Complex containing undenatured type II collagen and chitosan oligosaccharide and method for preparing same

Abstract

The present invention discloses a complex containing undenatured type II collagen and chitosan oligosaccharide and a method for preparing the same. The above preparation method includes the steps of: (1) activation: taking a certain amount of powder or microparticles containing undenatured type II collagen, adding an activator, mixing, and maintaining at 0 to 80°C for 1 to 300 minutes; (2) sieving: sieving the mixture obtained in step (1) to remove the activator that was not involved in the reaction; (3) coupling: adding chitosan oligosaccharide to the mixture obtained in step (2) from which the activator that was not involved in the reaction was removed, and performing dynamic mixing at 0 to 80°C for 1 to 300 minutes; and (4) drying: drying the mixture obtained in step (3) to a moisture content of 8% or less to obtain a product that is a complex containing undenatured type II collagen and chitosan oligosaccharide. This preparation method is simple to implement, has few work steps, and is efficient.

Description

[Technical field]

[0001] The present invention relates to a technique for preparing a protein complex in biotechnology, and more particularly to a complex containing undenatured type II collagen and chitosan oligosaccharide and a method for preparing the same. [Background technology]

[0002] Collagen is a natural biopolymer with a triple helical structure expressed by animal cells. As the main component of the extracellular matrix, it is present in almost all tissues, accounting for about one-third of the total proteins in the mammalian body, and is widely present in animal skin, Achilles tendons, cartilage and other connective tissues. Collagen plays a role in supporting and connecting tissues, and also affects cell differentiation, proliferation, migration, adhesion, etc., and has an important effect on tissue morphology, mechanical strength, and metabolism within tissues. Currently, 28 different types of collagen have been discovered in mammals. The various collagens have different distribution states, forms of existence, synthesis and metabolic pathways in the animal body.

[0003] Type II collagen is often found in tissues such as articular cartilage and bone, and is mainly secreted by animal chondrocytes. Type II collagen belongs to fibril-forming collagens that intertwine into a tough meshwork in the body to maintain the structural integrity of cartilage structures and provide tension and support to cartilage tissue. Type II collagen has specific molecular recognition signals that promote the adhesion, proliferation, and differentiation of chondrocytes, and contributes more to the phenotype and structural maintenance of chondrocytes than type I collagen. Abnormalities in the expression and metabolism of type II collagen, as well as degeneration of its molecular structure, can cause various diseases.

[0004] Rheumatoid arthritis is a chronic autologous disease characterized mainly by synovitis of the joints, and is a disease associated with an autoimmune response to type II collagen. The key to its pathological mechanism lies in recurrent inflammation of the joints. The pathological characteristics of rheumatoid arthritis mainly include proliferation of synovial lining cells, abundant infiltration of interstitial inflammatory cells, angiogenesis and formation, and destruction of cartilage and bone tissue. Rheumatoid arthritis belongs to immune diseases in humans and can cause various diseases in sites rich in type II collagen.

[0005] Induction of immune tolerance by oral administration of type II collagen has a significant effect on the prevention and treatment of rheumatoid arthritis. The immune determinants of type II collagen bind to antigens in vivo to form complexes, which are presented on the surface of antigen-presenting cells via the secretory pathway and are recognized by the surface receptors of receptor-specific immune-related cells to form complexes. This activates and proliferates specific immune-related cells, secretes various immune factors, and initiates an immune response. As a result, specific immunity in local gut-associated lymphoid tissue occurs, and systemic immune responses are suppressed, forming a form of peripheral immune tolerance.

[0006] When conventional type II collagen is orally administered, it is degraded in the human stomach, resulting in a reduced target release rate in the intestinal tract. At the same time, most of the amino acids on the surface of native type II collagen are negatively charged, which inhibits collagen phagocytosis by intestinal surface cells, resulting in limited efficacy. Undenatured type II collagen can improve the target release rate in the intestinal tract via an appropriate route and improve the phagocytosis rate by intestinal cells, leading to improved therapeutic efficacy. Summary of the Invention

[0007] The present invention provides a complex containing undenatured type II collagen and chitosan oligosaccharide and a method for preparing the same, in response to the limited therapeutic effect against arthritis due to the low release rate and low phagocytosis rate of conventional undenatured type II collagen.

[0008] According to one aspect of the present invention, (1) Activation: Take a certain amount of powder or microparticles containing undenatured type II collagen, add an activator, mix, and keep at 0 to 80°C for 1 to 300 minutes. (2) Sifting: Sifting the mixture obtained in step (1) to remove activator that has not participated in the reaction; (3) Coupling: adding chitosan oligosaccharide to the mixture obtained in step (2) from which the activator not involved in the reaction has been removed, and subjecting the mixture to dynamic mixing at 0 to 80°C for 1 to 300 minutes; (4) Drying: The mixture obtained in step (3) is dried so that the moisture content is 8% or less to obtain a product that is a complex containing undenatured type II collagen and chitosan oligosaccharide. The present invention provides a method for preparing a complex containing undenatured type II collagen and chitosan oligosaccharide, the method comprising the steps of:

[0009] Preferably, the activator added in step (1) above is daidzein, soy isoflavone, anthocyanidin, anthocyanin, tea polyphenol, dihydromyricetin, or a plant extract containing these.

[0010] Preferably, the mass ratio of the activator to the undenatured type II collagen is 100:1 to 1:10,000.

[0011] Preferably, the degree of polymerization of the chitosan oligosaccharide added in the above step (3) is 2-10.

[0012] Preferably, the chitosan oligosaccharide added in the above step (3) is a mixture of chitosan oligosaccharides having different degrees of polymerization, with an average degree of polymerization ranging from 2-10.

[0013] Preferably, the mass ratio of the chitosan oligosaccharide to the undenatured type II collagen added in the above step (3) is 100:1 to 1:200.

[0014] Preferably, in the above step (2), the mixture obtained in step (1) is sieved using a mesh screen.

[0015] According to another aspect of the present invention, there is provided a complex containing undenatured type II collagen and chitosan oligosaccharide prepared by the preparation method described in the above embodiment.

[0016] Preferably, in the above complex, the undenatured type II collagen and the chitosan oligosaccharide are present as a chemical bond.

[0017] Preferably, the ratio of undenatured type II collagen to chitosan oligosaccharide is in the range of 1:200 to 100:1.

[0018] The method for preparing the complex containing non-denatured type II collagen and chitosan oligosaccharide according to the embodiment of the present invention has the advantages of simple implementation process, few operation steps, and high efficiency. The embodiment of the present invention can use an activator to immobilize chitosan oligosaccharide on the surface of non-denatured type II collagen, thereby increasing the density of positive charges on the surface of non-denatured type II collagen. Therefore, the non-denatured type II collagen in the complex containing non-denatured type II collagen and chitosan oligosaccharide according to the embodiment of the present invention is less susceptible to in vivo protease degradation after oral administration, and can increase the target release rate of non-denatured type II collagen in the intestinal tract. Furthermore, the non-denatured type II collagen in the complex according to the embodiment of the present invention promotes the phagocytosis of collagen by cells on the surface of the intestine, thereby improving the phagocytosis rate of the non-denatured type II collagen by intestinal cells, and improving the therapeutic effect. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] Hereinafter, the present invention will be described in detail with reference to specific embodiments. However, it should be noted that the embodiments are merely illustrative of the complex comprising undenatured type II collagen and chitosan oligosaccharide according to the present invention and the preparation method thereof, and do not represent the entire patent of the present invention, much less should they be construed as limiting the scope of protection of the present invention. Some adjustments and improvements can be made without departing from the essential concept of the present invention, and all of these are included in the scope of protection of the present invention.

[0020] According to the general concept of the present invention, (1) Activation: Taking a certain amount of powder or microparticles containing undenatured type II collagen (e.g., powder or microparticles obtained by pretreating animal cartilage), adding an activator, mixing (e.g., uniformly), and holding at 0-80°C (e.g., 35-40°C or 50-55°C) for 1 min-300 min (e.g., 90 min or 200 min); (2) Sifting: Sifting the mixture obtained in step (1) to remove activator that has not participated in the reaction; (3) Coupling: adding chitosan oligosaccharide to the mixture obtained in step (2) from which the activator not involved in the reaction has been removed, and performing dynamic mixing at 0 to 80°C (e.g., 35 to 40°C or 50 to 55°C) for 1 to 300 minutes (e.g., 90 minutes or 120 minutes); (4) Drying: The mixture obtained in step (3) is dried so that the moisture content is 8% or less (e.g., 7% or less or 6% or less) to obtain a product that is a complex containing undenatured type II collagen and chitosan oligosaccharide. The present invention provides a method for preparing a complex containing undenatured type II collagen and chitosan oligosaccharide, the method comprising the steps of:

[0021] In the preparation method of the embodiment of the present invention, an activator is used to immobilize chitosan oligosaccharide on the surface of non-denatured type II collagen, so that non-denatured type II collagen is less susceptible to degradation by proteases in vivo after oral administration, and the target release rate of non-denatured type II collagen in the intestinal tract can be increased.Furthermore, by adding an activator, the density of positive charges on the surface of non-denatured type II collagen can be increased, so that the non-denatured type II collagen in the complex promotes the phagocytosis of collagen by cells on the surface of the intestine, thereby improving the phagocytosis rate of non-denatured type II collagen by intestinal cells.The complex obtained by the preparation method of the present invention improves the therapeutic effect of non-denatured type II collagen.

[0022] In an embodiment, the activator added in step (1) above is a flavone, anthocyanidin, a polyphenolic compound, or a plant extract containing these.

[0023] In an embodiment, the mass ratio of the activator to the undenatured type II collagen powder or microparticles is from 100:1 to 1:10000, such as 1:10.

[0024] In an embodiment, the degree of polymerization of the chitosan oligosaccharide added in above step (3) is 2-10, such as 3 or 4.

[0025] As an example, the chitosan oligosaccharide added in step (3) above is a mixture of chitosan oligosaccharides with different degrees of polymerization ranging from 2 to 10, such as an average degree of polymerization of 3 or 4.

[0026] As an example, the mass ratio of chitosan oligosaccharide to the powder or microparticles of undenatured type II collagen added in the above step (3) is 100:1 to 1:200, such as 2.5:1 or 1:5.

[0027] In an embodiment, in the above step (2), the mixture obtained in step (1) is sieved using a mesh screen. For example, the mixture obtained in step (1) may be sieved using a mesh screen of 200 to 500 mesh.

[0028] According to another aspect of the present invention, there is further provided a complex comprising undenatured type II collagen and chitosan oligosaccharide prepared by the preparation method according to any one of the above embodiments.

[0029] In an embodiment, in the above complex, the undenatured type II collagen and the chitosan oligosaccharide are present as a chemical bond.

[0030] In an embodiment, the ratio of undenatured type II collagen to chitosan oligosaccharide is in the range of 1:200 to 100:1.

[0031] Below, only some of the embodiments of the present invention are shown, which are used for illustrative purposes only, and other possible embodiments of the present invention will be understood by those skilled in the art, so they will not be repeated here. EXAMPLES

[0032] The raw material was microparticles containing 20% ​​undenatured type II collagen obtained by pretreatment of animal cartilage, with a water content of about 20% and an activator of soy isoflavone.

[0033] (1) Activation: 200 g of chicken cartilage powder with an average particle size of 300 mesh was taken, 4 g of soy isoflavone was added, and the mixture was mixed uniformly by a mechanical method. The mixture was then kept at 35°C to 40°C for 200 minutes.

[0034] (2) Sifting: The mixture obtained in step (1) was sieved using a 500 mesh screen and the residue was collected.

[0035] (3) Coupling: 100 g of chitosan oligosaccharide having an average degree of polymerization of 4 was added to the residue obtained in step (2), and the mixture was mixed by shaking at 35° C. to 40° C. for 200 minutes.

[0036] (4) Drying: The mixture obtained in step (3) was dried in vacuum at low temperature until the moisture content was 7% or less.

[0037] The mixture contains a complex of undenatured type II collagen and chitosan oligosaccharide, in which the chitosan oligosaccharide is covalently coupled to the surface of the undenatured type II collagen. Test results show that the ratio of undenatured type II collagen to chitosan oligosaccharide in the complex is 22.3:1. EXAMPLES

[0038] The raw material was a lyophilized powder of undenatured type II collagen extracted from chicken cartilage with a moisture content of approximately 10%, and the activator was blueberry extract containing 20% ​​dihydromyricetin with a moisture content of approximately 6%.

[0039] (1) Activation: 100 g of freeze-dried powder of non-denatured type II collagen was taken, 10 g of blueberry extract was added, and the mixture was mixed uniformly by a mechanical method and then kept at 50°C to 55°C for 90 minutes.

[0040] (2) Sieving: The mixture obtained in step (1) was sieved using a 200 mesh screen to collect the freeze-dried powder of non-denatured type II collagen.

[0041] (3) Coupling: 20 g of chitosan oligosaccharide having an average degree of polymerization of 3 was added to the lyophilized powder obtained in step (2), and the mixture was mixed by shaking at 50° C. to 55° C. for 120 minutes.

[0042] (4) Drying: The mixture obtained in step (3) was dried in vacuum at low temperature until the moisture content was 6% or less.

[0043] The mixture contains a complex of undenatured type II collagen and chitosan oligosaccharide. Test results show that the ratio of undenatured type II collagen to chitosan oligosaccharide in the complex is 20.6:1.

Claims

1. (1) Activation: A certain amount of powder or microparticles containing undenatured type II collagen is taken, an activator is added and mixed, and the mixture is kept at 0 to 80° C. for 1 to 300 minutes. (2) Sifting: Sifting the mixture obtained in step (1) to remove activator that has not participated in the reaction; (3) Coupling: adding chitosan oligosaccharide to the mixture obtained in step (2) from which the activator not involved in the reaction has been removed, and performing dynamic mixing at 0 to 80° C. for 1 to 300 minutes; (4) Drying: The mixture obtained in step (3) is dried so that the moisture content is 8% or less to obtain a product which is a complex containing undenatured type II collagen and chitosan oligosaccharide. The process comprises the steps of: The activator is characterized in that it is a soy isoflavone or a blueberry extract containing dihydromyricetin. A method for preparing a complex containing undenatured type II collagen and chitosan oligosaccharide.

2. The activator is characterized in that it is soy isoflavone.

2. The method of claim 1.

3. The mass ratio of the activator to the non-denatured type II collagen is 100:1 to 1:10000. The preparation method according to claim 2.

4. The degree of polymerization of the chitosan oligosaccharide added in the step (3) is 2 to 10. The preparation method according to any one of claims 1 to 3.

5. The chitosan oligosaccharide added in the step (3) is a mixture of chitosan oligosaccharides having different degrees of polymerization, the average degree of polymerization being 2 to 10. The preparation method according to any one of claims 1 to 3.

6. The mass ratio of the chitosan oligosaccharide to the undenatured type II collagen added in the step (3) is 100:1 to 1:

200. The preparation method according to any one of claims 1 to 3.

7. In the step (2), the mixture obtained in the step (1) is sieved using a mesh screen. The preparation method according to any one of claims 1 to 3.