rpm; 85°C to 90°C (high speed homogenizer, IKA T25 Digital Ultra Turrax, Mumbai, India). A viscous solution was obtained by mixing polyvinyl alcohol, xanthan gum and lignin in the ratio of 1:1:1, called as PXLG bio-polymer (F3); the mixture was stirred at 2000 rpm at 85°C to 90°C along with glycerol (1.5%) as plasticizer. Figure 9

[0111] ​Subsequently, α-lactalbumin (ALA), extracted from whey protein as described in the embodiments, was added to each of the three biopolymers, and the solutions were then mixed. Finally, the solutions were cast in a silicone mold (15 x 15 cm) and dried at 30°C for 48 hours. These biopolymers have the potential to revolutionize multiple fields, including pharmaceuticals, food, packaging, and leather alternatives. Further research is needed to explore their properties and potential applications in these and other fields.

[0112] Example 3

[0113] Characterization of lignin-based biopolymers

[0114] thickness

[0115] The width and thickness of the membranes were measured as follows: Five samples of similar size were randomly selected from each membrane, and then the lower jaws of a Mitutoyo Digimatic caliper were carefully clamped to fit the membrane. Each membrane was measured three times. Special care was taken to avoid excessive pressure when installing the caliper jaws, and the average value was calculated after multiple readings.

[0116] The membrane thickness should not be too thick to avoid disturbing the patient, while also having sufficient depth to effectively protect and accommodate the wound. Therefore, the membrane thickness was calculated by taking three readings and averaging the results. XGPG membrane was the thickest at 0.24 ± 0.01 mm, PXLG was in the middle at 0.22 ± 0.01 mm, and HLG was the thinnest at 0.21 ± 0.01 mm. Figure 10 ).

[0117] Foldability

[0118] To determine the foldability of the membrane, five samples of each membrane were cut into 3×3 cm pieces, fixed in the center with scissors, folded by hand, and then unfolded. This process was repeated multiple times until the membrane showed obvious cracks. The readings were recorded and the average value was calculated.

[0119] Any dressing used for wound dressings must have good folding resistance because it can be folded multiple times based on the wound area. For this purpose, the folding ability of the membranes was evaluated. Of all the membranes, HLG exhibited the best folding resistance, showing no cracks even after 500 folds, while PXLG showed slight bending and cracking at approximately 250 and 290 folds, respectively. The XGPG membrane also showed considerable folding resistance, but cracked at 190 folds. The following graph was plotted using the average values ​​for each membrane. Figure 11 ).

[0120] Texture analysis and elastic modulus

[0121] Three samples of each film were cut into 3 X 1 cm in size and analyzed using a CT3 Texture Analyzer (Brookfield Engineering Lab) at 30°C and 1 bar. Stress data, deformation at break point and elastic modulus were determined using data obtained from this study.

[0122] To determine the physical properties of the films, using the force applied on a known length of film, the elastic modulus was determined using the formula shown below.

[0123] E = F.L / (A.δ)

[0124] F is the force applied (load) in Newtons (N),

[0125] L is the original length of the sample in meters (m),

[0126] A is the cross-sectional area of the sample in square meters (m 2 ),

[0127] δ is the deformation of the sample (change in length) in meters (m).

[0128] PXLG showed the highest tensile strength followed by XGPG and HLG, the maximum load before break for PXLG, XGPG and HLG were 339 g, 270 g and 191 g respectively. Film PXLG also showed the maximum deformation at break point of 4.97 mm, followed by HLG of 2.75 mm and XGPG of 2.65 mm Figure 12 ).

[0129] Resistance to deformation is very important for any wound dressing as it should be able to maintain shape. Any shrinkage or elongation can expose the wound to the external environment and cause infection. This is also related to the level of physical protection that the dressing should be able to provide.

[0130] The load was converted from grams to Newtons (1 gram force is equal to 0.0098 N), using the aforementioned formula, the elastic modulus for PXLG, HLG and XGPG was determined as follows Figure 13 ).

[0131] Among all the films, PXLG had the highest TS due to the addition of PVA and glycerol cross-linked xanthan gum (GCX). Further dehydration should also increase its TS as the stability of GCX and PVA would be enhanced in the absence of water. This is because the presence of water would weaken the bonding of the hydroxyl group of glycerol to the hydrophilic functional groups of xanthan gum. The TS of XGPG film lagged behind PXLG due to the lower degree of cross-linking caused by PEG when PVA is present compared to glycerol. PEG remains mobile in the matrix when glycerol is immobilized (Domjan, Bajdik et al. 2009).

[0132] Film XGPG was more rigid than film PXLG and film HLG, while film PXLG and HLG were comparable in rigidity, with film HLG being slightly more rigid than film PXLG. Film XGPG was the most rigid, but film PXLG had the highest tensile strength. Film HLG had moderate rigidity but the lowest tensile strength. This correlation suggests that while rigidity (elastic modulus) and tensile strength can be correlated, they do not always increase proportionally. A material can be rigid but not necessarily strong, and vice versa.

[0133] Swelling degree

[0134] Swelling degree was measured by weighing 3 film samples each of 1X 1 cm (W1), then immersing them in a beaker containing 15 ml of phosphate buffer at pH 7.2 for 5 hours, weighing the weight of each sample every hour (W2). Using the following formula, the swelling degree was calculated.

[0135]

[0136] W1 is the initial weight of the film.

[0137] W2 is the weight of the film after being immersed in water.

[0138] Swelling degree is a measure of the degree of cross-linking and the ability to absorb in the absence of solubility itself. The uncross-linked portion plays a major role in swelling, so we confirmed the role of PEG as an external cross-linking agent, with XGPG having the highest swelling index of 430.56% after 5 hours, followed by PXLG 324.44%, which is due to the stability of GCX in the absence of water due to glycerol cross-linked xanthan gum functional groups, and HLG showing the lowest swelling degree of 14.28%, which is due to the extensive cross-linking between lignin and hydroxypropyl cellulose by glutaraldehyde Figure 14 ).

[0139] Water retention

[0140] The dry weight (W1) of the film of size 2 X 2 cm was taken and then it was placed in a beaker with 50 ml of water for 5 hours. After that, they were transferred to centrifuge tubes and centrifuged in a microcentrifuge (BioEra) at 500 rpm for 5 minutes. The supernatant was discarded and the wet weight (W2) of the film residue was weighed again following the method mentioned by Wang et al. (2019).

[0141] The water retention value (WRT) of the film was calculated using the following formula.

[0142]

[0143] The ability to retain moisture is most important for films targeted for wound dressings, as wounds exude fluids and the dressing must be able to withstand and contain this extracellular fluid. Moreover, it should withstand moisture from the outside without dissolving to suit the practical use.

[0144] HLG exhibited a hydrophobic tendency and had the lowest water retention capacity, while the other two films were able to retain 3 times more water than HLG due to the presence of xanthan gum in the films. Figure 15

[0145] Water vapor transmission rate

[0146] Three samples of each film were cut into a size of 1 X 1 cm and taped to the neck of a bottle using a Teflon tape, so that these films could safely plug the bottle opening of a vial of 10 ml of deionized water with a radius of 5 mm without coming into contact with the water. The vial was kept open so that the water vapor could pass freely. The vial was weighed initially (W1) and then again after 12 hours (W2). The water vapor transmission rate of each film was calculated using the formula shown below.

[0147] WVT = (W1 - W2) / (πr 2 ) · t

[0148] W1 is the initial weight of the vial with the film and tape.

[0149] W2 is the weight of the vial with the film and tape after 12 hours.

[0150] r is the radius of the vial opening.

[0151] t is the duration after which W2 is measured (i.e. 12 hours)

[0152] Films used as wound dressings should be able to create a moist wound environment to help promote cell migration and wound healing. The ideal range is approximately 2000 g / m 2 ​Of all the membranes, HLG has the lowest vapor transmission rate, followed by PXLG. Both of these membranes have low vapor transmission rates due to their good crosslinking properties, while XGPG, which uses PEG as an external crosslinking agent, has almost twice the vapor transmission rate of the other membranes. Figure 16 ).

[0153] ATR-IR

[0154] Place the sample under the ATR-IR sample holder and press lightly, then at 4000 cm⁻¹ -1 Up to 400cm -1 The sample was scanned within a certain range. The chemical composition of the polymer film was analyzed by ATR-IR spectroscopy using a Brookfield ATR-IR spectrophotometer.

[0155] PEG 400 acts similarly to an external crosslinking agent, but glutaraldehyde exhibits the best chemical crosslinking effect by introducing novel bonds in the form of hemiacetals and esters. Glycerol is known to impart membrane flexibility and better tensile strength at low concentrations (1% w / v to 3% w / v), while sorbitol tends to significantly increase flexibility at high concentrations (8% w / v to 10% w / v). These results also confirm our theory that excess and unreacted glutaraldehyde can be easily removed by photolytic degradation. Figure 17a ).

[0156] HLG, due to the OH group it binds, is at 3500 cm⁻¹ -1 Up to 3200cm -1 It exhibits a clear and broad peak, and at 2924 cm⁻¹, due to the CH stretching vibration of the methyl group (CH₃) present in the unsaturated aromatic system. -1 A peak is shown. 1462cm -1 The peak at 1043 cm⁻¹ is caused by the CH bending of the methyl group. -1 The new peaks in the hemiacetal and ester band regions are due to COC of the acetal or hemiacetal groups and are considered to be the result of chemical cross-linking. Figure 17b ).

[0157] PXLG analysis of the membrane revealed overlapping functional group spectra of lignin and xanthan gum. The reduced or absent peak intensities associated with unbound OH stretching vibrations indicated a decrease in free hydroxyl groups, possibly suggesting chemical modification of the material to reduce its interaction with water or its tendency to absorb water. (2950 cm⁻¹) -1 The splitting of the OH peak is due to protons in the conjugated system. The degree of binding between xanthan gum and glycerol is significant because, in an anhydrous environment, the degree of cross-linking is sufficient to overshadow cross-linking with other compounds. Figure 17c ).

[0158] The membrane XGPG spectrum showed significant changes. 3200 cm⁻¹ -1 Up to 3600cm -1 The intensity of the broad peak corresponding to the OH stretching vibration in the vicinity shows a significant decrease, indicating the consumption of hydroxyl groups during crosslinking. Other natural peaks in lignin, such as the new peaks in the carbonyl and CO stretching regions, and changes in aromatic ring vibrations, indicate the formation of new chemical bonds and structural changes in lignin. 2865 cm⁻¹ -1 The sharp peak at the [specific location] is due to -CH3 stretching, similar to the findings of Marcos et al. (2017). Compared to PXLG, lignin exhibits lower characteristic peak intensities; they either participate in crosslinking or are present in lower abundances than natural lignin. This confirms that PEG, as an external crosslinking agent, improves membrane stability by aiding in the formation of a polymer network. Figure 17d ).

[0159] XRD

[0160] The sample was cut into small squares and placed on an XRD cuvette for analysis using a Bruker D2 Phaser X-ray diffractometer. The voltage and current used were 40 kV and 30 mA, respectively, and XRD patterns were obtained in 2° increments of 0.02° and a scan rate of 5° / min within a range of 5° to 50°.

[0161] XRD analysis within the 2° range of 5° to 50° showed that the membranes were amorphous, with some HPC peaks masking the lignin. However, overall, the membranes remained amorphous. The absence of crystalline peaks indicates the absence of crystalline impurities that might arise during lignin extraction. Figure 18 ).

[0162] DSC

[0163] XGPG membranes loaded with α-lactalbumin were analyzed using differential scanning calorimetry (DSC Discovery 250, TA Instruments) to determine their thermal properties during curing. 2.3 mg of the biopolymer membrane sample was placed in an aluminum open pan and analyzed over a temperature range of 30°C to 325°C. DSC curves were recorded under a N2 atmosphere (50 mL / min) at a heating rate of 10°C / min.

[0164] DSC studies revealed that the film was able to protect a-lactalbumin from thermal degradation. The initial endothermic peak of a-lactalbumin was in the range of 70-80 °C, presumably due to protein denaturation. However, in the film, the slope was tilted, indicating that the film had protective properties towards a-lactalbumin. Due to the presence of xanthan gum in the film, no other peaks appeared in the film except for the peak at 125 °C. The exothermic peak of the film at 90-100 °C was due to the glass transition of the blend Figure 19

[0165] SEM

[0166] The matrix of the films was analyzed by scanning electron microscopy (JEOL JSM-6390LA). SEM was used to get a clear view of the surface morphology and structure of the films. SEM images of the surface of the polymer films were collected using SEM operated at 200Kv with a LaB6electron gun with a point resolution of 0.23 nm.

[0167] The SEM images show the surface of the XGPG film, where in the (a) and (b) parts of the first image, undissolved lignin particles can be seen, but the occurrence is very low, indicating that most of the lignin is associated with the xanthan gum film matrix Figure 20

[0168] Example 4

[0169] In vivo clinical evaluation in Swiss albino mice

[0170] ​​In this study, it was demonstrated that the combination of lignin and alpha-lactalbumin effectively promoted wound healing in Swiss albino mice. Moreover, the combination of lignin and alpha-lactalbumin was found to reduce scar formation at a faster rate and accelerate epithelial regeneration compared to the commercially available silver sulfadiazine cream. Notably, the hair follicles of animals treated with the film loaded with alpha-lactalbumin were more stimulated and the time of hair regeneration was earlier compared to other animals. Among them, alpha-lactalbumin (ALA) is a protein that is ubiquitous in the milk of almost all mammals and plays a key role in controlling lactose production. In its multimeric form (Eugenia Lucena, Alvarez et al. 2006, 2007), alpha-lactalbumin binds tightly to calcium and zinc ions, with potential bactericidal or antitumor effects (Sharma, Hanson et al. 2015). This affinity has been the focus of many studies that extract and isolate ALA (Eugenia Lucena, Alvarez et al. 2006, Eugenia lucena, Alvarez et al. 2007), which has a wide range of activities, such as bactericidal, antitumor effects (Krunic, Rakin et al. 2018) and wound healing capacity (Guo, Liu et al. 2020). Among all proteins, ALA has the highest percentage of tryptophan content (Layman, Various studies and hypotheses have proposed potential mechanisms by which the application of exogenous tryptophan promotes wound healing, including reducing cellular stress levels by inhibiting indoleamine 2,3-dioxygenase (IDO) and tumor necrosis factor-alpha (TNF-alpha) (Kiank, Zeden et al. 2010; Bandeira, Bortolot et al. 2015). It has previously been demonstrated that topical application of 1 mM tryptophan reduces pain and increases epithelial regeneration in patients with lower extremity ulcers, with the potential to become a natural ingredient for wound healing (Barouti, Mainetti et al. 2015). These findings suggest that the combination of lignin and alpha-lactalbumin has the potential to be an effective wound dressing for promoting wound healing and reducing scar formation. Histopathological analysis of wound tissue samples showed significant differences between the groups. The normal group showed well-preserved epidermal, dermal, and subcutaneous layer structures, including intact sebaceous glands and hair follicles. In contrast, the negative control group showed significant changes, such as hyperplasia, edema, and increased thickness of the epidermal layer. In addition, there was mild inflammatory cell infiltration with acanthosis and hyperkeratosis.

[0171] The blank film group maintained the typical structure of the epidermis and dermis, indicating normal structure but a clear lack of hair follicles. The standard and treatment groups both confirmed similar skin structure in the epidermal and dermal layers. However, the treatment group showed a significantly higher number of hair follicles than the standard group, indicating that alpha-lactalbumin can stimulate hair follicle growth. Figure 21 ).

[0172] (normal group - no damage inflicted, as a reference; negative control group was subjected to damage, but was not treated with anything; blank film group received a blank lignin-based biopolymer film that did not contain alpha-lactalbumin; standard group was also subjected to damage, but was treated with a commercially available silver sulfadiazine cream; treatment group was subjected to damage and treated with a film containing alpha-lactalbumin).

[0173] Example 5

[0174] Leather industry applications

[0175] Leather is a very versatile material used for a variety of purposes, including indoor furniture upholstery, clothing, footwear, luggage, handbags, accessories, and vehicle applications. Conventionally, leather is derived from animal hides. However, synthetic alternatives have emerged that involve coating natural or synthetic fibers with synthetic polymers. Although synthetic leather is popular, its production presents some environmental challenges, as it involves the use of non-biodegradable plastics that are not only difficult to process, but also take a considerable amount of time to decompose. Most synthetic leather consists of a polyester knit base coated with polyurethane and polyvinyl chloride. Furthermore, while synthetic leather can be less expensive than natural leather, it generally has lower quality and durability.

[0176] Prototypes of leather alternatives were manufactured using 2.5% glutaraldehyde as a cross-linking agent, with sulfuric acid added dropwise at a lower HPC to lignin (3:2) ratio, resulting in a free-flowing solution, designated HLG. A viscous solution was obtained using 10% concentration of polyethylene glycol, with a 1:1 ratio of xanthan gum and lignin, with high RPM stirring at high temperature, coded XGPG. A viscous solution was obtained using 1:1:1 ratio of polyvinyl alcohol, xanthan gum, and lignin, with 1.5% concentration of glycerol as a plasticizer, with 2000 RPM stirring at high temperature, designated PXLG. All solutions were then separately cast onto silica molds. Leather reinforcement materials obtained from fibers of coconut shell and / or coconut coir powder were uniformly attached to the cellulose water-based adhesive present in the leather alternative material.

[0177] Production of the prototypes involved complete bonding and sealing of the leather alternative material and the leather reinforcement material. This was achieved by placing a heavy object on top of the material for 48 hours at standard room temperature, followed by coating with a wax emulsion, softener, and wetting agent. The result was a completely sealed prototype, which could then be cut to specification.

[0178] The final product (as shown below in Figures 22-X GPG) exhibited excellent wear resistance and flexibility, particularly as a substitute for leather. Furthermore, it was not affected by weather and ultraviolet changes.

[0179] Elastic modulus was determined by force applied to a known length of finished leather prototype. Results are listed in Table 1.

[0180] Table 1: Elastic modulus of lignin-based leather substitute.

[0181] Number Sample Elasticity (10-100 N) 1 HLG 28.394±2.46 2 XGPG 31.746±1.72 3 PXLG 35.149±2.91 4 Standard leather 45.782±3.19

Claims

1. A natural biocompatible multifunctional biopolymer, comprising: a) Lignin extracted from coconut shell and / or coconut shell fiber pith powder obtained from the fruit of coconut (Cocos nucifera), a tropical plant belonging to the palm family; b) Copolymer; c) Crosslinking agent; d) Plasticizers; and e) At least one active agent.

2. The natural biocompatible multifunctional biopolymer as described in claim 1, wherein, At least one copolymer is selected from natural polymers or water-soluble synthetic polymers, wherein the natural polymers are, for example, hyaluronic acid and its derivatives, starch, modified starch, alginate, chitosan, chitin, natural gums; proteins such as gelatin, collagen, casein, zein, gluten, soy protein isolate, and whey protein isolate; terrestrial plant extracts, such as pectin; and combinations thereof, wherein the water-soluble synthetic copolymers include cross-linked polyacrylic acid chains, polyvinylpyrrolidone, and polyvinyl alcohol; modified cellulose ethers, such as hydroxypropyl methylcellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, and carboxymethyl cellulose; ion exchange resins and combinations thereof.

3. The natural biocompatible multifunctional biopolymer as described in claim 1, wherein, At least one crosslinking agent is selected from glutaraldehyde, formaldehyde, polyethylene glycol (PEG) and its derivatives, genipin, dextran, polyethyleneimine (PEI), ethylenediamine (EDA), and sugar alcohols / polyols, such as erythritol, xylitol, maltitol, mannitol, lactitol, polysaccharide alcohol, isomaltitol, sorbitol, glycerol, and combinations thereof, to promote the crosslinking chemical reaction.

4. The natural biocompatible multifunctional biopolymer as described in claim 1, wherein, At least one plasticizer is selected from sucrose ester derivatives, glycerin, propylene glycol, polyethylene glycol, magnesium stearate, tallow, beef hoof oil, lanolin, mineral oil, silicone oil, jojoba oil, castor oil, coconut oil, whale oil, squalene, grape seed oil, safflower oil, rapeseed oil, and almond oil, and combinations thereof.

5. The natural biocompatible multifunctional biopolymer as described in claim 1, wherein, The emulsifiers can be selected from xanthan gum, lanolin, sodium alginate, lecithin, casein, whey protein, pectin, phospholipids, gum arabic, and oleylamine.

6. The natural biocompatible multifunctional biopolymer as described in claim 1, wherein, It is used as a wound dressing or part of a wound dressing on a patient's severe burn wound to promote new tissue growth and replace damaged tissue on the burn wound, wherein at least one active agent may be loaded from turmeric extract (curcumin), honey, green tea extract, centella asiatica extract, α-lactalbumin, whey protein isolate, comfrey extract, tea tree oil, lavender oil, chamomile extract, calendula extract, aloe vera gel, silver sulfadiazine, bacitracin, hyaluronic acid, collagen, vitamin E, allantoin, sphingosine / phytosphingosine-1-phosphate, dextropanthenol (vitamin B5), and combinations thereof.

7. The natural biocompatible multifunctional biopolymer as described in claim 1, wherein, It is used as a wound dressing or part of a wound dressing on a patient's severe burn wound to promote new tissue growth and replace damaged tissue in the burn wound, wherein at least one active agent may be loaded with human growth factors and / or cytokines, such as epidermal growth factor (EGF), growth hormone / stoxin (hGH / STH), vasoactive intestinal peptide (VIP), fibroblast growth factor family proteins, namely fibroblast growth factor-1 (FGF1), fibroblast growth factor-2 (FGF2), fibroblast growth factor-7 / keratinocyte growth factor (FGF7; KGF), and fibroblast growth factor-10 / keratinocyte growth factor-2 (FGF10);KGF2), fibroblast growth factor-19 (FGF19), fibroblast growth factor-21 (FGF21), fibroblast growth factor-23 (FGF23), vascular endothelial growth factor (VEGF), thymosin β-4 (TMS), stem cell factor / stem factor (SCF), platelet-derived growth factor (PDGF), prolactin (PRL), placental prolactin (hPL), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), and ciliary neurotrophic factor (CNTF). Interleukin-1 receptor antagonists (IL-1Ra), interleukin family proteins, namely interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-13 (IL-13), interleukin-17 (IL-17), interleukin-18 (IL-18), etc., adiponectin (ApN), TSLP (thymic stromal lymphopoietin), bone morphogenetic protein family proteins, namely BMP-2, BMP-4, BMP-7, etc., differentiation cluster-34 (CD34), desmosome plaque protein (Dsp), erythropoietin (EPO), red blood cells, etc. Cell differentiation regulator 1 (ERDR1), fibronectin, follicle-stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), follicle-suppressant (FST), growth differentiation factor family proteins, namely growth differentiation factor 11 / bone morphogenetic protein-11 (GDF11 / BMP-11) and growth differentiation factor 15 (GDF15), irisin, kisspeptins, klotho, matrix peptides (extracellular matrix-derived peptides), and Scube3. (Signal peptide, CUB and EGF-like domain protein 3), osteopontin, oxytocin, pregnancy-associated plasma protein-A (PAPP-A), platelet factor 4 (PF4), Reelin, stem cell antigen-1 (Sca-1), calcitonin-1 (STC1), calcitonin-2 (STC2), transforming growth factor (TGF) family proteins, namely TGF-α, TGFβ-1, TGFβ-2, TGFβ-3, thyrotropin-releasing hormone (TRH), thyroid-stimulating hormone (TSH), heat shock protein family proteins, namely HSP90A and its derivatives, and combinations thereof.

8. The natural biocompatible multifunctional biopolymer as described in any one of claims 1, 6, and 7, wherein, The combination of lignin and α-lactalbumin has been shown to be effective in preparing wound dressings for severe burn wounds, wound healing, reducing scar formation and accelerating epithelial regeneration, as well as for restoring new tissue growth and replacing damaged burn wound tissue.

9. The natural biocompatible multifunctional biopolymer as described in claim 1, wherein, Its use in developing leather substitutes or a subset thereof, wherein prototypes of leather substitutes with or without α-lactalbumin or leather reinforcement materials obtained from coconut shells and / or coconut shell fiber pith powder exhibit the superior abrasion resistance and flexibility of natural leather.

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