Extraction process of non-denatured type Ⅱ collagen for relieving osteoarthritis and product and application thereof

Abstract

This invention discloses an extraction process for non-denatured type II collagen to alleviate osteoarthritis, along with its products and applications. It belongs to the field of non-denatured type II collagen extraction technology. The extraction process includes the following steps: (1) Pretreatment to obtain cartilage granules; (2) Enzyme activation: A mixture of wolfberry, ophiopogon japonicus, chicory, and honeysuckle is added to water for extraction to obtain an extract. Pepsin is then added and mixed evenly to obtain an activated enzyme; (3) Enzymatic hydrolysis: The activated enzyme is added to the cartilage granules for enzymatic hydrolysis to obtain a precipitate; (4) Post-treatment: The precipitate is dried and pulverized. The extraction process of this invention is simple, has high production efficiency, requires less enzyme, is low-cost, and is environmentally friendly, meeting the needs of large-scale production. Furthermore, the prepared non-denatured type II collagen has good improvement and therapeutic effects on osteoporosis and osteoarthritis.

Description

Technical Field

[0001] This invention belongs to the field of non-denatured type II collagen extraction technology. Specifically, it relates to an extraction process for non-denatured type II collagen to relieve osteoarthritis, as well as its products and applications. Background Technology

[0002] Undenatured type II collagen refers to type II collagen that retains the complete triple helix structure of macromolecular collagen after extraction. It has great development and application value in the prevention and treatment of rheumatoid arthritis and osteoarthritis.

[0003] Currently, the main extraction method for non-denatured type II collagen is enzymatic hydrolysis. For example, Chinese patent CN113563458A discloses a method for preparing non-denatured type II collagen, which uses chicken breast cartilage as raw material and involves steps such as material selection, cleaning, crushing, enzymatic hydrolysis, drying, crushing, and sieving. This invention provides rapid extraction with a total extraction time of no more than 4 hours, but the content of non-denatured type II collagen prepared by this method is low and it is not suitable for industrial application.

[0004] Wei Yongze et al. (Optimization and quality verification of extraction process of non-denatured type II collagen [J], Food Science, 2024, 49(06), 194-203) used fresh chicken breast cartilage as raw material. After cleaning and sterilization, the cartilage was crushed and pretreated by the co-action of neutral salt and alkali. Then, the non-denatured type II collagen was extracted by adding pepsin using a combination of acid and enzymatic extraction. The extraction rate of non-denatured type II collagen in this extraction process reached 68.9%. However, the extraction process is complicated and takes a long time, which is not suitable for industrial application.

[0005] Chinese patent CN116253791A discloses a method for preparing non-denatured type II collagen with the effect of improving bone and joint health, including the following steps: S1, mixing cartilage fragments with a mixed solution containing NaOH and NaCl, performing sterilization and impurity removal treatment, and separating the solid and liquid to obtain sterilized and impurity-removed cartilage fragments; S2, mixing the sterilized and impurity-removed cartilage fragments with water and performing homogenization treatment to obtain a homogenate; S3, adjusting the pH of the homogenate to 1.5-3, performing enzymatic hydrolysis treatment, and separating the solid and liquid to obtain a clear enzymatic hydrolysate; S4, adjusting the pH of the clear enzymatic hydrolysate to 4.0-6.5, performing solid-liquid separation, drying and pulverizing the solid phase to obtain non-denatured type II collagen. The protein content of the non-denatured type II collagen extracted by this method is above 68%, but its extraction time is 16-36 hours, which also has the problem of being time-consuming and not conducive to industrial application.

[0006] Currently, the extraction process for non-denatured type II collagen cannot simultaneously achieve both purity and extraction efficiency. It generally suffers from problems such as cumbersome production processes, long processing times, and high costs, and is only suitable for small-scale laboratory preparation. Therefore, developing an extraction process that is of high quality, has high reaction efficiency, and is low in cost to meet the needs of large-scale production of non-denatured type II collagen is a technical problem that urgently needs to be solved. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides an extraction process for non-denatured type II collagen, along with its products and applications, for alleviating osteoarthritis. This extraction process effectively improves reaction efficiency, reduces production costs while ensuring product quality, and provides technical support for the large-scale production of non-denatured type II collagen.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] In a first aspect, the present invention provides an extraction process for non-denaturing type II collagen to alleviate osteoarthritis, comprising the following steps:

[0010] (1) Pretreatment: The animal cartilage is washed and crushed to obtain cartilage particles;

[0011] (2) Enzyme activation: Mix wolfberry, ophiopogon japonicus, chicory and honeysuckle, extract with water, separate solid and liquid to obtain extract, cool, add pepsin and mix evenly, adjust pH to 1.5-3.5 to obtain activated enzyme;

[0012] (3) Enzymatic hydrolysis: Add the activated enzyme obtained in step (2) to the cartilage particles obtained in step (1) for enzymatic hydrolysis, adjust the pH to 5.0-5.5, separate the solid and liquid, and obtain the precipitate;

[0013] (4) Post-processing: Dry and crush the precipitate obtained in step (3) to obtain the final product.

[0014] In some implementations, the weight ratio of wolfberry, ophiopogon japonicus, chicory and honeysuckle in step (2) is 3-5:1:5-10:5-10; preferably 3-5:1:6-9:6-9; and more preferably 3-4:1:7-8:7-8.

[0015] In some implementations, the amount of water added in step (2) is 8-12 times the total amount of wolfberry, ophiopogon japonicus, chicory and honeysuckle; preferably 12-15 times; and more preferably 12-13 times.

[0016] In some implementations, the extraction in step (2) is performed as follows: extraction at 85-100℃ for 1-3 hours; preferably extraction at 90-100℃ for 1-3 hours; and more preferably extraction at 90-95℃ for 1-2 hours.

[0017] In some embodiments, the cooling in step (2) is cooling to 10-35°C; preferably 20-35°C; more preferably 30-35°C.

[0018] In some embodiments, the amount of pepsin added in step (2) is 10-30% of the weight of the extract; preferably 15-25%; more preferably 18-20%.

[0019] In some embodiments, the amount of activating enzyme added in step (3) is 0.5-2% of the weight of the cartilage particles, preferably 1-1.5%; more preferably 1.2-1.3%.

[0020] In some implementations, the enzymatic hydrolysis conditions in step (3) are: enzymatic hydrolysis at 30-37℃ for 2-10 hours; preferably enzymatic hydrolysis at 32-35℃ for 2-8 hours; and more preferably enzymatic hydrolysis at 32-35℃ for 2-6 hours.

[0021] In some implementations, the animal described in step (1) is a fish and / or a bird.

[0022] In some implementations, the cleaning in step (1) is: ultrasonic cleaning of animal cartilage with water.

[0023] Preferably, the temperature of the water is 10-35°C; more preferably, it is 20-30°C.

[0024] Preferably, the ultrasonic conditions are: ultrasonic treatment at a frequency of 30-50KHz for 20-30 minutes; more preferably, ultrasonic treatment at a frequency of 35-40KHz for 20-30 minutes.

[0025] In some embodiments, the crushing in step (1) is: crushing to a length of less than 1 cm after freezing. Preferably, the freezing temperature is -20 to -1°C.

[0026] In some embodiments, the drying in step (4) is drying to a moisture content of less than 10%. Preferably, the drying is vacuum drying or freeze drying.

[0027] The solid-liquid separation described in steps (2) and (4) of this invention is a conventional solid-liquid phase separation technique in the art, such as filtration, centrifugation or pressure filtration.

[0028] In some embodiments, the extraction process of the non-denaturing type II collagen for relieving osteoarthritis includes the following steps:

[0029] (1) Pretreatment: The animal cartilage is washed and crushed to obtain cartilage particles;

[0030] (2) Enzyme activation: mix wolfberry, ophiopogon japonicus, chicory and honeysuckle in a weight ratio of 3-4:1:7-8:7-8, add water to extract, filter to obtain extract, cool to 10-35℃, add pepsin and mix well, adjust pH to 1.5-3.5 to obtain activated enzyme;

[0031] The amount of pepsin added is 18-20% of the weight of the extract;

[0032] (3) Enzymatic hydrolysis: Add the activated enzyme obtained in step (2) to the cartilage particles obtained in step (1), hydrolyze at 32-35℃ for 2-6 hours, adjust the pH to 5.0-5.5, filter, and obtain the precipitate;

[0033] The amount of the activating enzyme added is 1.2-1.3% of the weight of the cartilage particles;

[0034] (4) Post-processing: Dry and crush the precipitate obtained in step (3) to obtain the final product.

[0035] Secondly, the present invention also provides non-denatured type II collagen prepared by the above extraction process.

[0036] Thirdly, this invention provides the application of water extracts of wolfberry, ophiopogon japonicus, chicory and honeysuckle in combination with pepsin in the preparation of non-denatured type II collagen.

[0037] In some embodiments, the water extract is prepared by mixing wolfberry, ophiopogon japonicus, chicory and honeysuckle in a weight ratio of 3-5:1:5-10:5-10, adding water for extraction, filtering, and cooling to 10-35°C to obtain the extract.

[0038] In some embodiments, the pepsin is 10-30% of the weight of the extract.

[0039] Fourthly, the present invention also provides the application of the above-mentioned non-denatured type II collagen in the preparation of pharmaceuticals.

[0040] The daily oral dose of the non-denatured type II collagen of the present invention when used in pharmaceuticals is 35-45 mg / kg; preferably 40 mg / kg.

[0041] In some embodiments, the application is the use of the above-mentioned non-denatured type II collagen in the preparation of medicines for the prevention and treatment of osteoporosis and / or osteoarthritis.

[0042] Fifthly, the present invention also provides a composition comprising non-denatured type II collagen prepared by the aforementioned extraction process.

[0043] In some embodiments, the non-denatured type II collagen of the present invention can also be used as a raw material to be mixed with pharmaceutically acceptable excipients to prepare corresponding pharmaceutical formulations.

[0044] Preferably, the pharmaceutical preparation is a tablet, capsule, granule, dry suspension, or pill.

[0045] The beneficial effects of this invention are as follows:

[0046] (1) The extraction process of the present invention is simple, has high production efficiency, requires less enzyme, has low cost, is green and environmentally friendly, meets the needs of large-scale production, and provides technical support for promoting the development of the non-denatured type II collagen extraction industry.

[0047] (2) In this invention, the aqueous extract of wolfberry, ophiopogon japonicus, chicory and honeysuckle can enhance the activity of pepsin after being mixed with pepsin, which greatly shortens the extraction time and improves the extraction efficiency of non-denatured type II collagen.

[0048] (3) The wolfberry, ophiopogon japonicus, chicory and honeysuckle used in this invention are all food and medicine homologous foods with good safety. At the same time, by utilizing the synergistic effect of the active ingredients among the components, the conditions for enzymatic hydrolysis can be reduced, the amount of enzyme used can be reduced, the cost can be lowered, and it is green and environmentally friendly.

[0049] (4) Experiments have shown that the non-denatured type II collagen prepared by this invention can increase bone density and improve bone strength. It can also reduce joint inflammation and slow down bone and cartilage destruction by reducing the production of inflammatory factors. Detailed Implementation

[0050] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0051] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention.

[0052] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0053] The “composition” described in this invention comprises non-denatured type II collagen and pharmaceutically acceptable excipients. In a specific embodiment, the non-denatured type II collagen described in this invention is provided in the composition in an effective amount (e.g., a therapeutically effective amount).

[0054] The "acceptable" ingredients described in this invention are substances suitable for human and / or animal use without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., having a reasonable benefit / risk ratio. The "pharmaceuticalally acceptable excipients" include inert diluents, dispersants and / or granulators, surfactants and / or emulsifiers, disintegrants, binders, preservatives, buffers, lubricants, and / or oils. The compositions may also contain excipients (e.g., cocoa butter and suppository waxes), colorants, coating agents, sweeteners, and flavoring agents.

[0055] The “compositions” described in this invention can be prepared by any method known in pharmaceutical science. Generally, these preparation methods involve associating the undenatured type II collagen (hereinafter referred to as the active ingredient) with a carrier or excipient and / or one or more other auxiliary ingredients, and then, if desired and / or expected, shaping and / or packaging the product into desired single-dose or multi-dose units.

[0056] The compositions of the present invention can be prepared according to known methods, such as those described in the General Rules for Preparation of the Chinese Pharmacopoeia 2020, the Japanese Pharmacopoeia 16th Edition, the United States Pharmacopoeia, and the European Pharmacopoeia 9th Edition. The specific preparation method depends on the dosage form.

[0057] The active ingredient and pharmaceutically acceptable excipients in the "composition" described in this invention will vary depending on the identity, body type, and / or condition of the treated subject and further on the route of administration of the active ingredient. The pharmaceutical composition may contain between 0.1% and 100% (w / w) of the active ingredient.

[0058] The "composition" described in this invention may optionally include other therapeutic ingredients for combined use in order to better exert the therapeutic effect on osteoporosis or osteoarthritis, such as "other drugs for treating osteoporosis" or "other drugs for treating osteoarthritis", wherein the "other drugs for treating osteoporosis" or "other drugs for treating osteoarthritis" may be chemical drugs, traditional Chinese medicine or traditional Chinese medicine preparations.

[0059] As used herein, “treatment” means, unless otherwise stated, reversing or alleviating the condition or disease to which the term applies, or one or more symptoms of such condition or disease, inhibiting the progression of said condition or disease or one or more symptoms thereof, or preventing said condition or disease or one or more symptoms thereof. As used in this invention, the term “treatment” refers to a therapeutic act, as defined above.

[0060] The term "effective amount" as used in this invention refers to an amount sufficient to elicit the desired biological response. The effective amount of the active ingredient of this invention may vary depending on factors such as the desired biological endpoint, the pharmacokinetics of the compound, the condition being treated, the administration pattern, and the age and health status of the subject. In some embodiments, the effective amount is a therapeutically effective amount. The effective amount is the amount of the sole active ingredient described in this invention in a single dose. In some embodiments, the effective amount is a combination of the sole active ingredient described in this invention in multiple doses.

[0061] The term "therapeuticly effective amount" as used in this invention refers to an amount sufficient to provide therapeutic benefit in the treatment of a condition or sufficient to delay or minimize one or more symptoms associated with that condition. A therapeutically effective amount of an active ingredient means an amount in which the therapeutic agent, alone or in combination with other therapies, provides therapeutic benefit in the treatment of a condition. The term "therapeuticly effective amount" may encompass amounts that improve overall therapy, reduce or avoid symptoms, signs, or causes of a condition, and / or enhance the therapeutic efficacy of another therapeutic agent. In some embodiments, a therapeutically effective amount is an amount sufficient to treat any of the diseases or conditions described.

[0062] It is worth noting that the pepsin used in this invention was purchased from Shanghai Maclean Biochemical Technology Co., Ltd. (1:30000). Unless otherwise specified, all raw materials used are common commercially available products, therefore their sources are not specifically limited. Unless otherwise specified, all operations are carried out at room temperature, specifically 20-25°C.

[0063] Basic Implementation

[0064] An extraction process for non-denaturing type II collagen to alleviate osteoarthritis, comprising the following steps:

[0065] (1) Pretreatment: Add water to chicken breast cartilage and stir to wash it to remove blood and impurities; then add purified water at a material-to-liquid mass ratio of 1:1.2-2 and ultrasonically wash (40KHz) for 0.5h to collect the cartilage; freeze and crush the cartilage into cartilage particles with a particle size not exceeding 1cm.

[0066] (2) Enzyme activation: Mix wolfberry, ophiopogon japonicus, chicory and honeysuckle in a weight ratio of 3-5:1:5-10:5-10, add 8-12 times the amount of water, extract at 85-100℃ for 1-3 hours, filter, remove the filter residue, and obtain the extract. Cool the extract to 10-35℃, add pepsin and mix well, and then adjust the pH to 1.5-3.5 with hydrochloric acid solution to obtain the activated enzyme;

[0067] The amount of pepsin added is 10-30% of the weight of the extract;

[0068] (3) Enzymatic hydrolysis: Add the activated enzyme obtained in step (2) to the cartilage particles obtained in step (1), hydrolyze at 32-35℃ for 2-6 hours, adjust the pH to 5.0-5.5 with sodium hydroxide solution, filter, remove the liquid, and obtain the precipitate;

[0069] The amount of the activating enzyme added is 0.5-2% of the weight of the cartilage particles;

[0070] (4) Post-processing: freeze-dry the precipitate obtained in step (3), pulverize it and pass it through an 80-mesh sieve to obtain the final product.

[0071] Example 1

[0072] An extraction process for non-denaturing type II collagen to alleviate osteoarthritis, comprising the following steps:

[0073] (1) Pretreatment: Add water to chicken breast cartilage and stir to wash it to remove blood and impurities; then add purified water at a material-to-liquid mass ratio of 1:1.5 and ultrasonically wash (40KHz) for 0.5h to collect the cartilage; freeze and crush the cartilage at -20℃ into cartilage particles with a particle size not exceeding 1cm.

[0074] (2) Enzyme activation: mix wolfberry, ophiopogon japonicus, chicory and honeysuckle in a weight ratio of 4:1:7:7, add 12 times the amount of water, extract at 95℃ for 2 hours, filter, remove the filter residue, obtain the extract, cool the extract to room temperature, add pepsin and mix evenly, then adjust the pH to 2.5 with 2M hydrochloric acid solution to obtain the activated enzyme.

[0075] The amount of pepsin added is 19% of the weight of the extract;

[0076] (3) Enzymatic hydrolysis: Add the activated enzyme obtained in step (2) to the cartilage particles obtained in step (1), hydrolyze at 32±2℃ for 4h, adjust the pH to 5.0 with 2M sodium hydroxide solution, filter, remove the liquid, and obtain the precipitate;

[0077] The amount of the activating enzyme added is 1.2% of the weight of the cartilage particles;

[0078] (4) Post-processing: freeze-dry the precipitate obtained in step (3) (pre-freeze at -60℃, vacuum degree 0.1mbar), pulverize it through an 80-mesh sieve, and obtain the final product.

[0079] Example 2

[0080] An extraction process for non-denaturing type II collagen to alleviate osteoarthritis, comprising the following steps:

[0081] (1) Pretreatment: Add water to chicken breast cartilage and stir to wash it to remove blood and impurities; then add purified water at a material-to-liquid mass ratio of 1:1.5 and ultrasonically wash (40KHz) for 0.5h to collect the cartilage; freeze and crush the cartilage at -20℃ into cartilage particles with a particle size not exceeding 1cm.

[0082] (2) Enzyme activation: mix wolfberry, ophiopogon japonicus, chicory and honeysuckle in a weight ratio of 3:1:5:10, add 8 times the amount of water, extract at 85℃ for 3h, filter, remove the filter residue, obtain the extract, cool the extract to room temperature, add pepsin and mix evenly, then adjust the pH to 1.5 with 2M hydrochloric acid solution to obtain the activated enzyme.

[0083] The amount of pepsin added is 30% of the weight of the extract;

[0084] (3) Enzymatic hydrolysis: Add the activated enzyme obtained in step (2) to the cartilage particles obtained in step (1), hydrolyze at 35±2℃ for 2h, adjust the pH to 5.0 with 2M sodium hydroxide solution, filter, remove the liquid, and obtain the precipitate;

[0085] The amount of the activating enzyme added is 2% of the weight of the cartilage particles;

[0086] (4) Post-processing: freeze-dry the precipitate obtained in step (3) (pre-freeze at -60℃, vacuum degree 0.1mbar), pulverize it through an 80-mesh sieve, and obtain the final product.

[0087] Example 3

[0088] An extraction process for non-denaturing type II collagen to alleviate osteoarthritis, comprising the following steps:

[0089] (1) Pretreatment: Add water to chicken breast cartilage and stir to wash it to remove blood and impurities; then add purified water at a material-to-liquid mass ratio of 1:1.5 and ultrasonically wash (40KHz) for 0.5h to collect the cartilage; freeze and crush the cartilage at -20℃ into cartilage particles with a particle size not exceeding 1cm.

[0090] (2) Enzyme activation: mix wolfberry, ophiopogon japonicus, chicory and honeysuckle in a weight ratio of 5:1:10:5, add 12 times the amount of water, extract at 100℃ for 1h, filter, remove the filter residue, obtain the extract, cool the extract to room temperature, add pepsin and mix evenly, then adjust the pH to 3.5 with 2M hydrochloric acid solution to obtain the activated enzyme.

[0091] The amount of pepsin added is 10% of the weight of the extract;

[0092] (3) Enzymatic hydrolysis: Add the activated enzyme obtained in step (2) to the cartilage particles obtained in step (1), hydrolyze at 34±2℃ for 6h, adjust the pH to 5.5 with 2M sodium hydroxide solution, filter, remove the liquid, and obtain the precipitate;

[0093] The amount of the activating enzyme added is 2% of the weight of the cartilage particles;

[0094] (4) Post-processing: freeze-dry the precipitate obtained in step (3) (pre-freeze at -60℃, vacuum degree 0.1mbar), pulverize it through an 80-mesh sieve, and obtain the final product.

[0095] Comparative Example 1

[0096] The difference between this comparative example and Example 1 is that the weight ratio of wolfberry, ophiopogon japonicus, chicory, and honeysuckle is different.

[0097] Specifically, the weight ratio of wolfberry, ophiopogon japonicus, chicory, and honeysuckle is 5:5:1:10.

[0098] The remaining steps are the same as in Example 1.

[0099] Comparative Example 2

[0100] The difference between this comparative example and Example 1 is that the weight ratio of wolfberry, ophiopogon japonicus, chicory, and honeysuckle is different.

[0101] Specifically, the weight ratio of wolfberry, ophiopogon japonicus, chicory, and honeysuckle is 8:1:7:2.

[0102] The remaining steps are the same as in Example 1.

[0103] Comparative Example 3

[0104] The difference between this comparative example and Example 1 is that the enzyme activation step (2) is omitted.

[0105] Specifically, the extraction process includes the following steps:

[0106] (1) Pretreatment: Add water to chicken breast cartilage and stir to wash it to remove blood and impurities; then add purified water at a material-to-liquid mass ratio of 1:1.5 and ultrasonically wash (40KHz) for 0.5h to collect the cartilage; freeze and crush the cartilage at -20℃ into cartilage particles with a particle size not exceeding 1cm.

[0107] (2) Enzymatic hydrolysis: Add pepsin to the cartilage particles obtained in step (1), hydrolyze at 32±2℃ for 4h, adjust the pH to 5.0 with 2M sodium hydroxide solution, filter, remove the liquid, and obtain the precipitate;

[0108] The amount of pepsin added is 1.2% of the weight of the cartilage particles;

[0109] (4) Post-processing: freeze-dry the precipitate obtained in step (3) (pre-freeze at -60℃, vacuum degree 0.1mbar), pulverize it through an 80-mesh sieve, and obtain the final product.

[0110] Comparative Example 4

[0111] The difference between this comparative example and Example 1 is that the process parameters for enzyme activation and enzymatic hydrolysis are different.

[0112] Specifically, the extraction process includes the following steps:

[0113] (1) Pretreatment: Add water to chicken breast cartilage and stir to wash it to remove blood and impurities; then add purified water at a material-to-liquid mass ratio of 1:1.5 and ultrasonically wash (40KHz) for 0.5h to collect the cartilage; freeze and crush the cartilage at -20℃ into cartilage particles with a particle size not exceeding 1cm.

[0114] (2) Enzyme activation: mix wolfberry, ophiopogon japonicus, chicory and honeysuckle in a weight ratio of 4:1:7:7, add 12 times the amount of water, extract at 95℃ for 2 hours, filter, remove the filter residue, obtain the extract, cool the extract to room temperature, add pepsin and mix evenly, then adjust the pH to 2.5 with 2M hydrochloric acid solution to obtain the activated enzyme.

[0115] The amount of pepsin added is 5% of the weight of the extract;

[0116] (3) Enzymatic hydrolysis: Add the activated enzyme obtained in step (2) to the cartilage particles obtained in step (1), hydrolyze at 32±2℃ for 4h, filter, remove the liquid, and obtain the precipitate;

[0117] The amount of the activating enzyme added is 12% of the weight of the cartilage particles;

[0118] (4) Post-processing: freeze-dry the precipitate obtained in step (3) (pre-freeze at -60℃, vacuum degree 0.1mbar), pulverize it through an 80-mesh sieve, and obtain the final product.

[0119] I. Product Physicochemical Properties

[0120] The protein content was determined using GB5009.5-2016 National Food Safety Standard - Determination of Protein in Food - Method I, with a conversion factor of 5.79.

[0121] The method for detecting the content of non-denatured type II collagen is sampling high performance liquid chromatography, as detailed in the draft group standard for non-denatured type II collagen (including type II collagen cartilage powder).

[0122] Table 1. Physicochemical properties and structure of non-denatured type II collagen in each group

[0123]

[0124] As can be seen from the above, the protein content of the non-denatured type II collagen obtained by the extraction process in Examples 1-3 of the present invention is 60.8-63.2 g / 100g, and the content of non-denatured type II collagen is 25.6-28.1 g / 100g.

[0125] Compared to Example 1, the protein content and non-denatured type II collagen content of the undenatured type II collagen prepared by the extraction process in Comparative Example 3 were significantly reduced, at 20.7 g / 100 g and 6.4 g / 100 g, respectively. This indicates that in the claimed technical solution of this invention, using only 1.2% of the cartilage granule weight of pepsin for 4 hours for enzymatic hydrolysis results in low protein and non-denatured type II collagen content, which cannot meet the requirements for large-scale production.

[0126] Compared with Comparative Example 3, the protein content and non-denatured type II collagen prepared by the extraction processes of Comparative Examples 1 and 2 were increased to some extent, reaching 25.3-28.8 g / 100g and 8.3-9.6 g / 100g, respectively. This indicates that the extract prepared by combining wolfberry, ophiopogon japonicus, chicory, and honeysuckle can activate pepsin and enhance its activity to a certain extent, but it still cannot meet the requirements for large-scale production.

[0127] Compared with Comparative Examples 1 and 2, it can be seen that the extract prepared by Example 1 using a specific weight ratio of wolfberry, ophiopogon japonicus, chicory and honeysuckle is beneficial to the synergistic effect of the effective components among each component, which can effectively enhance the activity of pepsin. While reducing the amount of enzyme used, it greatly shortens the enzymatic hydrolysis time. Moreover, the extract of the present invention is relatively mild and can effectively prevent the denaturation of type II collagen. The prepared non-denatured type II collagen has a high protein content and a high non-denatured type II collagen content, which meets the requirements of large-scale production.

[0128] II. In vitro efficacy experiments of non-denatured type II collagen

[0129] 1. Test cells

[0130] Mouse macrophages (RAW264.7).

[0131] 2. Experimental Methods

[0132] RAW264.7 cells were cultured in a 5 wt% non-denatured type II collagen solution and lipopolysaccharide (LPS, 1 μg / mL) for 24 h. 100 μL of the cell culture supernatant was collected for NO production measurement. A blank control group (without LPS and non-denatured type II collagen) and an LPS control group (containing LPS but without non-denatured type II collagen) were set up. cellular NO production was measured using a spectrophotometer, and the results are shown in Table 2.

[0133] RAW 264.7 cells were cultured in 5 wt% non-denaturing type II collagen solution for 1 hour, and then stimulated with 100 ng / mL lipopolysaccharide (LPS) solution for 18 hours. Cell culture medium was collected, and a blank control group (without LPS and non-denaturing type II collagen) and an LPS control group (containing LPS but without non-denaturing type II collagen) were set up. The concentrations of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were measured by enzyme-linked immunosorbent assay (ELISA), and the results are shown in Table 3.

[0134] Table 2. Effects of different non-denatured type II collagens on NO production in RAW264.7 cells (mean ± deviation, n=6)

[0135]

[0136] Note: Different letters in the same column indicate significant differences between groups (P < 0.05).

[0137] The results showed that, compared with the blank control group, the NO content in RAW264.7 cells in the LPS control group was significantly increased (P < 0.05). Compared with the LPS control group, the NO content in RAW264.7 cells in Examples 1-3 was significantly decreased (P < 0.05), while the changes in NO content in Comparative Examples 1-4 were not statistically significant (P > 0.05). This indicates that the non-denatured type II collagen extracted in Examples 1-3 of this invention can reduce NO production in RAW264.7 cells, while the non-denatured type II collagen extracted in Comparative Examples 1-4 did not have the above effect.

[0138] Table 3. Effects of different non-denatured type II collagens on the production of IL-6 and TNF-α in RAW264.7 cells (mean ± deviation, n=6)

[0139]

[0140] Note: Different letters in the same column indicate significant differences between groups (P < 0.05).

[0141] The results showed that, compared with the blank control group, the levels of IL-6 and TNF-α in RAW264.7 cells in the LPS control group were significantly increased (P < 0.05). Compared with the LPS control group, the levels of IL-6 and TNF-α in RAW264.7 cells in Examples 1-3 were significantly decreased (P < 0.05), while the changes in IL-6 and TNF-α levels in Comparative Examples 1-4 were not statistically significant (P > 0.05). This indicates that the non-denatured type II collagen extracted in Examples 1-3 of this invention can reduce the production of IL-6 and TNF-α in RAW264.7 cells, while the non-denatured type II collagen extracted in Comparative Examples 1-4 did not have the above effect.

[0142] In summary, the non-denatured type II collagen extracted in Examples 1-3 of this invention can significantly reduce NO content in cells and significantly downregulate IL-6 and TNF-α levels. By reducing the production of inflammatory factors, it can alleviate joint inflammation and slow down bone and cartilage destruction.

[0143] III. Animal experiments on non-denatured type II collagen

[0144] 1. Laboratory animals

[0145] Male C57bl / 6 mice, weighing 22-25g, were purchased from Beijing Huafukang Laboratory Animal Technology Co., Ltd.

[0146] 2. Experimental Methods

[0147] Mice were randomly divided into four groups according to body weight: a normal control group, a hormone group, experimental group 1, and comparative group 1, with eight mice in each group. Except for the normal control group, all groups received intramuscular injections of dexamethasone 1.2 mg / kg, while the normal control group received an equal volume of physiological saline, twice a week for six weeks. One week after injection, experimental group 1 and comparative group 1 received non-denatured type II collagen extracted from the corresponding examples and comparative groups via gavage at 1.8 g / kg (the recommended human dose is 40 mg / kg, five times the recommended human dose), while the normal control group and hormone group received an equal volume of physiological saline via gavage, continuously for five weeks. All groups maintained normal diet and activity during the experiment.

[0148] After the experiment, the mice were euthanized by decapitation, and the right femur and tibia were dissected. The femur was frozen for later use after removing surrounding tissues, and the tibia was covered with gauze soaked in physiological saline. Bone strength was measured within 1 day.

[0149] Bone mineral density determination: A sample of the right femur was taken and its length was measured using calipers; after drying, its weight and density were measured using a solid density meter.

[0150] Bone biomechanics determination: A sample of the right femur was taken and a three-point bending test was performed using a bone biomechanics measuring instrument to determine the maximum load and bending strength.

[0151] The results are shown in Table 4.

[0152] Table 4. Results of bone mineral density, maximum load, and flexural strength measurements in each group of mice.

[0153]

[0154] Note: Different letters in the same column indicate significant differences between groups (P < 0.05).

[0155] The results showed that, compared with the normal control group, the bone mineral density, maximum load, and bending strength of the hormone-treated mice were significantly reduced (P < 0.05). This indicates that the dexamethasone-induced osteoporosis model was successful.

[0156] Compared to the hormone group, the mice in Example 1 showed significantly increased bone mineral density, maximum load, and flexural strength (P < 0.05), while there was no significant difference in Comparative Example 1 (P > 0.05). This indicates that the non-denatured type II collagen extracted in this invention can increase bone mineral density and improve bone strength, while the non-denatured type II collagen provided in the comparative example had a poor effect on improving bone mineral density and bone strength.

[0157] The above description, in conjunction with specific embodiments, further illustrates the present invention. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions to the details and form of the technical solutions of the present invention can be made without departing from the spirit and scope of the invention, and all such modifications and substitutions fall within the protection scope of the present invention.

Claims

1. An extraction process for non-denatured type II collagen to relieve osteoarthritis, characterized in that, Includes the following steps: (1) Pretreatment: The animal cartilage is washed and crushed to obtain cartilage particles; (2) Enzyme activation: Mix wolfberry, ophiopogon japonicus, chicory and honeysuckle, extract with water, separate solid and liquid to obtain extract, cool, add pepsin and mix evenly, adjust pH to 1.5-3.5 to obtain activated enzyme; (3) Enzymatic hydrolysis: Add the activated enzyme obtained in step (2) to the cartilage particles obtained in step (1) for enzymatic hydrolysis, adjust the pH to 5.0-5.5, separate the solid and liquid, and obtain the precipitate; (4) Post-processing: Dry and pulverize the precipitate obtained in step (3) to obtain the final product; The weight ratio of wolfberry, ophiopogon japonicus, chicory and honeysuckle in step (2) is 3-5:1:5-10:5-10; The amount of pepsin added in step (2) is 10-30% of the weight of the extract; The amount of activating enzyme added in step (3) is 0.5-2% of the weight of cartilage particles.

2. The extraction process according to claim 1, characterized in that, The weight ratio of wolfberry, ophiopogon japonicus, chicory and honeysuckle in step (2) is 3-5:1:6-9:6-9.

3. The extraction process according to claim 1, characterized in that, The amount of water added in step (2) is 8-12 times the total amount of wolfberry, ophiopogon japonicus, chicory and honeysuckle.

4. The extraction process according to claim 1, characterized in that, The extraction described in step (2) is performed at 85-100℃ for 1-3 hours.

5. The extraction process according to claim 1, characterized in that, The amount of pepsin added in step (2) is 15-25% of the weight of the extract; the amount of activating enzyme added in step (3) is 1-1.5% of the weight of the cartilage particles.

6. The extraction process according to claim 1, characterized in that, The conditions for enzymatic hydrolysis in step (3) are: 2-10 h at 30-37℃.

7. Application of aqueous extracts of wolfberry, ophiopogon japonicus, chicory and honeysuckle in combination with pepsin in the preparation of non-denatured type II collagen. The aqueous extract is prepared by mixing wolfberry, ophiopogon japonicus, chicory and honeysuckle in a weight ratio of 3-5:1:5-10:5-10, adding water for extraction, filtering, and cooling to 10-35℃. The pepsin is 10-30% of the weight of the extract.

8. Non-denatured type II collagen prepared by the extraction process according to any one of claims 1-6.

9. The use of the non-denatured type II collagen according to claim 8 in the preparation of a medicine for preventing and treating osteoporosis or osteoarthritis.

10. A composition, characterized in that, Includes the non-denatured type II collagen as described in claim 8.

Images