KR20240050823A - Novel Method for Preparing Sericin for Osteogenesis and Uses Thereof - Google Patents

Abstract

The present invention relates to a novel method for producing sericin for bone regeneration and its use. Sericin isolated from silkworm cocoons through 4-hexylresorcinol pretreatment and refining, which is the method of the present invention, is a protein derived from natural substances. It is safe, has little degeneration, is biocompatible, and has excellent bone regeneration effects. Therefore, 4HR-pretreated sericin according to the method of the present invention can not only be an excellent alternative to conventional agents related to bone disease/injury treatment, but can also contribute to economic growth and economic growth by recycling sericin, which is discarded as a by-product of silk protein. It can also contribute to social benefits.

Description

Novel Method for Preparing Sericin for Osteogenesis and Uses Thereof}

The present invention relates to a novel method for producing sericin for bone regeneration and its use.

In addition to the mechanical function of supporting the human body and performing movements, bones play a role in regulating the concentration of calcium ions in the body, and perform an important physiological function of producing red and white blood cells necessary for the human body in the bone marrow. Bones are made up of cortical bone, a dense structure with high mechanical strength, and trabecular bone, an internal sponge-like structure. As age increases, bone density decreases, making it easier to fracture even with a small impact. Bone damage is caused by osteoporosis and arthritis, and when fractures occur due to such physiological and physical damage, bone regeneration becomes difficult.

In addition, treatment methods for bone regeneration include natural treatments, treatment methods using drugs or physical therapy, and treatment methods using orthopedic surgery. When the degree of damage to the bone is large and recovery is difficult, normal tissue is protected. Damaged tissue is removed.

Recently, attempts have been made to replace or regenerate damaged bone tissue with normal bone tissue, and accordingly, interest in the development of bone tissue for transplantation into the body or agents that promote bone regeneration thereof is increasing.

Accordingly, research is in progress to develop artificial bone from living tissue to increase the bone formation effect by using cells such as bone marrow-derived stem cells to compensate for damaged bone tissue. Methods of transplanting bone from another person or an animal or the patient's own bone are in progress. There is also a method of transplanting tissues, but there are problems such as transplanting other tissues, which may cause immunological rejection or the damaged area is large, so there is not enough material available in the patient's body.

On the other hand, silk sericin is a component of silk and is generated as a by-product during the refining process. Although it is a natural ingredient, unlike fibroin, which has been recognized for its value as a fiber, various functional foods, and medicinal purposes, it cannot be used for various purposes and is disposed of as a separate waste. The situation was being disposed of through a process.

However, as silk sericin has recently come into the spotlight as a medicinal resource as it is used as a raw material for dressings, cosmetics, and food, for its medical application, compounds that may pose a problem to the human body are used to a minimum during the refining process and sericin's unique protein structure is used. A new refining method is needed that can preserve as much as possible, do not denature, and extract it in a biologically inert state.

Therefore, if sericin, a natural substance, can be structurally stabilized and obtained with high efficiency without using ingredients harmful to the human body such as acids and bases, it will complement the problems associated with conventional bone disease treatments and provide an excellent graft material for repairing and regenerating bone damage. Not only can it be used as a by-product of silk protein, but by utilizing sericin, which is discarded as a by-product of silk protein, it will be able to contribute to significant economic growth and social benefits.

Accordingly, the present inventors sought to develop a degumming method to obtain sericin, a natural active ingredient that can promote bone regeneration and is easily supplied, biocompatible, and safe for the human body, from silkworm cocoons in a structurally stable and efficient manner. I made a diligent research effort. When using the technology previously registered by our research team (registration number: 1022235440000, registration date: 2021.02.26), sericin is extracted using an ultrasonic cleaner at a low temperature of 60℃ or less to separate only those with high molecular weight. , it was possible to prepare highly functional sericin that is rich in β-sheet structure and increases the expression of BMP-2 (bone morphogenic protein-2), which is known to increase bone regeneration. However, this method had a very low yield compared to the administered cocoons, less than 1% of the administered weight, and a cellulose filter was used to extract only those with high molecular weight, but the filter was expensive, which had the disadvantage of being less economical. In order to overcome the shortcomings of the existing technology, it has become necessary to develop a technology that can simply extract sericin rich in β-sheet structure with higher yield. Accordingly, when the present inventors pretreated silkworm cocoons with 4HR (4-Hexylresorcinol)- to hydrothermally extract (refine) sericin, they confirmed that 4HR-pretreatment improved the structural stability of silk sericin and increased the β-sheet structure. This method dramatically increased the yield compared to the existing method, with a yield of over 20% of the administered weight. In addition, the present invention was completed by demonstrating that bone regeneration is promoted by upregulating the expression of BMP-2, and that when bone defects are filled in a bone damage model, bone damage and defects are rapidly regenerated.

Therefore, one object of the present invention is to provide a method for producing sericin for bone regeneration.

Additionally, another object of the present invention is to provide a method for producing a therapeutic agent for bone regeneration containing sericin protein.

In addition, another object of the present invention is to provide a therapeutic agent for bone regeneration, manufactured according to a method for producing a therapeutic agent for bone regeneration containing sericin protein.

In addition, another object of the present invention is to provide a pharmaceutical composition for preventing and treating bone diseases containing sericin protein as an active ingredient.

In addition, another object of the present invention is to provide a food composition for bone regeneration containing sericin as an active ingredient.

In addition, another object of the present invention is to provide a feed composition for bone regeneration containing sericin as an active ingredient.

Other objects and advantages of the present invention will become clearer from the following detailed description, claims, and drawings.

The terms used herein are for descriptive purposes only and should not be construed as limiting. Singular expressions include plural expressions unless the context clearly dictates otherwise. In this specification, terms such as “comprise” or “have” are intended to designate the presence of features, numbers, steps, operations, components, parts, or combinations thereof described in the specification, but are not intended to indicate the presence of one or more other features. It should be understood that it does not exclude in advance the possibility of the existence or addition of elements, numbers, steps, operations, components, parts, or combinations thereof.

Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as generally understood by a person of ordinary skill in the technical field to which the embodiments belong. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of the related technology, and should not be interpreted in an ideal or excessively formal sense unless explicitly defined in the present application. No.

Hereinafter, the present invention will be described in detail.

According to one aspect of the present invention, the present invention provides a method for producing sericin for bone regeneration, comprising the following steps:

(a) cutting the cocoon into pieces;

(b) preparing a 4-hexylresorcinol ethanol solution by dissolving 4-hexylresorcinol in ethanol;

(c) pretreating the shredded silkworm cocoon of step (a) by mixing the 4-hexylresorcinol ethanol solution of step (b), followed by drying; and

(d) obtaining sericin by refining the dried 4HR-pretreated silkworm cocoons of step (c) and then drying them.

Additionally, in this specification, sericin refers to sericin obtained according to the method of the present invention, and is used interchangeably with “sericin protein,” “Sericin + 4HR (Sericin + 4HR),” or “4HR-pretreated sericin.”

In other words, the feature of the method of the present invention is to transform the molecular structure by pre-treating the silkworm cocoon with 4HR before refining, thereby securing the structural stability of the sericin protein denatured during the refining process, thereby providing sericin for bone regeneration with increased physiological activity. .

Therefore, the sericin in step (d) has a modified molecular structure compared to sericin that has not been 4HR-pretreated, and the modification of the molecular structure includes an increase in beta-sheet secondary structure and alpha-helix ( It may be one or more types selected from the group consisting of reduction of α-helix secondary structure, reduction of beta-turn secondary structure, and reduction of random coil secondary structure, but maximizes the effect of increasing bone regeneration. In that it can be done, it is preferable to increase the beta-sheet.

Additionally, sericin in step (d) increases the expression of BMP (Bone morphogenic protein)-2 compared to sericin that is not 4HR-pretreated.

In the present invention, the shredded silkworm cocoons in step (c) are mixed to constitute 90 to 99.99% by weight and the 4-hexylresorcinol ethanol solution is 0.01 to 10% by weight, preferably 0.01 to 0.5% by weight. .

The IUPAC name of 4-hexylresorcinol, which is included as a pretreatment agent in the present invention, is 4-hexylbenzene-1,3-diol and is a compound having the following formula: Conventionally, it has been used as a functional material that can be mixed with bone graft material, but in the present invention, for the purpose of increasing the physiological activity of sericin, it is dissolved in 100% ethanol and added to the silkworm cocoon before refining. In the examples of the present specification, 4- It is mixed with hexylresorcinol ethanol solution,

[Formula 1]

In other words, when the weight of 4-hexylresorcinol is less than 0.01% by weight, it is difficult to expect the desired effect, and when it exceeds 10% by weight, the increase in effect due to increase in content is very weak, while There is a high possibility of inducing an apoptosis response in normal cells.

It can be manufactured so that the total composition of 4-hexylresorcinol and silkworm cocoon composed as above is 100% by weight, but bioapplicable materials, which are biocompatible tissue engineering materials, can be added to the total weight to the extent that they do not harm the physical properties of sericin. It can also be manufactured by adding less than 30% by weight.

Any bioapplicable material may be added as the bioapplicable material, including collagen, gelatin, chitin, chitosan, keratin, cellulose, fibronectin, elastin, fibrinogen, fibromodulin, laminin, tenascin, vitronectin, alginate, Hyaluronic acid, silk proteins and their derivatives, agarose, polylactic acid (PLA), polyglycolic acid (PGA), copolymer of polylactic acid and polyglycolic acid (PLGA), polycaprolactone; PCL), poly{poly(ethylene oxide) terephthalate-co-butylene terephthalate}(PEOT/PBT), polyphosphoester (PPE), polyphosphazene (PPA), polyanhydride; PA), polyortho ester {polyortho ester; POE}, pluronic, glycerin, poly(propylene fumarate)-diacrylate {poly(propylene fumarate)-diacrylate; PPF-DA} and polyethylene glycol diacrylate; It is preferable to add any one or more of PEG-DA}.

In addition, various additives consisting of one or more selected from antibiotics, antiviral agents, antibacterial agents, nucleic acids, peptides, and proteins may be added as long as they do not negatively affect the structure and function of sericin of the present invention.

The protein is characterized as being selected from the group consisting of hormones, cytokines, enzymes, antibodies, growth factors, transcriptional regulators, vaccines, structural proteins, ligand proteins, receptors, cell surface antigens, and receptor antagonists.

In addition, the method used for the extraction (refining) of sericin is not limited as long as the purpose of the present invention can be achieved, but in the present invention, the refining in step (d) is performed by extracting 4HR-pretreated silkworm cocoons from 100 to 100%. 4HR-pretreated sericin is obtained by hydrothermal extraction at 150°C for 30 minutes to 3 hours, preferably at 120°C for 30 minutes to 1 hour.

In other words, if manufactured at a temperature below 120℃ for more than 3 hours, the manufacturing time is too long and there is a risk that the active ingredients contained in the silkworm cocoon itself may be destroyed, and if manufactured at a temperature exceeding 120℃ for less than 30 minutes. This is because the temperature is too high and there is a risk of adversely affecting the active ingredients contained in the silkworm cocoon itself.

Therefore, the sericin of the present invention prepared by the above method is preferably sericin prepared by 4HR-pretreatment followed by hot water extraction at 120°C for 1 hour, and the protein structure is stable, biocompatible, and promotes bone regeneration. It has excellent functionality in enhancing the expression level of BMP-2, which is known to increase.

In addition, according to another aspect of the present invention, the present invention provides a method for producing a therapeutic agent for bone regeneration containing Sericin, and a therapeutic agent for bone regeneration prepared thereby, comprising the following steps:

(a) cutting the cocoon into pieces;

(b) preparing a 4-hexylresorcinol ethanol solution by dissolving 4-hexylresorcinol (4HR) in ethanol;

(c) The shredded silkworm cocoon in step (a) is pretreated by mixing the ethanol solution of 4-hexylresorcinol in step (b), and then 4-hexylresorcinol in free form not bound to sericin is added. Washing with 100% ethanol, removing and drying; and

(d) obtaining sericin by refining the dried 4HR-pretreated silkworm cocoons of step (c) and then drying them.

Sericin included in the bone regeneration treatment of the present invention exerts the effect of increasing the expression of BMP-2 (Bone morphogenic protein-2).

The therapeutic agent for bone regeneration of the present invention can promote bone remodeling and mineralization, and when filled in bone defects, it rapidly regenerates bone damage and defects.

As used herein, the term “bone regeneration” or “bone regeneration” refers to the process in which bone is destroyed and resorbed by osteoclasts and then bone is formed again by osteoblasts. The term “bone mineralization” refers to the phenomenon of deposition of minerals such as calcium in bone cells or extracellular matrix.

The therapeutic agent for bone regeneration of the present invention not only increases the expression of BMP (Bone morphogenic protein)-2 by acting on damaged bone areas with sericin protein, which has excellent biocompatibility, but also affects bone cells to prevent bone cell shedding. there is. Therefore, it can also be applied to manufacture bone cement with properties suitable for the purpose.

In addition, the therapeutic agent for bone regeneration of the present invention may additionally include one or more selected from the group consisting of biocompatible binders, antibiotics, vitamins, glucosamine, cytokines, and growth factors.

The biocompatible binder is from the group consisting of gelatin, hyaluronic acid, hydroxyapatite, collagen, fibrin, fibrinogen, thrombin, muscle adhesive protein, elastin, casein, albumin, keratin, chitin and chitosan. There may be one or more selected types, but it is not limited thereto.

In addition, the biocompatible binder is starch, polylactic acid, polyglycolic acid, polylactic-co-glycolic acid, polydioxanone, polycaprolactone, polycarbonate, polyoxoester, polyamino acid, polyanhydride, poly At least one selected from the group consisting of hydroxybutyrate, polyhydroxyvalylate, poly(propylene glycol-co-fumaric acid), tyrosine-based polycarbonate, polyvinylpyrrolidone, cellulose, ethyl cellulose, and carboxymethyl cellulose. may, but is not limited to this.

In addition, the therapeutic agent for bone regeneration of the present invention can be injected directly into the bone defect site or filled into a graft material in a bioinjectable form to achieve the intended effect.

In one embodiment of the present invention, when the sericin of the present invention was treated with a gelatin sponge and implanted into a bone defect model, it was observed that the level of BMP-2 protein was significantly higher. These results show that the sericin protein filled in the micropore structure of the gelatin sponge is dissolved by biological fluids and interacts with new biological bone generated from adjacent bone tissue to maintain high BMP-2 levels, thereby improving bone regeneration efficacy. .

Additionally, the present invention can provide a bone graft containing sericin.

Sericin, the active ingredient of the present invention, may be included in the bone graft material at a concentration of 0.01-1000 μg/ml, more preferably 0.1-600 μg/ml, and even more preferably 0.5-300 μg/ml. Most preferably, it is 1-100 μg/ml.

Sericin protein, the active ingredient of the present invention, may be included in an amount of 0.01-40% by weight, more preferably 0.1-30% by weight, and most preferably 1-20% by weight, based on the total weight of the bone graft material.

The term "bone graft material" herein refers to a general term for graft materials used to fill the space within the bone tissue by replacing this part when part of the bone tissue is damaged or missing due to various diseases or accidents and to promote bone new growth. It is defined in the sense that

The bone graft material is selected from the group consisting of growth factors that promote bone growth, fibrin, bone morphogenetic factors, bone growth agents, chemotherapy agents, antibiotics, analgesics, bisphosphonates, strontium salts, fluoride salts, magnesium salts, and sodium salts. It may additionally contain biologically active substances.

The above growth factors include PDGF (Platelet-derived growth factor), TGF-beta (Transgenic growth factor), IGF-I (Insulin-like growth factor), IGF-II, FGF (Fibroblast growth factor), and BGDF-II (beta). -2-microglobulin) etc. can be used. As the bone tissue formation enhancing peptides and proteins, various peptides containing the RGD sequence and various proteins such as collagen and fibrogen can be used. As the bone morphogenesis factor, osteocalcin, bonesialo protein, or osteogenin can be used. The bone growth agent can be used without limitation as long as it is harmless to the human body and promotes bone growth. Nucleic acids that promote bone formation, antagonists of substances that inhibit bone formation, etc. can be used.

According to another preferred embodiment of the present invention, the compounds for formulating the bone graft material of the present invention include hyaluronic acid, hydroxyapatite, collagen, calcium carbonate, calcium phosphate, calcium sulfate, and monocalcium phosphate. , tricalcium phosphate, and silica may additionally be included.

The bone graft material of the present invention can be molded into the form of putty, paste, moldable strips, blocks, chips, etc. using methods such as compression, compression, pressurized contact, packing, compression, and hardening, and can be used by using the above compounds. Therefore, it can be formulated and used in the form of gel, powder, paste, tablet, pellet, etc. It is also possible to use it in powder form.

The bone area to which the bone graft material of the present invention can be applied is not particularly limited.

Therefore, the sericin protein of the present invention alone or the therapeutic agent for bone regeneration containing the sericin protein of the present invention and an appropriate biocompatible binder, antibiotics, vitamins, glucosamine, cytokines and/or growth factors can be injected directly into the bone defect site. When injected by filling a bio-injectable type of graft material, the bone regeneration effect at the site of bone damage or defect is excellent.

Based on the above, the therapeutic agent for bone regeneration of the present invention can be used for various fractures, osteonecrosis diseases, or bone regeneration.

In addition, according to another aspect of the present invention, the present invention provides a pharmaceutical composition for preventing and treating bone diseases containing the sericin as an active ingredient.

The bone disease may be one selected from the group consisting of osteoporosis, osteomalacia, rickets, osteitis fibrous, aplastic bone disease, metabolic bone disease, and bone damage, but is not limited thereto, as long as the bone regeneration effect is exerted.

In addition to the above ingredients, the pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

As used herein, the term "pharmaceutically acceptable carrier" refers to those commonly used in preparation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, Includes, but is limited to, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. It doesn't work.

The pharmaceutical composition of the present invention can be administered orally or parenterally, and in case of parenteral administration, it can be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, etc.

The appropriate dosage of the pharmaceutical composition of the present invention varies depending on factors such as formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and reaction sensitivity. Usually, a skilled physician can easily determine and prescribe an effective dosage for the desired treatment or prevention.

According to one embodiment of the present invention, the daily dosage of the pharmaceutical composition of the present invention is 0.0001-1000 mg/kg.

The pharmaceutical composition of the present invention is formulated in a pharmaceutically effective amount using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily performed by a person skilled in the art to which the invention pertains. It can be manufactured in dosage form or by placing it in a multi-dose container. At this time, the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, granule, tablet, or capsule, and may additionally contain a dispersant or stabilizer.

As used herein, the term “therapeutically effective amount” means that the amount of the inhibitor administered alleviates to some extent one or more symptoms of bone disease, which is the disease to be treated. Therefore, a pharmaceutically effective amount means that (1) reverses the rate of progression of bone disease, (2) halts further progression of bone disease to some extent, and (3) causes one or more symptoms associated with bone disease. It refers to an amount that has the effect of reducing (preferably, eliminating) to some extent.

In addition, according to another aspect of the present invention, the present invention provides a food composition for bone regeneration containing the sericin as an active ingredient.

The food composition includes compositions for all types of foods, such as health functional foods, nutritional supplements, food additives, special medicinal foods, and dietary supplements. do.

In the present invention, health functional food refers to a food group or food composition that provides added value to a food by using physical, biochemical, biotechnological methods, etc. to function and express the function of the food for a specific purpose, or a food group that regulates the biological defense rhythm and diseases. It refers to food that has been designed and processed to fully express the body's regulatory functions related to prevention and recovery.

The health functional food of the present invention includes forms such as tablets, capsules, pills, or liquid, and there is no particular limitation on the type of the health functional food. Examples include drinks, dairy products, baked goods, condiments, beverages, snacks, candies, ice cream and frozen desserts, breakfast cereals, nutritional bars, snack bars, chocolate products, processed foods, grain products, pasta, soups, sauces and dressings, and confectionery products. , oil and fat products, dairy drinks, milk drinks, soy milk, soy dairy-like products, frozen foods, prepared foods, alternative foods, meat products, cheese, yogurt, bread, rolls, yeast products , cakes, cookies, and crackers, but is not limited thereto, and includes all health foods in the conventional sense.

In the present invention, the food composition can be manufactured by a method commonly used in the art, and can be manufactured by adding raw materials and components commonly added in the art. Additionally, the formulation of the food composition can be manufactured without limitation as long as it is a formulation recognized as a food composition.

Foods according to the present invention include, for example, various foods, beverages, gum, tea, vitamin complexes, functional foods, etc. Additionally, foods include special nutritional foods (e.g., milk formula, infant and baby food, etc.), processed meat products, fish products, tofu, jelly, noodles (e.g., ramen, noodles, etc.), breads, health supplements, seasonings (e.g., soy sauce) , soybean paste, red pepper paste, mixed paste, etc.), sauces, confectionery (e.g., snacks), candy, chocolate, gum, ice cream, dairy products (e.g., fermented milk, cheese, etc.), other processed foods, kimchi, pickled foods (various types of kimchi) , pickles, etc.), beverages (e.g., fruit drinks, vegetable drinks, soy milk, fermented drinks, etc.), natural seasonings (e.g., ramen soup, etc.), food additives, etc., but are not limited thereto. The food, beverage or food additive can be manufactured by conventional manufacturing methods.

When using the composition of the present invention as a health functional food additive, the composition can be added as is or used together with other health functional food ingredients, and can be used appropriately according to conventional methods. The mixing amount of the active ingredient can be appropriately determined depending on the purpose of use. In general, when producing a food or beverage, the composition of the present invention can be added in an amount of preferably 50 parts by weight or less, more preferably 25 parts by weight or less, based on the raw materials. However, in the case of long-term intake for the purpose of health control and hygiene, the amount may be below the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range.

In addition to containing the active ingredient sericin of the present invention, the food or health functional food composition of the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients like a typical food composition. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose, etc.; Disaccharides such as maltose, sucrose, etc.; and polysaccharides, such as common sugars such as dextrin, cyclodextrin, etc., and sugar alcohols such as xylitol, sorbitol, and erythritol. The above-described flavoring agents include natural flavoring agents (thaumatin), stevia extracts (e.g. rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.).

In addition, the food composition contains, in addition to sericin of the present invention, various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavors, colorants and thickening agents (cheese, chocolate, etc.), pectic acid, and salts thereof. , alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the food composition of the present invention may contain pulp for the production of natural fruit juice and fruit juice drinks and vegetable drinks.

Since sericin of the present invention shows an effect of improving bone function, it can be usefully used, for example, as a health functional food for the elderly or a health functional food for promoting growth in the growth layer. At the same time, it can be used as a special medicinal food for patients suffering from decreased bone function.

At this time, the sericin can be used in an amount of 0.01 to 100% by weight in the food composition of the present invention. Food compositions of this type can be prepared in various forms according to conventional methods known in the art. For example, health foods can be manufactured in the form of gum, vitamin complex, tea, juice, and drinks containing sericin, and food compositions containing sericin as an active ingredient can be granulated, encapsulated, or powdered for consumption. . The functional food composition of the present invention may include ingredients commonly added during food production, and may include, for example, proteins, carbohydrates, fats, nutrients, and seasonings.

For example, when manufactured as a drink, citric acid, high fructose corn syrup, sugar, glucose, acetic acid, malic acid, fruit juice, jujube extract, licorice extract, etc. may be additionally included in addition to the sericin of the present invention.

The food composition includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavors, colorants and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, and organic acids. , protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, it may contain extracts for the production of natural fruit juice, fruit juice drinks, and vegetable drinks. These ingredients can be used independently or in combination.

In the case of long-term intake for the purpose of health and hygiene or health control, the food composition of the present invention can be taken for a long period of time because there is no problem in terms of safety.

In addition, according to another aspect of the present invention, the present invention provides a feed composition for bone regeneration containing the sericin as an active ingredient.

The feed composition of the present invention may be a feed additive or compound feed.

Sericin of the present invention exhibits an overall improvement effect by improving bone function, so it can be included in a feed composition as a growth promoter for animals or livestock.

Specifically, the feed containing sericin as an active ingredient according to the present invention can be manufactured from various types of feed known in the art, and may preferably include concentrate feed, roughage feed, or special feed.

Concentrated feed includes seeds and fruits, including grains such as wheat, oats, and corn; bran, which includes rice bran, bran, and barley bran as by-products obtained from refining grains; soybeans; oil, sesame seeds, linseed, and coco oil; Fish soluble (fish solution) is a concentrated product of fresh liquid obtained from fish meal, fish waste, fish meal, fish waste, etc. soluble), animal feed such as meat meal, blood meal, feather meal, skim milk powder, dried whey, which is the residue obtained when producing cheese from milk, and casein from skim milk, yeast, chlorella, and seaweed.

Forage includes raw grass feed such as wild grasses, grasses, green cuttings, etc., root vegetables such as feed turnips, feed beets, root vegetables such as rutterbearger, a type of turnip, raw grass, green cuttings, grains, etc., which are stored feeds filled in silos and fermented with lactic acid. These include silage, field grass, hay made by cutting and drying grass, straw from breeding crops, and leaves from legumes.

Special feeds include mineral feeds such as shells and rock salt, urea feeds such as urea and its derivative diureide isobutane, and trace amounts in mixed feeds to supplement ingredients that are likely to be lacking when mixing only natural feed raw materials, or to increase the storability of the feed. There are substances added as feed additives, dietary supplements, etc.

The feed composition according to the present invention may contain various feed additives.

In the present invention, the term "feed additive" refers to a substance added to feed for the purpose of various effects such as nutrient supplementation and weight loss prevention, improvement of digestibility of fiber in feed, improvement of milk quality, prevention of reproductive disorders and improvement of conception rate, and prevention of high temperature stress in summer. says The feed additive of the present invention corresponds to supplementary feed under the Feed Management Act, and includes mineral preparations such as sodium bicarbonate (sodium bicarbonate), bentonite, magnesium oxide, and complex minerals, and trace minerals such as zinc, copper, cobalt, and selenium. Preparations, vitamin preparations such as kerotene, vitamin E, vitamins A, D, E, nicotinic acid, and vitamin B complex, protective amino acid preparations such as methionine and lysic acid, protective fatty acid preparations such as fatty acid calcium salts, probiotics (lactic acid bacteria), yeast culture Water, live bacteria such as mold fermentation products, yeast, etc. may be additionally included.

The feed composition according to the present invention is not particularly limited and can be applied to any individual as long as it is aimed at promoting overall bone function. The subjects include animals, such as non-primates (e.g., cows, pigs, horses, cats, dogs, rats, and mice) and primates (e.g., monkeys, such as cynomolgous monkeys and Refers to mammals, including chimpanzees. The object is a livestock animal (eg, horse, cow, pig, etc.) or a pet animal (eg, dog or cat).

The dosage of the feed composition according to the present invention will depend on a number of factors such as the species, size, weight, and age of the animal. In principle, typical dosages may range from 0.001 to 10 g per animal/day, but are not limited to this.

Since the composition of the present invention contains the above-described sericin protein as an active ingredient, the description of common content between the two is omitted to avoid excessive complexity of the present specification.

Sericin isolated from silkworm cocoons through 4-hexylresorcinol pretreatment and refining, which is the method of the present invention, is not only safe, has little denaturation, and is biocompatible because it is a protein derived from natural substances, but also has an excellent bone regeneration effect. Therefore, 4HR-pretreated sericin according to the method of the present invention can not only be an excellent alternative to conventional agents related to bone disease/injury treatment, but can also contribute to economic growth and economic growth by recycling sericin, which is discarded as a by-product of silk protein. It can also contribute to social benefits.

Figure 1 is a schematic diagram showing the process of obtaining 4HR-pretreated sericin according to the method of the present invention.
Figure 2 shows the sericin yield results at each refining temperature according to the method of the present invention.
Figure 3 shows FT-IR results analyzing the structural stability of 4HR-pretreated sericin according to the method of the present invention.
Figure 4A shows the level of BMP-2 expression by 4HR-pretreated silk sericin of the present invention in RAW264.7 cells. Figure 4b shows the level of BMP-2 expression by silk sericin without 4HR-pretreatment in RAW264.7 cells. Figure 4c shows the expression level of BMP-2 by silk sericin (Sericin) extracted at low temperature and 4HR-pretreated silk sericin (Sericin + 4HR) of the present invention.
Figures 5a to 5c show the mechanism of BMP-2 induction in RAW264.7 cells by 4HR-pretreated sericin according to the method of the present invention.
Figure 6 shows the bone regeneration effect of 4HR-pretreated sericin according to the method of the present invention in a bone damage model.
Figure 7 shows the bone regeneration effect of 4HR-pretreated sericin according to the method of the present invention in a bone damage model through MT staining (Bar = 1.0 mm).
Figure 8 shows that the bone regenerative effect of 4HR-pretreated sericin according to the method of the present invention in a bone damage model is due to increased expression of BMP-2 and TLR-2 (Counterstain: DAPI, original magnification x200).
Figure 9 shows the results of confirming the bone regeneration effect of 4HR-pretreated sericin according to the method of the present invention in a bone damage model through increased expression of bone regeneration marker proteins BMP-2 and runx2 in tissue specimens (*P<0.005) .

Hereinafter, the examples are only for illustrating the present invention in more detail, and it is understood by those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. It will be self-evident.

Example

Experiment method

Sericin Extraction

Bombyx mori (white hyacinth) cocoons were cut into pieces. To treat the shredded cocoons with 4-hexylresorcinol, 4-hexylresorcinol was first dissolved in 100% ethanol and diluted with distilled water at a ratio of 1:9. The 4-hexylresorcinol ethanol solution prepared above is added to the chopped silkworm cocoon and mixed, so that 0.5% by weight based on the total weight is the 4-hexylresorcinol ethanol solution and the remainder is made of the chopped silkworm cocoon. After mixing and stirring, it was dried to remove ethanol and water. To remove 4-hexylresorcinol that failed to bind to the sericin protein, washing was performed three times for 5 minutes using a 100% ethanol solution. After drying it, distilled water is added to the dried 4HR-pretreated silkworm cocoon, mixed, heated (refined) at 120° C. under 1.5 pressure for 1 hour, and then the supernatant is collected and dried to obtain the active ingredient of the present invention. Phosphorus 4HR-pretreated sericin was obtained (Figure 1).

Fourier-transform infrared (FT-IR) spectroscopy

FT-IR absorption spectra were measured using a Fourier transform spectrometer (Vertex 80, Bruker Optics, Germany) equipped with an attenuated total reflectance (ATR) accessory (MIRacle, PIKE technologies). Spectra were recorded in the spectral range of 1000 to 1800 cm ^-1 with a spectral resolution of 4 cm ^-1 with a deuterated L-alanine doped triglycene sulphate (DLATGS) detector, and 128 repeated scans were averaged for each spectrum.

Cell culture and sericin treatment

RAW264.7 murine macrophages (Korean Cell Line Bank No. 40071) were suspended in culture medium. Cells were placed in a 6-well culture plate and treated with each sericin at concentrations of 1, 5, and 10 μg/mL. After 2, 8 and 24 hours of culture, cells were collected. Cells from control cultures were treated with the same volume of solvent as required for sericin.

animal model

Eight-week-old Crl:CD (Sprague-Dawley) specific pathogen-free (SPF)/VAF inbred rats (Orientbio Inc., Seongnam, Korea) were used. All procedures were performed in accordance with laboratory animal care guidelines and were approved by the Gangneung-Wonju Animal Research Institute (GWNU-2022-7, approved April 29, 2022). Twenty rats (2-3 per cage) were housed under a 12-h light/12-h dark cycle in a controlled environment at 20-22°C and 40% humidity for 1 week for acclimatization before experimentation. Rats had ad libitum access to food and water and were fed a controlled semi-synthetic diet according to classical recommendations (74% carbohydrates from soy vegetable oil, 14% protein from casein, supplemented with a standard vitamin and mineral mix).

Considering the high mortality rate of surgery for cranial defects of critical size, additional animals were assigned to each group.

Each transplant experimental group used in the bone defect model is as follows:

Unfilled control (U);

Group treated with gelatin sponge (Cutanplast Dental®, Uniplex, Sheffield, UK) alone (G);

Gelatin sponge-treated group containing sericin (B); and

A group treated with a gelatin sponge containing 4HR-pretreated sericin of the present invention (B+4HR).

The amount of sericin in each graft was 50 μg.

The anesthetic solution was a combination of 0.5 mL of Zoletil (125 mg/mL; Bayer Korea, Seoul, Korea) and 0.5 mL of Xylazine (Rompun; Bayer Korea). The injection volume was 0.3 mL per rat. After sterilizing the skin, a midsagittal incision was made in the calf. A trephine bur (diameter: 8.0 mm) was used to create a critical size defect in the calvaria. After inserting the graft, the skin and periosteum were simultaneously sutured using 3-0 black silk. After surgery, antibiotics (gentamicin; Daesung, Yiwang, Korea) and painkillers (tolfenamic acid; Samyang anipharm, Seoul, Korea) were administered. The doses of gentamicin and tofenamic acid were 5 mg/day and 4 mg/day, respectively. The drug was administered for 48 hours after surgery. Two animals died in group B during the first 24 hours. After 8 weeks, all rats were sacrificed and samples of the calvaria were processed for further analysis.

Micro-computerized tomography (mCT)

ttisellen, Switzerland)을 사용하여 얻었다. 초기 수술 결손의 크기는 관심 영역(ROI)을 결정하기 위해 참조되었다. ROI의 골 부피(bone volume, BV)는 mCT 소프트웨어(CT Analyzer V.1.17.7.2+, Skyscan)를 사용하여 분석되었다. 이미지는 각각 48 및 255로 설정된 낮은 회색조 임계값과 상위 회색조 임계값으로 분석되었다.For mCT analysis of calvarial specimens (10 Х 10 Х 0.5 mm), the SkyScan1173 system (Skyscan, Kontich, Belgium) was used. The source voltage was set at 90kVp. An aluminum filter was used, and the image pixel size was 9.04 μm. Scanned images were reconstructed using Nrecon software (V.1.7.0.4, Micro Photonics Inc., Allentown, PA, USA). Additional images were obtained from the Gangneung-Wonju National University Science and Instrumentation Center (Gangneung, Korea) using a μCT50 (Scanco Medical, Br) for repeat confirmation.

ttisellen, Switzerland). The size of the initial surgical defect was referenced to determine the region of interest (ROI). Bone volume (BV) of the ROI was analyzed using mCT software (CT Analyzer V.1.17.7.2+, Skyscan). Images were analyzed with the lower and upper grayscale thresholds set to 48 and 255, respectively.

MT staining, immunochemical staining, and Western blot.

Histochemical analysis of calvarial specimens (10 Х 10 Х 0.5 mm) and Western blot were performed on decalcified tissue. After microcomputed tomography, the tissue pieces were placed in decalcification solution and decalcified for 5 days at room temperature. The decalcified tissue was divided in half at the center, half was embedded in paraffin, and immunochemical staining for MT stain and BMP-2 and TLR-2 was performed. Immunochemical staining used FITC-tagged BMP-2 and TLR-2 antibodies, all of which were purchased from SantaCruz biotech (Santa Cruz, CA, USA). Counter-staining for these stains was performed using DAPI. Proteins were extracted from the remaining tissue pieces as in the cell experiment and analyzed by western blot for BMP-2 and runx2, a transcription factor related to osteogenesis.

Example 1. Sericin yield according to refining temperature

In order to extract sericin in high yield, the present inventors pretreated 4HR to shredded silkworm cocoons before refining according to the present invention, and incubated the 4HR-pretreated shredded silkworm cocoons at various temperatures (37, 70, 100, and 120°C). ) was refined.

As a result, as shown in Figure 2, it was confirmed that the yield of sericin improved as the temperature increased. The yield was expressed as the ratio of sericin obtained to the weight of administered cocoons.

Example 2. FT-IR analysis

The present inventors established a method of treating 4HR on shredded silkworm cocoons before refining in order to extract and obtain sericin that was not denatured during hot water extraction, and the 4HR pretreatment improved the structural stability of the 4HR-pretreated silk sericin obtained after refining. Actual improvement was confirmed through FT-IR analysis.

As a result, as shown in Figure 3a, the 4HR-pretreated sericin protein of the present invention is characterized in the mid-infrared region of 1600-1700 (amide I), 1480-1580 (amide II) and 1230-1300 cm ^-1 (amide III) A typical absorption band was observed. In particular, the amide I band corresponding to C=O stretching is known to change very sensitively depending on the shape of the protein secondary structure. Therefore, careful peak assignment and abundance assessment of amide I subbands are very useful to obtain detailed information about protein secondary structures such as β-sheet, α-helix, β-turn and random coil. .

Additionally, as shown in Figure 3b, the enlarged spectrum of the amide I band showed a broad vibrational absorption centered at 1644 cm ^-1 for the investigated 4HR-pretreated sericin protein. When pretreated with 4HR, it was observed that the protein structure of silk sericin showed an additional increase in absorption at ~1620 cm ^-1 .

Additionally, as shown in Figure 3c, the broad amide I band can be divided into several fine peaks by calculating the second derivative of the infrared absorption spectrum, thereby corresponding to β-sheet, random, helical, and β-turn structures. A subtle peak could be assigned.

Interestingly, at ~1620 cm ^-1 , compared to the 4HR-untreated sericin protein, the β-sheet secondary structure of the 4HR-pretreated sericin protein was significantly increased, while other secondary structures of random, helical, and β-turn structures ( 1649, 1660, and 1682 cm ^-1 , respectively) decreased significantly.

This demonstrates that the 4HR pretreatment before refining of the present invention can effectively obtain a structurally stabilized sericin protein by increasing the β-sheet structure of the refined silk sericin protein.

In other words, in the case of the conventional hydrothermal extraction method of sericin, the protein is denatured by high temperature. In order to develop a graft material with excellent bone regeneration ability due to the efficacy of sericin itself, which is a natural substance, the unique protein structure of sericin must be preserved as much as possible to prevent denaturation. Compounds that may pose a problem to the human body during the refining process require at least the refining technology used. Accordingly, the 4HR pretreatment method of the present invention is a novel sericin extraction method that can obtain undenatured sericin, and it overcomes the denaturation problem of the hot water extraction method and improves the conventional method.

Example 3. BMP-2 expression in RAW264.7 cells

The present inventors investigated the expression level of BMP-2, which is known to increase bone regeneration, when RAW264.7 cells were treated with 4HR-pretreated silk sericin of the present invention. As a result, in RAW264.7 cells, 4HR-pretreated silk sericin of the present invention significantly increased BMP-2 expression (Figure 4a), while silk sericin without 4HR-pretreated did not increase BMP-2 expression. (Figure 4b). Additionally, compared to silk sericin extracted at low temperature, the expression level of BMP-2 was higher in the 4HR-pretreated silk sericin (Sericin + 4HR) of the present invention (FIG. 4c). In other words, 4HR-pretreatment can significantly increase the expression of BMP-2 by improving the production of biologically active sericin compared to general refining (room temperature extraction) product treatment, and this action is believed to promote bone formation.

Additionally, the present inventors investigated the mechanism of BMP-2 induction when RAW264.7 cells were treated with the 4HR-pretreated silk sericin of the present invention. In the case of 4HR, induction of BMP-2 is known to be mainly mediated by histone deacetylase (HDAC) inhibition. However, as shown in Figure 5a, the 4HR-pretreated silk sericin of the present invention induced BMP-2 through the Toll-like receptor (TLR)-mediated pathway. TRIF is a signaling mediator of the TLR signaling pathway. When TRIF inhibitory peptide (Pepinh-TRIF) was treated before 4HR-pretreated silk sericin, the expression level of BMP-2 was greatly reduced in RAW264.7 cells. Therefore, the mechanism of BMP-2 induction in response to 4HR-pretreated silk sericin appears to be primarily mediated by the TLR signaling pathway. Additionally, 4HR is a potent inhibitor of HDAC. As shown in Figure 5b, there was no significant difference in HDAC activity between groups when 4HR-pretreated silk sericin was administered to RAW264.7 cells (P>0.05). Additionally, Sparstolonin B is known to be an inhibitor of TLR2 and TLR4. 4HR can also induce BMP-2 in RAW264.7 cells. 4HR has anti-inflammatory and antioxidant activities. Since the induction of BMP-2 in RAW264.7 cells after 4HR-pretreated silk sericin administration was believed to be mediated by the TLR signaling pathway, the expression level of BMP-2 was significantly affected by spastolonin B pretreatment in the 4HR-pretreated silk sericin group. will be significantly reduced. However, increased BMP-2 expression by 4HR administration is a TLR-independent pathway, and application of sparstolonin B (a TLR inhibitor) will be less effective in inhibiting BMP-2 expression. As shown in Figure 5c, in fact, in the case of spastolonin B pretreatment, the 4HR group showed a higher BMP-2 expression level than the 4HR-pretreated silk sericin (Sericin + 4HR) group. Interestingly, spastolonin B pretreatment itself reduced BMP-2 expression in RAW264.7 cells compared to 4HR without pretreatment. Spastolonin B can inhibit BMP-2 expression in cells. Therefore, this decrease may be due to the inhibitory action of sparstolonin B.

In other words, the inducing effect of BMP-2 is not achieved by 4-hexylresorcinol, which is mostly lost during the refining process and only a trace amount remains in sericin, but is structurally stabilized by pretreatment with 4-hexylresorcinol. BMP-2 is induced by sericin, which means that the 4HR-pretreated sericin induces BMP-2 through the Toll-like receptor pathway.

Example 4. Bone regeneration effect by 4HR-pretreated silk sericin of the present invention in bone damage model

The present inventors analyzed the bone regeneration effect of treating the 4HR-pretreated silk sericin of the present invention in a bone damage model using micro-CT.

As a result, as shown in Figure 6, the bone volume (BV) of the gelatin sponge-treated group (B+4HR) containing 4HR-pretreated sericin of the present invention was significantly higher than that of the other comparative control groups (P<0.001) .

That is, 4HR pretreated sericin grafts significantly increased bone formation in a critical-size rat calvarial defect model compared to untreated sericin grafts.

Additionally, as shown in Figure 7, the bone regeneration effect was confirmed even in demineralized tissue specimens. In other words, the group treated with the 4HR-pretreated sericin-containing gelatin sponge showed greater bone regeneration compared to the non-pretreated sericin-containing gelatin sponge (bar = 1 mm).

Additionally, as shown in Figure 8, when tissue specimens were subjected to immunofluorescence staining, the expression of BMP-2 and TLR2 was increased in the gelatin sponge-treated group containing 4HR-pretreated sericin.

In addition, as shown in Figure 9, when Western blotting was performed on these tissue specimens, the expression of BMP-2 and runx2 in the group treated with the 4HR-pretreated sericin-containing gelatin sponge was higher than that of the non-pretreated sericin-containing gelatin. It was confirmed that there was a statistically significant increase compared to sponge (P=0.001 & 0.002, respectively).

In summary, in the present invention, silkworm cocoons were pretreated with 4-hexylresorcinol to stabilize the structure of the sericin protein before refining, and in the case of sericin obtained by refining after the 4-hexylresorcinol-pretreatment, it was a stable protein. It has a structure that increases BMP-2 expression and has a significant effect in promoting bone formation, making it suitable for use as a treatment for bone diseases/damage.

The above-described present invention has been examined with a focus on preferred embodiments and experimental examples, and those skilled in the art may implement forms different from the detailed description of the present invention within the essential technical scope of the present invention. Examples and experimental examples may be implemented. Here, the essential technical scope of the present invention is indicated in the patent claims, and all differences within the equivalent scope should be construed as being included in the present invention.

Claims (14)

Method for producing Sericin for bone regeneration, comprising the following steps:
(a) cutting the cocoon into pieces;
(b) preparing a 4-hexylresorcinol ethanol solution by dissolving 4-hexylresorcinol (4HR) in ethanol;
(c) pretreating the shredded silkworm cocoon of step (a) by mixing the 4-hexylresorcinol ethanol solution of step (b), followed by drying; and
(d) obtaining sericin by refining the dried 4HR-pretreated silkworm cocoons of step (c) and then drying them.
According to paragraph 1,
A method characterized in that the shredded silkworm cocoons in step (c) are mixed to contain 90% by weight and the 4-hexylresorcinol ethanol solution at 0.5% by weight.
According to paragraph 1,
The refining method in step (d) is characterized in that the 4HR-pretreated silkworm cocoon is subjected to hot water extraction at 100 to 150° C. for 30 minutes to 3 hours.
According to paragraph 1,
The method wherein the sericin in step (d) has a modified molecular structure compared to sericin that has not been 4HR-pretreated.
According to clause 4,
The modification of the molecular structure includes an increase in beta-sheet secondary structure, a decrease in alpha-helix secondary structure, a decrease in beta-turn secondary structure, and random coil (random coil). coil) method, characterized in that it is one or more selected from the group consisting of reduction of secondary structure.
According to paragraph 1,
The sericin in step (d) is characterized in that it increases the expression of BMP (Bone morphogenic protein)-2 compared to sericin that is not 4HR-pretreated.
Method for manufacturing a therapeutic agent for bone regeneration containing Sericin, comprising the following steps:
(a) cutting the cocoon into pieces;
(b) preparing a 4-hexylresorcinol ethanol solution by dissolving 4-hexylresorcinol (4HR) in ethanol;
(c) pretreating the shredded silkworm cocoon of step (a) by mixing the 4-hexylresorcinol ethanol solution of step (b), followed by drying; and
(d) obtaining sericin by refining the dried 4HR-pretreated silkworm cocoons of step (c) and then drying them.
A therapeutic agent for bone regeneration prepared according to the method of claim 7.
According to clause 8,
The therapeutic agent for bone regeneration is characterized in that it additionally includes at least one selected from the group consisting of biocompatible binders, antibiotics, vitamins, glucosamine, cytokines, and growth factors.
According to clause 8,
The therapeutic agent for bone regeneration is characterized in that it can be injected directly into the bone defect site or by filling it into a graft material in a bioinjectable form.
A pharmaceutical composition for preventing and treating bone disease containing Sericin as an active ingredient, prepared according to the production method of any one of claims 1 to 6.
According to clause 11,
The composition, wherein the bone disease is at least one selected from the group consisting of bone damage, bone defect disease, osteoporosis, osteoarthritis, rheumatoid arthritis, osteomalacia, rickets, osteitis fibrositis, aplastic bone disease, and metabolic bone disease. .
A food composition for bone regeneration containing sericin as an active ingredient, manufactured according to the production method of any one of claims 1 to 6.
A feed composition for bone regeneration containing sericin as an active ingredient, manufactured according to the production method of any one of claims 1 to 6.