Preparation method of non-denatured II-type collagen for promoting growth of bones and cartilages

Abstract

The invention discloses a preparation method of non-denatured II-type collagen for promoting bone and cartilage growth, and belongs to the technical field of II-type collagen extraction.The preparation method comprises the following steps that 1, pretreatment is conducted; (2) soaking in a salt solution: adding the salt solution into the cartilage particles for soaking treatment to obtain a precipitate; (3) soaking in an organic solvent; and (4) drying and crushing the solid matter to obtain the traditional Chinese medicine composition. The salt solution is prepared from the following components in percentage by mass: 1 to 5 percent of sodium chloride, 0.2 to 0.8 percent of lecithin, 0.3 to 1 percent of plant extract and the balance of water; the plant extract is a water extract of rhizoma kaempferiae, chicory and houttuynia cordata; the organic solvent is an ethanol water solution and pullulan polysaccharide. According to the method disclosed by the invention, the saline solution is improved, so that the structural stability of the collagen is improved while the dissolution of non-collagen components is promoted, the denaturation of the type II collagen is avoided, and the extraction efficiency of the non-denatured type II collagen is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of non-denatured type II collagen extraction, and in particular relates to a method for preparing non-denatured type II collagen for promoting bone and cartilage growth. Background Art

[0002] Type II collagen (CII), with its dense fibrous structure, is the primary organic component of the cartilage matrix and a characteristic protein of cartilage tissue. Tightly bound to polysaccharides, it imparts cartilage's flexibility, allowing it to absorb shock and withstand loads. Research results indicate that non-denatured type II collagen (type II collagen that maintains its intact triple helical structure) is highly effective in preventing and improving arthritis. Its convenience, safety, non-toxicity, and antigen-specificity make it a promising new functional raw material for the prevention and improvement of arthritis.

[0003] CN111662375A discloses a preparation method for extracting nutrients from animal cartilage tissue, which process includes the following steps: soaking-cleaning-ultrasonic cleaning-crushing-salt solution soaking-filtration-ethanol soaking-filtration-vacuum drying-vortex crushing-packaging. The non-denatured type II collagen (composite collagen) extracted by the invention has a complete molecular structure and can well retain the biological activity of nutrients. It has low fat content, high collagen content, and a macromolecular mucopolysaccharide content of more than 20%. It also contains mineral nutrients such as calcium, magnesium, and phosphorus. Studies have shown that the composite collagen prepared by the invention can increase serum osteocalcin levels and increase bone density (Zhang Dandan et al., Clinical efficacy of type II collagen combined with calcium carbonate and calcitriol in the treatment of glucocorticoid-induced osteoporosis [J], Chinese Medical Journal, 2018, 9 (53), 989-992). At the same time, studies have found that the composite collagen can increase the number and connection density of trabeculae, and has therapeutic potential for treating glucocorticoid-induced osteoporosis.

[0004] However, the ethanol soaking process used in this invention easily causes protein denaturation, resulting in a low content of non-denatured type II collagen in the extracted product. CN113563458A discloses a method for preparing non-denatured type II collagen and a product thereof. Using chicken sternal cartilage as raw material, the method involves selecting, cleaning, pulverizing, enzymatic hydrolysis, drying, pulverizing, and screening. The product produced by this invention has a non-denatured type II collagen content of between 16% and 24%. However, the reaction time is long and the low content of non-denatured type II collagen still exists.

[0005] In view of the problems existing in the prior art, it is of great significance to provide a method for preparing non-denatured type II collagen with a short reaction time and a high extraction rate. Summary of the Invention

[0006] In order to solve the above technical problems, the present invention provides a method for preparing non-denatured type II collagen that promotes bone and cartilage growth. The preparation method improves the salt solution, thereby promoting the dissolution of non-collagen components while improving the structural stability of collagen, avoiding the denaturation of type II collagen caused by subsequent organic solvent treatment, and improving the extraction efficiency of non-denatured type II collagen.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions: In a first aspect, the present invention provides a method for preparing non-denatured type II collagen for promoting bone and cartilage growth, comprising the following steps: (1) Pretreatment: Clean and crush the animal cartilage to obtain cartilage particles; (2) Soaking in a salt solution: adding a salt solution to the cartilage particles obtained in step (1) for soaking, and performing solid-liquid separation to obtain a precipitate; (3) Soaking in an organic solvent: soaking the precipitate obtained in step (2) in an organic solvent, separating the solid and the liquid to obtain a solid; (4) Drying and crushing the solid obtained in step (3) to obtain the product.

[0008] In some embodiments, the salt solution in step (2) comprises, by mass percentage, 1-5% sodium chloride, 0.2-0.8% lecithin, 0.3-1% plant extract, and the balance water.

[0009] In some embodiments, the plant extract is an aqueous extract of Kaempferia galanga, Cichorium incisum, and Houttuynia cordata.

[0010] In some embodiments, the mass ratio of the cartilage particles to the saline solution in step (2) is 1:2-6.

[0011] In some embodiments, the soaking treatment time in step (2) is 30-120 minutes.

[0012] In some embodiments, the organic solvent in step (3) is an ethanol aqueous solution and pullulan; preferably, the mass ratio of the ethanol aqueous solution to pullulan is 3-8:0.1; and the mass fraction of the ethanol aqueous solution is 30-60%.

[0013] In some embodiments, the mass ratio of the precipitate to the organic solvent in step (3) is 1:2-6.

[0014] In some embodiments, the soaking treatment time in step (3) is 30-120 minutes.

[0015] In some embodiments, the cleaning in step (1) is: ultrasonic cleaning of the animal cartilage with water; preferably, the ultrasonic conditions are: ultrasonic treatment at a frequency of 30-50 KHz for 20-30 min; more preferably, ultrasonic treatment at a frequency of 35-40 KHz for 20-30 min.

[0016] In some embodiments, the animal in step (1) is fish and / or poultry.

[0017] In some embodiments, the crushing in step (1) is performed by freezing. Preferably, the crushing is performed to a length of less than 1 cm after freezing. Further preferably, the freezing temperature is between -20°C and -1°C.

[0018] In some embodiments, the drying in step (4) is drying to a moisture content of less than 10%.

[0019] The solid-liquid separation involved in the present invention is a conventional solid-liquid separation technique in the art, such as filtration, centrifugation or filter pressing.

[0020] In a second aspect, the present invention further provides the use of a salt solution containing a plant extract and an organic solvent containing pullulan in combination for preparing non-denatured type II collagen.

[0021] The salt solution of the present invention comprises, by weight percentage, 1-5% sodium chloride, 0.2-0.8% lecithin, 0.3-1% plant extract, and the balance water; wherein the plant extract is an aqueous extract of 3-8 parts by weight of galangal, 10-15 parts by weight of chicory, and 10-20 parts by weight of houttuynia cordata; The organic solvent is an ethanol aqueous solution and pullulan in a mass ratio of 3-8:0.1, and the mass fraction of the ethanol aqueous solution is 30-60%.

[0022] In a third aspect, the present invention also provides non-denatured type II collagen prepared by the above preparation method.

[0023] In a fourth aspect, the present invention further provides the use of the non-denatured type II collagen in the preparation of health products or medicines.

[0024] The daily oral dosage of the non-denatured type II collagen of the present invention when used in health products or medicines is 300-500 mg / kg, preferably 400-500 mg / kg.

[0025] In some embodiments, the application is the application of the non-denatured type II collagen in the preparation of a health product for increasing bone density.

[0026] In some embodiments, the application is the application of the non-denatured type II collagen in the preparation of drugs for preventing and treating osteoporosis and / or osteoarthritis.

[0027] In a fifth aspect, the present invention further provides a composition comprising the non-denatured type II collagen prepared by the aforementioned preparation method.

[0028] In some embodiments, the non-denatured type II collagen of the present invention can also be used as a raw material and mixed with health care products or pharmaceutically acceptable excipients to prepare corresponding health care products or pharmaceutical preparations.

[0029] Preferably, the health care product is in the form of tablets, capsules, oral liquids, ointments, granules or powders.

[0030] Preferably, the pharmaceutical preparation is a tablet, capsule, granule, dry suspension or pill.

[0031] The beneficial effects of the present invention are: (1) The present invention improves the salt solution, thereby promoting the dissolution of non-collagen components while improving the structural stability of collagen, avoiding the denaturation of type II collagen caused by subsequent organic solvent treatment, and improving the extraction efficiency of non-denatured type II collagen.

[0032] (2) The galangal, chicory and houttuynia cordata used in the present invention are all medicinal and edible foods with good safety. Compared with the existing preparation methods, the extraction efficiency of the non-denatured type II collagen of the present invention is higher, and the endotoxin content of the prepared product is lower. DETAILED DESCRIPTION

[0033] The following describes the embodiments of the present invention through specific examples. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. The present invention may also be implemented or applied through various other specific embodiments, and the details in this specification may be modified or altered based on different viewpoints and applications without departing from the spirit of the present invention.

[0034] Before further describing the 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 terms used in the examples of the present invention are for describing specific embodiments rather than for limiting the scope of protection of the present invention.

[0035] When numerical ranges are given in the examples, it should be understood that, unless otherwise specified herein, 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 those of ordinary skill in the art to which the present invention belongs.

[0036] The "composition" described in the present invention comprises non-denatured type II collagen and health products or pharmaceutically acceptable excipients. In a specific embodiment, the non-denatured type II collagen described in the present invention is provided in the composition in an effective amount (e.g., a therapeutically effective amount).

[0037] For purposes of this invention, "acceptable" ingredients are substances suitable for use in humans and / or animals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), that is, substances with a reasonable benefit / risk ratio. "Acceptable excipients for health products" include fillers, binders, preservatives, antioxidants, etc. "Pharmaceutically acceptable excipients" include inert diluents, dispersants and / or granulating agents, surfactants and / or emulsifiers, disintegrants, binders, preservatives, buffers, lubricants, and / or oils. Excipients (such as cocoa butter and suppository wax), colorants, coating agents, sweeteners, and flavoring agents may also be present in the composition.

[0038] The "compositions" described herein can be prepared by any method known in the art of health care or pharmacy. Generally speaking, these preparation methods include combining the non-denatured 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 necessary and / or desired, shaping and / or packaging the product into a desired single-dose or multi-dose unit.

[0039] The compositions of the present invention can be prepared according to known methods, such as those described in the general rules for preparation in the Chinese Pharmacopoeia (2020 edition), the Japanese Pharmacopoeia (16th edition), the United States Pharmacopoeia (USP), and the European Pharmacopoeia (EP), 9th edition. The specific preparation method depends on the dosage form.

[0040] The active ingredient and pharmaceutically acceptable excipients in the "compositions" of the present invention will vary depending on the identity, size, and / or condition of the subject being treated and further depending on the route of administration of the active ingredient. The composition may contain between 0.1% and 100% (w / w) active ingredient.

[0041] The "composition" described in the present invention may optionally contain other therapeutic ingredients for use in combination in order to better exert the efficacy of treating osteoporosis or osteoarthritis, such as "other drugs for treating osteoporosis" or "other drugs for osteoarthritis", wherein the "other drugs for treating osteoporosis" or "other drugs for osteoarthritis" may be chemical drugs, traditional Chinese medicines or Chinese patent medicine preparations.

[0042] As used herein, "treating," unless otherwise indicated, means reversing, alleviating, inhibiting the progression of, or preventing the condition or disorder to which the term applies, or one or more symptoms of such condition or disorder. The term "treating," as used herein, refers to the act of treating, as "treating" is defined immediately above.

[0043] As used herein, an "effective amount" refers to an amount sufficient to elicit a desired biological response. The effective amount of the active ingredients of the present invention may vary depending on factors such as the desired biological endpoint, the pharmacokinetics of the compound, the condition being treated, the mode of administration, and the age and health of the subject. In certain embodiments, an effective amount is a therapeutically effective amount. An effective amount is the amount of the only active ingredient described herein in a single dose. In certain embodiments, an effective amount is the combined amount of the only active ingredients described herein in multiple doses.

[0044] As used herein, a "therapeutically effective amount" is an amount sufficient to provide a therapeutic benefit in the treatment of a condition or sufficient to delay or minimize one or more symptoms associated with the condition. A therapeutically effective amount of an active ingredient means an amount of a therapeutic agent, alone or in combination with other therapies, that provides a therapeutic benefit in the treatment of a condition. The term "therapeutically effective amount" may encompass an amount that improves overall therapy, reduces or avoids symptoms, signs, or causes of a condition, and / or enhances the therapeutic efficacy of another therapeutic agent. In certain embodiments, a therapeutically effective amount is an amount sufficient to treat any of the diseases or conditions described.

[0045] The raw materials used in the present invention are all common commercially available products, and therefore their sources are not specifically limited.

[0046] It is worth noting that, unless otherwise specified, all operations in this embodiment are performed at room temperature, wherein room temperature is 20-25°C. The amount in this embodiment is relative to the weight of the decoction ingredients. For example, 8-15 times the amount of water means that the amount of water is 8-15 times the total weight of Kaempferia galangal, chicory, and Houttuynia cordata. The animal cartilage in this embodiment is chicken breast cartilage.

[0047] [Method for preparing plant extract] The plant extract is a water extract of Kaempferia galanga, Cichorium incisum and Houttuynia cordata; The specific preparation method is: weigh galangal, chicory and houttuynia cordata, add 8-15 times the amount of water and boil for 1-3 hours, separate the solid and liquid, and concentrate and dry the filtrate.

[0048] In some embodiments, the amount of galangal is 3-8 parts by weight, preferably 4-6 parts, and more preferably 5 parts.

[0049] In some embodiments, the amount of chicory is 10-15 parts by weight, preferably 11-13 parts by weight, and more preferably 12 parts by weight.

[0050] In some embodiments, the amount of Houttuynia cordata is 10-20 parts by weight, preferably 14-16 parts, and more preferably 15 parts.

[0051] In some embodiments, the decocting step is: adding 10-12 times the amount of water and decocting for 2 hours.

[0052] In some embodiments, the drying is performed until the moisture content is less than 10%. Preferably, the drying is performed by vacuum drying, spray drying, freeze drying or oven drying.

[0053] [Method for preparing non-denatured type II collagen] The steps include: (1) Pretreatment: The animal cartilage is ultrasonically cleaned with water and then frozen and crushed to obtain cartilage particles.

[0054] (2) Soaking in a salt solution: adding a salt solution to the cartilage particles obtained in step (1) and soaking for 30-120 min, separating the solid and the liquid to obtain a precipitate; The salt solution comprises, by mass percentage, 1-5% sodium chloride, 0.2-0.8% lecithin, 0.3-1.0% plant extract and the balance water; The mass ratio of the cartilage particles to the saline solution is 1:2-6.

[0055] (3) Soaking in an organic solvent: soaking the precipitate obtained in step (2) in an organic solvent for 30-120 min, separating the solid and the liquid to obtain a solid; The organic solvent is an ethanol aqueous solution and pullulan in a mass ratio of 3-8:0.1, and the mass fraction of the ethanol aqueous solution is 30-60%; The mass ratio of the precipitate to the organic solvent is 1:2-6.

[0056] (4) Drying (40°C) and crushing the solid obtained in step (3) to obtain the product.

[0057] In some embodiments, the salt solution in step (2) comprises, by mass percentage, 1-3% sodium chloride, 0.3-0.5% lecithin, 0.5-0.7% plant extract, and the balance water; preferably, it comprises 2% sodium chloride, 0.4% lecithin, 0.6% plant extract, and the balance water.

[0058] In some embodiments, the mass ratio of the cartilage particles to the saline solution in step (2) is 1:3-4; preferably 1:3.

[0059] In some embodiments, the soaking treatment time in step (2) is 60-90 minutes, preferably 60 minutes.

[0060] In some embodiments, the mass ratio of the ethanol aqueous solution to pullulan in step (3) is 4-6:0.1; preferably 5:0.1.

[0061] In some embodiments, the mass fraction of the ethanol aqueous solution in step (3) is 48-52%, preferably 50%.

[0062] In some embodiments, the mass ratio of the precipitate to the organic solvent in step (3) is 1:3-5; preferably 1:4.

[0063] In some embodiments, the soaking treatment time in step (3) is 60-90 minutes, preferably 60 minutes.

[0064] Example 1 Preparation method of non-denatured type II collagen The steps include: (1) Pretreatment: The animal cartilage was cleaned with water ultrasound (40 kHz) for 25 min, and then frozen and crushed at -15°C to obtain cartilage particles with a length of less than 1 cm.

[0065] (2) Soaking in salt solution: adding salt solution to the cartilage particles obtained in step (1) at a mass ratio of 1:3, soaking for 60 minutes, centrifuging, and obtaining a precipitate; The salt solution comprises, by mass percentage, 2% sodium chloride, 0.4% lecithin, 0.6% plant extract and the balance water; The preparation method of the plant extract is as follows: taking 5 parts of Kaempferia galanga, 12 parts of chicory and 15 parts of houttuynia cordata by weight, adding 11 times the amount of water, boiling for 2 hours, filtering, and concentrating and drying the filtrate.

[0066] (3) Soaking in an organic solvent: the precipitate obtained in step (2) was added to an organic solvent at a material-liquid mass ratio of 1:5 and soaked for 60 min, followed by centrifugation to obtain a solid; The organic solvent is an ethanol aqueous solution and pullulan in a mass ratio of 5:0.1, and the mass fraction of the ethanol aqueous solution is 50%.

[0067] (4) The solid obtained in step (3) is dried (40°C) and crushed through an 80-mesh sieve to obtain the product.

[0068] Example 2 Preparation method of non-denatured type II collagen The steps include: (1) Pretreatment: The animal cartilage was cleaned with water ultrasound (40 kHz) for 25 min, and then frozen and crushed at -15°C to obtain cartilage particles with a length of less than 1 cm.

[0069] (2) Soaking in salt solution: adding salt solution to the cartilage particles obtained in step (1) at a material-liquid mass ratio of 1:2, soaking for 30 minutes, centrifuging, and obtaining a precipitate; The salt solution comprises, by mass percentage, 1% sodium chloride, 0.8% lecithin, 1.0% plant extract and the balance water; The preparation method of the plant extract comprises the following steps: weighing 3 parts of Kaempferia galanga, 10 parts of chicory and 10 parts of houttuynia cordata by weight, adding 8 times the amount of water, boiling for 1 hour, filtering, and concentrating and drying the filtrate.

[0070] (3) Soaking in an organic solvent: the precipitate obtained in step (2) was added to an organic solvent at a material-liquid mass ratio of 1:2 and soaked for 120 min, followed by centrifugation to obtain a solid; The organic solvent is an ethanol aqueous solution and pullulan in a mass ratio of 3:0.1, and the mass fraction of the ethanol aqueous solution is 30%.

[0071] (4) The solid obtained in step (3) is dried (40°C) and crushed through an 80-mesh sieve to obtain the product.

[0072] Example 3 Preparation method of non-denatured type II collagen The steps include: (1) Pretreatment: The animal cartilage was cleaned with water ultrasound (40 kHz) for 25 min, and then frozen and crushed at -15°C to obtain cartilage particles with a length of less than 1 cm.

[0073] (2) Soaking in salt solution: adding salt solution to the cartilage particles obtained in step (1) at a material-liquid mass ratio of 1:6, soaking for 120 minutes, centrifuging, and obtaining a precipitate; The salt solution comprises, by mass percentage, 5% sodium chloride, 0.2% lecithin, 0.3% plant extract and the balance water; The preparation method of the plant extract comprises the following steps: weighing 8 parts of Kaempferia galanga, 15 parts of chicory and 20 parts of houttuynia cordata by weight, adding 15 times the amount of water, boiling for 3 hours, filtering, and concentrating and drying the filtrate.

[0074] (3) Soaking in an organic solvent: the precipitate obtained in step (2) was added to an organic solvent at a material-liquid mass ratio of 1:6 and soaked for 30 min, followed by centrifugation to obtain a solid; The organic solvent is an ethanol aqueous solution and pullulan in a mass ratio of 8:0.1, and the mass fraction of the ethanol aqueous solution is 60%.

[0075] (4) The solid obtained in step (3) is dried (40°C) and crushed through an 80-mesh sieve to obtain the product.

[0076] Comparative Example 1 The difference between this comparative example and Example 1 is that the weight proportions of Kaempferia galanga, chicory and houttuynia cordata in the plant extracts are different; Specifically, the preparation method of the plant extract is as follows: take 15 parts of Kaempferia galangal, 8 parts of chicory and 9 parts of Houttuynia cordata by weight, add 11 times the amount of water, and boil for 2 hours, filter, and concentrate the filtrate to dryness.

[0077] Other steps and parameters are the same as in Example 1.

[0078] Comparative Example 2 The difference between this comparative example and Example 1 is that the weight proportions of Kaempferia galanga, chicory and houttuynia cordata in the plant extracts are different; Specifically, the preparation method of the plant extract is as follows: take 2 parts of Kaempferia galangal, 12 parts of chicory and 25 parts of houttuynia cordata by weight, add 11 times the amount of water, and boil for 2 hours, filter, and concentrate the filtrate to dryness.

[0079] Other steps and parameters are the same as in Example 1.

[0080] Comparative Example 3 The difference between this comparative example and Example 1 is that the weight ratio of the ethanol aqueous solution and pullulan in the organic solvent is different; Specifically, the organic solvent is an ethanol aqueous solution and pullulan in a mass ratio of 2.5:0.1, and the mass fraction of the ethanol aqueous solution is 10%.

[0081] Other steps and parameters are the same as in Example 1.

[0082] Comparative Example 4 The difference between this comparative example and Example 1 is that the organic solvent does not contain pullulan; Specifically, the organic solvent is an ethanol aqueous solution, and the mass fraction of the ethanol aqueous solution is 50%.

[0083] Other steps and parameters are the same as in Example 1.

[0084] Product physical and chemical properties The protein content was detected using the first method of GB5009.5-2016 National Food Safety Standard for the Determination of Protein in Food, with a conversion factor of 5.79.

[0085] The method for detecting the content of non-denatured type II collagen is sampling high performance liquid chromatography. For details, please refer to the draft for comments on the group standard non-denatured type II collagen (including type II collagen cartilage powder).

[0086] For the detection of endotoxins, refer to the 2025 edition of the Chinese Pharmacopoeia, General Chapter 1143 (Bacterial Endotoxin Test Method).

[0087]

[0088] As can be seen from the above, the protein content of the non-denatured type II collagen obtained by the preparation methods of Examples 1-3 of the present invention is 60.1-65.0 g / 100 g, the content of non-denatured type II collagen is 30.1-33.7 g / 100 g, and the endotoxin content is 10-17 EU / mg.

[0089] Compared to Example 1, the protein content of the non-denatured type II collagen obtained by the preparation methods of Comparative Examples 1 and 2 met the requirements, but the non-denatured type II collagen content and endotoxin were significantly reduced. This shows that in the technical solution claimed in the present invention, the plant extract obtained by aqueous extraction of specific amounts of Kaempferia galanga, chicory, and Houttuynia cordata can improve the structural stability of collagen, avoid denaturation of collagen, improve the extraction efficiency of non-denatured type II collagen, and at the same time, reduce endotoxins in non-denatured type II collagen by utilizing the interaction between the active ingredients of each component.

[0090] Compared with Example 1, the non-denatured type II collagen protein obtained by the preparation methods of Comparative Examples 3 and 4 was soaked in a salt solution using a plant extract prepared by compounding a specific amount of galangal, chicory and houttuynia cordata. However, due to the change in the organic solvent used, the extraction efficiency of the non-denatured type II collagen decreased, indicating that the change in the organic solvent will have a certain impact on the structural stability of the collagen. As long as the plant extract and the specific organic solvent in the present invention are used in combination with each other, better results can be achieved.

[0091] The above further describes the present invention in conjunction with specific embodiments. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solutions of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, and such modifications and replacements shall fall within the scope of protection of the present invention.

Claims

1. A method for preparing non-denatured type II collagen for promoting bone and cartilage growth, characterized in that: The steps include: (1) Pretreatment: Clean and crush the animal cartilage to obtain cartilage particles; (2) Soaking in a salt solution: adding a salt solution to the cartilage particles obtained in step (1) for soaking, and performing solid-liquid separation to obtain a precipitate; (3) Soaking in an organic solvent: soaking the precipitate obtained in step (2) in an organic solvent, separating the solid and the liquid to obtain a solid; (4) drying and crushing the solid obtained in step (3); The salt solution in step (2) comprises, by weight percentage, 1-5% sodium chloride, 0.2-0.8% lecithin, 0.3-1% plant extract, and the balance water; wherein the plant extract is an aqueous extract of 3-8 parts by weight of Kaempferia galanga, 10-15 parts by weight of chicory, and 10-20 parts by weight of houttuynia cordata; The organic solvent in step (3) is an ethanol aqueous solution and pullulan in a mass ratio of 3-8:0.1, and the mass fraction of the ethanol aqueous solution is 30-60%.

2. The preparation method according to claim 1, characterized in that The salt solution comprises, by mass percentage, 2% sodium chloride, 0.4% lecithin, 0.6% plant extract and the balance water.

3. The preparation method according to claim 1, characterized in that The preparation method of the plant extract comprises the following steps: taking 3-8 parts of Kaempferia galanga, 10-15 parts of chicory and 10-20 parts of houttuynia cordata by weight, adding water and boiling, separating the solid and liquid, and concentrating and drying the filtrate.

4. The preparation method according to claim 3, characterized in that The amount of water added is 8-15 times the total amount of galangal, chicory and houttuynia cordata; and the decocting time is 1-3 hours.

5. The preparation method according to claim 1, characterized in that The mass ratio of the ethanol aqueous solution to pullulan is 5:0.1, and the mass fraction of the ethanol aqueous solution is 50%.

6. The preparation method according to claim 1, characterized in that The mass ratio of the cartilage particles to the saline solution in step (2) is 1:2-6.

7. The preparation method according to claim 1, characterized in that The mass ratio of the precipitate to the organic solvent in step (3) is 1:2-6.

8. The preparation method according to claim 1, characterized in that The soaking time in step (2) is 30-120 minutes: And / or the soaking treatment time in step (3) is 30-120 minutes.

9. Use of a salt solution containing a plant extract and an organic solvent containing pullulan in the preparation of non-denatured type II collagen, wherein the salt solution comprises, by mass percentage, 1-5% sodium chloride, 0.2-0.8% lecithin, 0.3-1% plant extract, and the balance water; wherein: The plant extract is an aqueous extract of 3-8 parts by weight of Kaempferia galanga, 10-15 parts by weight of chicory, and 10-20 parts by weight of houttuynia cordata; The organic solvent is an ethanol aqueous solution and pullulan in a mass ratio of 3-8:0.1, and the mass fraction of the ethanol aqueous solution is 30-60%.

10. Use of the preparation method according to any one of claims 1 to 7 in the preparation of health products or medicines.

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