Method for preparing high-activity small-molecule cartilage extract from animal cartilage tissue, cartilage extract and application

Abstract

The invention relates to the technical field of biology, in particular to a method for preparing a high-activity small-molecule cartilage extract from animal cartilage tissue, the cartilage extract and application of the cartilage extract, and the method for preparing the high-activity small-molecule cartilage extract from the animal cartilage tissue comprises the following steps: S1, soaking; s2, bone breaking; s3, removing cartilage fat by adopting a skimming and centrifugal separation process; s4, enzymolysis: performing compound shearing on compound protease to obtain enzymatic hydrolysate; s5, filtering; and S6, freeze drying. Through the application of various new technologies, the content of type II collagen in the prepared cartilage extract reaches 65% or above, the content of GAGS reaches 30% or above, the content of hyaluronic acid reaches 17% or above, and the problem that in the prior art, biological activity is damaged due to the processes of degreasing through a chemical degreasing agent, physical high temperature, chemical degradation and high-temperature drying is solved; the biological activity of the natural extract is completely reserved by adopting a physical degreasing and multi-enzyme multi-level directional composite enzymolysis technology and a freeze-drying technology at the temperature of minus 40 DEG C or below.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a method for preparing a highly active small molecule cartilage extract from animal cartilage tissue, a cartilage extract and its application. Background Art

[0002] Meat processing belongs to the field of food industry. During the slaughtering process, a large amount of waste is generated, which can be processed as by-products, such as bones (including hard bones and cartilage), tendons, blood, viscera and skin. These wastes are the inedible parts of meat and are useless in processing meat products.

[0003] At present, by-products such as seed coat and bone are not selling well in the market, have low bioavailability, and are very likely to cause waste and pollution. However, these by-products contain a large amount of collagen, glycosaminoglycans (GAGS) and other ingredients. If they are deeply processed using modern biotechnology to become small molecule cartilage extracts, they will be extremely valuable and can not only solve the current environmental pollution problems, but also improve people's health level as a product in the field of big health and achieve good social benefits.

[0004] Patent CN101711591B "A method for preparing a fish cartilage extract and the resulting product" discloses a method for preparing a fish cartilage extract, the steps of which include subjecting the fish cartilage to alkaline protease enzymolysis to obtain an enzymolysis solution, followed by flavor protease enzymolysis, followed by enzyme inactivation and impurity removal, and separation of collagen and chondroitin sulfate through molecular sieves, thereby achieving product purification and separation, and obtaining low-cost and high-content single-component products of collagen peptides and chondroitin sulfate.

[0005] Patent CN113599397A "A cartilage extract and its preparation method and application" discloses a cartilage extract and its preparation method and application. A cartilage extract composed of low molecular weight chondroitin sulfate and low molecular weight polypeptides is prepared by a two-step enzymatic hydrolysis method. The specific steps are: mixing cartilage with 0.5-1% by mass of papain and enzymolyzing it at 50-55°C and pH 6.5-6.8 for 4 hours to obtain a first enzymatic hydrolyzate; mixing the enzymatic hydrolyzate with chondroitin sulfate enzyme at 1215U / g and enzymolyzing it at 37°C and pH 4.0-7.2 for 4 hours to obtain a cartilage extract solution, which is then purified and dried to obtain a finished product, the composition ratio of which is 53-63% collagen, 17-21% glucosamine, and 20-35% chondroitin sulfate, wherein the mass of collagen peptides with a molecular weight less than 100OD is 97% of the total mass of the type II collagen, and the molecular weight of chondroitin sulfate is 3000Da-3500Da. However, the glucosamine in the patented ingredient is a monosaccharide component that does not contain glycosaminoglycans and hyaluronic acid, and its related functions cannot be proven by animal experiments or clinical evidence.

[0006] Patent CN115120618A "Cartilage extract with improved immune response, its preparation method and use" provides a cartilage extract with improved immune response, its preparation method and use. The product is made of animal cartilage as raw material, defatted for 6 hours using a degreasing agent, homogenized and then added with at least one selected from elastase, ficin and ginger protease for enzymolysis, filtered, and then added with sodium hyaluronate and lentinan in a mass ratio of 4:1 to 1:1, freeze-dried and crushed to obtain a cartilage extract of undenatured type II collagen, which can simultaneously prevent the microbial corruption of the cartilage extract and the denaturation of type II collagen during storage, thereby improving the safety and storage stability of the product. The obtained cartilage extract of the present invention has the advantages of high safety and strong effectiveness, and has the effects of improving immune response, increasing the content of serum anti-inflammatory factors such as TGF-β, and reducing the content of inflammatory factors such as TNF-α. The patent is for non-denatured macromolecular collagen with a complete triple helix structure and a molecular weight of 300,000 Da. It is not a small molecule of <1000 Da that is easily absorbed in the category of bioactive peptides.

[0007] The preparation methods of cartilage extracts in current technologies have the following disadvantages: (1) A variety of technologies are used to prepare small molecule cartilage extracts, including physical degradation, chemical degradation, enzyme degradation, biofilm molecular sieve separation and high-temperature spray drying processes. Most of the processes for preparing collagen and chondroitin sulfate as single components are used. Chemical degradation, chemical degreasing, high-temperature drying and other processes have a great damage to the biological activity of small molecules, and do not reflect the biological utility value of the high activity of small molecules in natural extracts; (2) Chemical degreasing agents are mostly used to degrease the cartilage for post-processing, which may cause chemical pollution and residual hazards. (3) Most of the products prepared are large-molecule collagen, not the traditional small-molecule highly active cartilage extracts; (4) The efficacy of the products in the current technology is mainly the efficacy experiment of degenerative osteoarthritis (OA), which does not reflect the comprehensive efficacy of cartilage extracts (GAGS "oral gel") including skin improvement function; (5) Although the current technology can achieve the preparation of cartilage extracts through different routes, there are major deficiencies in the retention of activity and no series of activity experiments have been carried out in the application, and no systematic and comprehensive activity and efficacy verification has been carried out. Summary of the invention

[0008] In view of the above-mentioned shortcomings of the prior art, the purpose of the present application is to provide a method, cartilage extract and application of cartilage extract which uses animal cartilage tissue as raw material, adopts pure physical method for defatting, multi-level composite directional enzymatic cutting and low-temperature freeze-drying process to retain the biological activity of small molecule extracts to the greatest extent.

[0009] To achieve the above objectives, this application is implemented through the following technical solutions:

[0010] The method for preparing a high-activity small molecule cartilage extract from animal cartilage tissue is characterized by comprising the following steps:

[0011] S1 Soaking: Put the dried pig and cattle nose bones into pure water, keep the system temperature at 20-30°C, soak for 2-3 hours, and after being fully covered with water, the volume of the nose bones will fully expand;

[0012] S2 bone crushing: the wet bones are taken out and crushed by a grinder, sieved, and then purified water is added and mixed to obtain cartilage liquid A;

[0013] S3 defatting: keeping the cartilage liquid A at 70-80°C, removing the cartilage fat by skimming centrifugal separation process, and obtaining cartilage liquid B;

[0014] S4 enzymatic hydrolysis: adjusting the pH value of cartilage liquid B, adding compound protease to cartilage liquid B for compound shearing to obtain enzymatic hydrolyzate;

[0015] S5 filtration: filtering the enzymatic hydrolysate to obtain a clarified solution;

[0016] S6 freeze drying: freeze-dry the clarified liquid under reduced pressure to obtain highly active cartilage extract freeze-dried powder.

[0017] Preferably, in step S2, a 4-8 mm sieve plate is used for sieving, and the volume of pure water is 1-1.5 times of the crushed wet bone.

[0018] Preferably, in step S4, the pH value of the cartilage liquid B is adjusted to 6.5-8.5, the temperature is 45-65°C, the composite protease is a mixture of at least two of flavor protease, alkaline protease, neutral protease, papain and bromelain, and the enzymolysis time is 4-6h.

[0019] Preferably, the amount of the composite protease used is 0.01-0.1% of the dry matter in the cartilage liquid B.

[0020] Preferably, in step S5, the filtering step includes:

[0021] S51 carbon filtration removal: add 0.7-1% composite activated carbon to the enzymatic hydrolyzate, adsorb for 40 minutes at 70°C to obtain carbon filtrate;

[0022] S52 plate and frame filtration: The carbon filtrate is pressurized to 3kg and enters the plate and frame through a pressure pump for filtration to obtain clarified liquid.

[0023] In addition, the present invention also provides a cartilage extract, which is prepared according to any of the above methods for preparing a high-activity small-molecule cartilage extract from animal cartilage tissue.

[0024] Preferably, the average molecular weight of the cartilage extract is < 1000 Da.

[0025] Preferably, the content of type II collagen in the cartilage extract is ≥65%, and the content of glycosaminoglycan is ≥30%.

[0026] The hyaluronic acid content in the glycosaminoglycan is ≥17%.

[0027] Finally, the present invention also provides the application of the above cartilage extract, which is applied to strengthen joints and cartilage structures, improve joints, improve joint health and improve skin.

[0028] Compared with the known public technologies, the technical solution provided by this application has the following beneficial effects:

[0029] (1) The present application adopts multi-enzyme multi-level directional composite enzymatic hydrolysis and a directional enzymatic hydrolysis process with mild conditions to avoid the destruction of GAGS & collagen activity by strong physical and chemical conditions such as high temperature cooking and chemical dissociation. At the same time, the directional enzyme is selected to selectively obtain the target peptides: DVGEKGPEGA (HYP) PGKDGAR, GESGSP (HYP) GENGSP (HYP) GPM, GPAGPSGPRGPP (HYP) GPVGPS, SGSP (HYP) GENGSP (HYP) GPM, GDRGP (HYP) PGPVGP (HYP) PGLT, GESGSP (HYP) GENGSP (HYP) GPM, GAP (HYP) GDKGEAG PP(HYP)GPAGPT, GNSGKP(HYP)GEQGPEGEAGPT, GDQGPP(HYP)GNNGPAGPR, GARGQP(HYP)GVM, SGSP(HYP)GENGSPGP(HYP)M, GDRGPP(HYP)GPVGPP(HYP)GLT, AGP P(HYP)GADGQP(HYP)GAK, TGPIGPP(HYP)GPAGPN, GPSGPRGPP(HYP)GPVGPS, GPAGPP(HYP)GEKGEPGEDGPAGPDGPP(HYP)GPQ, GDRGP(HYP)PGPVGPP(HYP)GLTGPA;

[0030] (2) This application uses composite activated carbon for decolorization and debittering. Compared with the existing removal system, the carbon filtration removal process provided by this technology innovates the removal system, using composite activated carbon for color and taste synergistic improvement with enzymes, which has significant improvements in color, smell and taste;

[0031] (3) The present application adopts freeze-drying technology to retain the biological activity of GAGS (glycosaminoglycans) and collagen peptides in cartilage extract to the greatest extent, avoiding the destruction of biologically active components by high temperature in the spray drying process;

[0032] (4) The present application, through the application of a variety of new technologies, obtains a cartilage extract with a type II collagen content of more than 65%, a GAGS content of more than 30%, and a hyaluronic acid content of more than 17%. This not only solves the problems of the prior art in the use of chemical degreasing agents for degreasing, physical high temperature, chemical degradation, and high-temperature drying processes that cause the destruction of biological activity; it also creatively adopts physical degreasing, mild process, multi-enzyme, multi-level directional composite enzymatic hydrolysis technology, and adopts freeze-drying technology below -40°C to completely retain the biological activity of the natural extract, and retains the collagen peptides and GAGS functional active ingredients in the cartilage to the greatest extent. The cartilage extract of the present application has been subjected to animal experiments and has confirmed the effectiveness of the activity of specific type II collagen peptides and GAGS components selected by directional enzymatic hydrolysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application, and for ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0034] Figure 1 This is the chromatogram of the BⅡP of the highly active cartilage extract freeze-dried powder prepared in Example 1 of the present invention;

[0035] Figure 2 is a CⅡP chromatogram of the highly active cartilage extract freeze-dried powder prepared in Example 2 of the present invention;

[0036] Figure 3 It is a picture of the modeling surgery of the present invention;

[0037] Figure 4 This is a weight record chart of SD rats of the present invention;

[0038] Figure 5 This is the fur status of SD rats in the 12th week of the present invention;

[0039] Figure 6 It is a Micro-CT scanning image of osteophyte formation in the knee joints of rats in different groups at week 12 of the present invention;

[0040] Figure 7 The present invention is a three-dimensional model of the roughness of the knee cartilage surface of rats in different groups at week 12;

[0041] Figure 8 a is a safranin fast green stained section of the cartilage of the knee joint of SD rats in different groups at week 12 of the present invention;

[0042] Figure 8 b is a quantitative analysis diagram of cartilage in safranin fast green stained sections of cartilage of knee joints of SD rats in different groups at week 12 of the present invention;

[0043] Fig. 9 a is a toluidine blue stained section of the knee joints of rats in different groups at week 12 of the present invention;

[0044] Fig. 9 b is a quantitative analysis diagram of glycosaminoglycans in toluidine blue stained sections of the knee joints of rats in different groups at week 12 of the present invention. DETAILED DESCRIPTION

[0045] In order to make the purpose, technical scheme and advantages of the embodiments of the present application clearer, the technical scheme in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0046] Example 1

[0047] The steps of preparing the highly active small molecule cartilage extract BⅡP include:

[0048] S1 Soaking: Put the dried pig and cattle nose bones into 9 times the volume of drinking water, keep the system temperature at 20-30℃, soak for 2-3h, and after being fully covered with water, the volume of the nose bones will be fully expanded;

[0049] S2 bone crushing: The wet bones were taken out and crushed with a grinder, sieved through a 5 mm sieve plate, added with 1.5 times the volume of purified water, and mixed evenly to obtain cartilage liquid A.

[0050] S3 defatting: keep cartilage liquid A warm at 70-80℃, remove cartilage fat by centrifugal separation to obtain cartilage liquid B.

[0051] S4 enzymatic hydrolysis: detect the dry matter content of the material, adjust the pH value of cartilage liquid B to 7.5-8.5, temperature 50°C, add 0.05% compound protease to the material, compound shear, enzymatic hydrolysis time 4.5h.

[0052] In this embodiment, the composite enzyme protease includes flavor protease, alkaline protease, neutral protease, papain and bromelain, wherein the content of flavor protease, alkaline protease, neutral protease, papain and bromelain is 2:2:1:1:1.

[0053] S51 carbon filtration removal: add 0.7-1% composite activated carbon to the enzymatic hydrolyzate, adsorb for 40 minutes at 70°C to obtain carbon filtrate;

[0054] S52 Plate and frame filtration: The carbon filtrate is pressurized to 3kg and then enters the plate and frame through a pressure pump for filtration to obtain a clarified liquid;

[0055] S6 freeze drying: The clarified liquid is quickly frozen at -40°C, and then freeze dried under reduced pressure at 10 Pa and -50°C to obtain highly active cartilage extract freeze-dried powder.

[0056] Example 1-1

[0057] The difference between this example and Example 1 is that in step S4, the composite enzyme protease includes alkaline protease, neutral protease and papain, wherein the content of alkaline protease, neutral protease and papain is 1:1:1.

[0058] Example 1-2

[0059] The difference between this example and Example 1 is that in step S4, the pH value of cartilage liquid B is adjusted to 6.5-7, and the composite enzyme protease includes neutral protease, papain, and bromelain, wherein the content of neutral protease, alkaline protease, neutral protease, papain, and bromelain is 2:1:2.

[0060] Examples 1-3

[0061] The difference between this example and Example 1 is that in step S4, the compound enzyme protease includes flavor protease and alkaline protease, wherein the ratio of flavor protease to alkaline protease is 2:1.

[0062] Comparative Example 1-1

[0063] The difference between this ratio and Example 1 is that in step S4, no composite protease is used, but a single flavor protease is used.

[0064] Comparative Example 1-2

[0065] The difference between this ratio and Example 1 is that in step S4, no composite protease is used, and a two-step enzymatic hydrolysis method is used. Specifically, the pH value of cartilage liquid B is adjusted to 6.5-6.8, 0.5% papain is added to cartilage liquid B and mixed at 50-55°C for 4 hours to obtain a first enzymatic hydrolyzate; the first enzymatic hydrolyzate is mixed with chondroitin sulfate enzyme at 1215 U / g at 37°C and pH value of 4.0-7.2 for 4 hours to obtain a second enzymatic hydrolyzate.

[0066] Comparative Examples 1-3

[0067] The difference between this comparative example and Example 1 is that in step S6, spray drying is adopted, and its process parameters are an inlet air temperature of 160°C and an outlet air temperature of 85°C.

[0068] The physical and chemical indicators of Example 1 (BⅡP), Example 1-1 to Example 1-2, and Comparative Example 1-1 to Comparative Example 1-3 are shown in Table 1, and the molecular weight distribution of BⅡP is shown in Tables 2 and Figure 1

[0069] Table 1

[0070]

[0071]

[0072] Table 2

[0073] Molecular weight distribution result Molecular weight <500Da 71.01％ Molecular weight 2000-500Da 22.29％ Molecular weight 2000-3000Da 2.33％ Molecular weight 3000-5000Da 2.21％ Molecular weight 5000-10000Da 1.73％ Molecular weight>10000Da 0.43％

[0074] Example 2

[0075] The steps of preparing the highly active small molecule cartilage extract CⅡP include:

[0076] S1 Soaking: Put the dried pig and cattle nasal bones into 8 times the volume of drinking water, keep the system temperature at 20-30℃, soak for 2-3 hours, and after being fully covered with water, the volume of the nasal bones will be fully expanded;

[0077] S2 bone crushing: The wet bones were taken out and crushed with a grinder, sieved through a 7 mm sieve plate, added with 1.3 times the volume of purified water, and mixed evenly to obtain cartilage liquid A.

[0078] S3 defatting: keep cartilage liquid A warm at 70-80℃, remove cartilage fat by centrifugal separation to obtain cartilage liquid B.

[0079] S4 enzymatic hydrolysis: detect the dry matter content of the material, adjust the pH value of cartilage liquid B to 7.5-8.2, the temperature to 55°C, add 0.075% compound protease to the material, perform compound shearing, and the enzymatic hydrolysis time is 4h.

[0080] In this embodiment, the composite enzyme protease includes flavor protease, alkaline protease, neutral protease, papain and bromelain, wherein the content of flavor protease, alkaline protease, neutral protease, papain and bromelain is 2:2:1:1:1.

[0081] S51 carbon filtration removal: add 0.75% composite activated carbon to the enzymatic hydrolyzate, adsorb for 40 minutes at 70°C to obtain carbon filtrate;

[0082] S52 Plate and frame filtration: The carbon filtrate is pressurized to 3kg and then enters the plate and frame through a pressure pump for filtration to obtain a clarified liquid;

[0083] S6 freeze drying: The clarified liquid is quickly frozen at -40°C, and then freeze dried under reduced pressure at 10 Pa and -50°C to obtain highly active cartilage extract freeze-dried powder.

[0084] In the present application, HCl or NaOH is used to adjust the pH value of cartilage solution B.

[0085] The physical and chemical indicators and molecular weight data of Example 2 (CⅡP), Example 1-1-Example 1-2, Comparative Example 1-1-Comparative Example 1-3 are shown in Table 3, Table 4 and Figure 2

[0086] Table 3

[0087] CⅡP Average molecular weight 826Da Moisture% 5.5％ protein% 68.1％ GAGS 32.5％ Hyaluronic acid 17.7％ Total colony count <10cfu / g Mold and Yeast <10cfu / g Escherichia coli Not detected Staphylococcus aureus Not detected

[0088] Table 4

[0089]

[0090]

[0091] Effect Experiment

[0092] In the experiment of improving skin and osteoarthritis symptoms by oral administration of highly active small molecule cartilage extracts, CⅡP and BⅡP were used as test subjects, and a rat OA model was established. The effects of CⅡP and BⅡP on rat skin and knee cartilage damage were first studied.

[0093] 1. Experimental Materials

[0094] 1.1 Sample Information

[0095] Animal experiments on the effects of high-activity small molecule cartilage extract dry powder (batch number BⅡP&CⅡP) on improving osteoarthritis and skin.

[0096] 1.2 Experimental animals

[0097] Selection of rats: Healthy clean SD rats, 180g-200g, 125 rats, 12 weeks old, female. Rearing period: 12 weeks. Raised at 24±1℃, 12h light-dark cycle, provided with sufficient maintenance feed and drinking water every day. Weigh and record every three days.

[0098] Gavage and drive requirements: Each experimental group was gavaged before modeling according to the grouping requirements, and gavage was continued for 2 weeks, once a day. After modeling, all rats were gavaged daily for 12 weeks. Gavage time: 10:00-11:00 am every day. Drive time: 11:00-11:30; 14:00-14:30; 16:30-17:00 every day.

[0099] The experimental groups are as follows:

[0100] (1) Normal group, 12 rats, without surgery, gavage with 1 mL of normal saline daily;

[0101] (2) Sham operation group, 12 rats, the joint cavity was opened without cutting the ligament or removing the meniscus, and after suturing, 1 mL of normal saline was gavaged daily;

[0102] (3) Model group, 12 rats, modeling, daily gavage with normal saline;

[0103] (4) drug control group, 12 rats, modeling, daily gavage of 0.675 mg / kg meloxicam tablets;

[0104] (5) Experimental group

[0105] CⅡP-L, low-dose group (50 mg / kg / d);

[0106] CⅡP-M, medium-dose group (200 mg / kg / d);

[0107] CⅡP-H, high-dose group (500 mg / kg / d);

[0108] BⅡP-L, low-dose group (50 mg / kg / d);

[0109] BⅡP-M, medium-dose group (200 mg / kg / d);

[0110] BⅡP-H, high-dose group (500 mg / kg / d).

[0111] The test substances of different doses were divided into groups, with 12 rats in each group, and the model was established. The pictures of the modeling operation are shown in Figure 3 .

[0112] 1.3 Experimental design

[0113] After 1 week of adaptive feeding, female SD rats were randomly divided into groups according to 1.2, and the experimental group rats were pre-gavaged for 2 weeks. After the pre-gavage, the rats underwent corresponding modeling surgery according to the grouping requirements, and samples were collected 12 weeks after modeling. The rats were fasted for 12 hours before sampling, and blood was collected from the abdominal aorta after anesthesia. The blood was centrifuged at 4°C and 4500rpm for 30 minutes, and the serum was separated for PⅡCP detection and subsequent exploration of the effect of the test substance on OA targets; then the knee joint tissues of the killed rats were quickly separated, and some were fixed in 10% neutral formaldehyde for paraffin section staining, CT and Micro-CT scanning; the remaining knee joint tissues were temporarily stored in liquid nitrogen and transferred to a -80°C refrigerator for long-term storage.

[0114] 1.4 Experimental reagents and instruments

[0115] Table 5 Experimental reagents

[0116] Reagent and material name factory Chloral hydrate Dalian Meilun Biotechnology Co., Ltd. penicillin Sichuan Pharmaceutical Preparations Co., Ltd. 75% alcohol Bengbu Jingcheng Chemical Co., Ltd. Acetic acid Sinopharm Chemical Reagent Co., Ltd. Chloramine T Sinopharm Chemical Reagent Co., Ltd. Hydroxyproline Standards Beijing Solable Technology Co., Ltd. Sodium hydroxide Sinopharm Chemical Reagent Co., Ltd. EDTA Beijing Solable Technology Co., Ltd. Citric acid Sinopharm Chemical Reagent Co., Ltd. hydrochloric acid Sinopharm Chemical Reagent Co., Ltd. Formaldehyde solution Sinopharm Chemical Reagent Co., Ltd. Perchloric acid Sinopharm Chemical Reagent Co., Ltd. Rat PⅡCP Kit Suzhou Calvin Biotechnology Co., Ltd.

[0117] Table 6 Experimental instruments

[0118]

[0119]

[0120] 1.5 Experimental Results

[0121] 1.5.1 Figure 4 It is the SD rat weight record chart. Within two weeks of adaptive feeding and pre-oral gavage for 2 weeks, the weight of each group of rats increased steadily, indicating that gavage did not affect rats' feeding and life. And except that the rats in the normal group had a decrease in weight after surgery, it was shown that surgery would cause certain damage to the rat body and affect its feeding situation. But after about a week of wound recovery, the weight gradually stabilized after gradually increasing steadily, indicating that the damage caused by surgery would not cause serious long-term effects on the feeding of rats. Among them, the normal group had the fastest weight growth, and the drug group had the slowest weight growth. It is speculated that the drug meloxifen would affect rats' appetite to a certain extent, and the two samples had less impact on the appetite of rats.

[0122] Figure 5 The fur status of rats in different groups was compared after 12 weeks of modeling. The fur of rats in the normal group was smooth, soft and shiny (e.g. Figure 5 a), the hair of the rats in the model group was rough and messy, and there was more serious hair loss (such as Figure 5 b). Compared with the model group, the hair roughness of the experimental groups was reduced, and the hair loss phenomenon was improved to varying degrees, among which the CⅡP-M group and the BⅡP-M group showed the best improvement, while the CⅡP-L group and the BⅡP-L group still had a slight degree of hair loss.

[0123] 1.5.2 Micro-CT scanning analysis (osteophyte analysis)

[0124] Osteophytes are often described as bony overgrowths, but are actually formed by the proliferation of cells in the periosteum or synovium at the joint margin that are activated, differentiate into chondrocytes, and then undergo chondrocyte hypertrophy and mineralization through endochondral bone formation. Mature osteophytes contain trabeculae adjacent to existing subchondral bone.

[0125] Figure 6 The observation of osteophyte formation in the knee joints of rats in different groups at week 12 (Micro-CT scan images) showed that the articular cartilage surface of the normal group and the sham operation group was smooth and flat, the joint cavity gap was large, and no osteophyte formation was observed; the joint cavity gap of the model group was narrowed, and there was obvious sharp osteophyte formation; compared with the model group, there was no obvious osteophyte formation in the drug group, which showed an improvement effect. Compared with the model group, the CⅡP test substance CⅡP had a narrower joint cavity and obvious osteophyte formation in the CⅡP-L group, and no significant improvement; the distance of the joint cavity gap in the CⅡP-M group was significantly improved, and there was no obvious osteophyte formation; the joint cavity gap in the CⅡP-H group was not significantly improved, but there was no obvious osteophyte formation, and the cartilage edge was relatively flat. Therefore, it was judged that the CⅡP medium and high dose groups had a better intervention effect. Compared with the model group, the BⅡP test substance BⅡP had obvious osteophyte formation in the BⅡP-L group, which was sharp, and there was no significant improvement; there was no obvious osteophyte formation in the BⅡP-M group, and the cartilage surface was smooth and flat; there was no obvious osteophyte formation in the BⅡP-H group. Therefore, it can be judged that the medium and high dose groups of BⅡP can effectively improve the deterioration of OA.

[0126] Figure 7 The roughness of the cartilage surface of the knee joint of rats in different groups at week 12 (three-dimensional model construction). As the disease worsens, the cartilage wears and the bone proliferates, and the surface of the cartilage tissue will gradually become rough. After re-simulating the three-dimensional model, it can be observed that the surface cartilage tissue of the tibia in the normal group is smooth and flat; there is no significant difference between the sham operation group and the normal group; the surface roughness of the model group is the most serious compared with the normal group; the surface of the drug group is relatively smooth and flat compared with the model group, which has been significantly improved. Compared with the model group, the CⅡP test substance CⅡP has a certain improvement effect in the CⅡP-L group, but it is not significant; the surface of the CⅡP-M group and the CⅡP-H group is relatively smooth and flat, which has a significant improvement effect. Compared with the model group, the surface smoothness of the BⅡP test substance BⅡP in the BⅡP-L group has improved; the surface of the BⅡP-M group and the BⅡP-H group is relatively smooth, and the improvement effect is better, which is similar to the effect of the drug group.

[0127] 1.5.3 Safranin Fast Green Staining Analysis (Cartilage Damage and Degeneration Analysis)

[0128] In the safranin fast green staining, the alkaline dye safranin combines with cartilage to show red, and the acidic dye fast green combines with bone to show green or blue, thus distinguishing cartilage tissue from bone tissue. It is mainly used to judge the degree of cartilage tissue damage.

[0129] Figure 8 This is the cartilage damage of the knee joints of SD rats in different groups at week 12. Figure 8 a is a safranin fast green stained section of the knee joint of rats in different groups, in which cartilage is stained red and osteoblasts are stained green; Figure 8 b is the quantitative analysis of cartilage in safranin fast green stained sections, and the degree of knee cartilage damage in different groups was determined by calculating the proportion of the red stained area. ## indicates that the model group has a significant difference with the normal group, p < 0.01; * indicates that the test group has a significant difference with the model group, p < 0.05.

[0130] like Figure 8 As shown in the figure, there was no significant difference between the normal group and the sham operation group, the cartilage matrix was uniformly stained red, the cartilage layer was thicker in the tibia, and the boundary with the subchondral bone was clearly discernible, and the tide line in the femur was clearer. After 12 weeks of modeling, the red-stained area in the model group was less, and the matrix staining became lighter and uneven, which was significantly different from the normal group (p < 0.01), indicating that the modeling effect was significant.

[0131] After 12 weeks of modeling, the staining distribution area of ​​the CⅡP-M group was larger, and some areas were unevenly stained; the cartilage matrix of the femur in the CⅡP-H group was uniformly stained, with clear tide lines, and the cartilage part of the tibia was slightly thinner, which was significantly different from the model group (p < 0.05). Compared with the model group, the red staining area of ​​the BⅡP-L group was still small, with no significant improvement, and even significantly worsened; the matrix staining of the BⅡP-M group was darker, which was significantly different from the model group (p < 0.05); the matrix staining of the BⅡP-H group was darker, and the red staining area was widely distributed, which was significantly different from the model group (p < 0.05).

[0132] It can be seen from this that both CⅡP and BⅡP can inhibit cartilage damage caused by OA, among which the medium and high dose groups of CⅡP and BⅡP have better effects. As OA develops over time, the high dose group better achieves the purpose of alleviating OA by inhibiting cartilage damage.

[0133] 1.5.4 Toluidine blue staining analysis Toluidine blue staining makes the glycosaminoglycan in chondrocytes dark blue and the background light blue. It is usually used to observe cartilage tissue damage.

[0134] Fig. 9 This is the effect of CⅡP and BⅡP on glycosaminoglycans in the cartilage matrix of knee joint cartilage of SD rats at the 12th week. Fig. 9 a is the toluidine blue stained sections of the knee joints of rats in different groups, in which the glycosaminoglycans in the cartilage matrix were stained dark blue; Fig. 9b is the quantitative analysis of glycosaminoglycans in toluidine blue stained sections. ## indicates that the model group has a significant difference with the normal group, p < 0.01; * indicates p < 0.05, ** indicates p < 0.01, indicating that the test group has a significant difference with the model group.

[0135] like Fig. 9 As shown in the figure, the cartilage matrix of the normal group was stained evenly, the tide line was clear, the cartilage layer was thicker, and the tissue structure was intact; the cartilage matrix of the sham operation group was slightly lighter in staining, but the cartilage tissue was intact and there was no significant difference from the normal group.

[0136] After 12 weeks of modeling, cartilage damage in the CⅡP-L and CⅡP-M groups gradually increased, and the glycosaminoglycan content gradually decreased. Only the glycosaminoglycan content in the CⅡP-H group was significantly increased compared with the model group.

[0137] The glycosaminoglycan content in the BⅡP-L group decreased significantly, with no significant difference compared with the model group; while the matrix blue-stained areas in the BⅡP-M and BⅡP-H groups were darker, and although some parts were unevenly stained, the overall dark-stained areas were widely distributed. Among them, the BⅡP-M group had a significant difference from the model group (p < 0.05), and the BⅡP-H group had a significant difference (p < 0.01).

[0138] It can be seen that the intervention effect of CⅡP and BⅡP is dose-dependent, and the high-dose group has better effects over time, and the loss of glycosaminoglycans in cartilage is more significantly alleviated, which corresponds to the above-mentioned staining section results. The test substance slowly reduces the loss of glycosaminoglycans in the cartilage matrix component, thereby alleviating OA.

[0139] 1.5.5 Experimental conclusion:

[0140] This chapter studies the effects of CⅡP and BⅡP on OA by means of fur status, CT scanning, and histological staining, and the following results are obtained:

[0141] (1) Compared with the model group, the hair roughness of the experimental group rats was reduced, and the hair loss phenomenon was improved to varying degrees. Among them, the CⅡP-M group and the BⅡP-M group showed the best improvement, indicating that medium and high doses of this technology product have a significant effect on skin repair and improvement.

[0142] (2) Both oral medications can inhibit the formation of osteophytes caused by OA, and the construction of a three-dimensional model shows that they can significantly improve the smoothness of the cartilage surface. Among them, the medium and high dose groups of CⅡP and BⅡP have better effects.

[0143] (3) Histological observations showed that after oral administration of the two test substances, the number of chondrocytes in the knee joints of rats with OA, as well as the content of collagen and proteoglycan in the cell matrix, increased. This indicates that both test substances can improve cartilage damage caused by OA.

[0144] This technology is different from other technical inventions that only focus on joint care or collagen peptide supplementation. It uses a composite directional enzymatic cleavage technology of multiple enzymes and low-temperature freeze-drying technology to retain the biological activity of small molecule peptides and GAGS in cartilage extracts to the greatest extent. While improving joints and OA bone joint intervention, it also has a good improvement effect on the skin.

[0145] The above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit them. Although the present application has been described in detail with reference to the aforementioned embodiments, a person skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements will not cause the essence of the corresponding technical solutions to deviate from the protection scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for preparing a highly active small molecule cartilage extract from animal cartilage tissue, characterized in that: The following steps are involved: S1 Soaking: Put the dried pig and cattle nose bones into pure water, keep the system temperature at 20-30°C, soak for 2-3 hours, and after being fully covered with water, the volume of the nose bones will fully expand; S2 bone crushing: the wet bones are taken out and crushed by a grinder, sieved, and then purified water is added and mixed to obtain cartilage liquid A; S3 defatting: keeping the cartilage liquid A at 70-80°C, removing the cartilage fat by skimming centrifugal separation process, and obtaining cartilage liquid B; S4 enzymatic hydrolysis: adjusting the pH value of cartilage liquid B, adding compound protease to cartilage liquid B for compound shearing to obtain enzymatic hydrolyzate; S5 filtration: filtering the enzymatic hydrolysate to obtain a clarified solution; S6 freeze drying: freeze-dry the clarified liquid under reduced pressure to obtain highly active cartilage extract freeze-dried powder.

2. The method for preparing a high-activity small molecule cartilage extract from animal cartilage tissue according to claim 1, characterized in that: In step S2, a 4-8 mm sieve plate is used for sieving, and the volume of pure water is 1-1.5 times of the crushed wet bone.

3. The method for preparing a high-activity small molecule cartilage extract from animal cartilage tissue according to claim 1, characterized in that: In step S4, the pH value of the cartilage liquid B is adjusted to 6.5-8.5, the temperature is 45-65° C., the composite protease is a mixed enzyme of at least two of flavor protease, alkaline protease, neutral protease, papain, and bromelain, and the enzymolysis time is 4-6 hours.

4. The method for preparing a high-activity small molecule cartilage extract from animal cartilage tissue according to claim 3, characterized in that: The dosage of the composite protease is 0.01-0.1% of the dry matter in the cartilage liquid B.

5. The method for preparing a high-activity small molecule cartilage extract from animal cartilage tissue according to claim 1, characterized in that: In step S5, the filtering step includes: S51 carbon filtration removal: add 0.7-1% composite activated carbon to the enzymatic hydrolyzate, adsorb for 40 minutes at 70°C to obtain carbon filtrate; S52 plate and frame filtration: The carbon filtrate is pressurized to 3kg and enters the plate and frame through a pressure pump for filtration to obtain clarified liquid.

6. The method for preparing a high-activity small molecule cartilage extract from animal cartilage tissue according to claim 1, characterized in that: In step S6, the clear liquid is quickly frozen at -40°C and then freeze-dried under reduced pressure at 10 Pa and -50°C.

7. A cartilage extract, characterized in that The preparation is carried out according to the method for preparing high-activity small molecule cartilage extract from animal cartilage tissue as described in any one of claims 1-6.

8. The cartilage extract according to claim 7, characterized in that The average molecular weight of cartilage extract is less than 1000 Da.

9. The cartilage extract according to claim 7, characterized in that The content of type II collagen in the cartilage extract is ≥65%, and the content of glycosaminoglycan is ≥30%.

10. The cartilage extract according to claim 9, characterized in that The hyaluronic acid content in the glycosaminoglycan is ≥17%.

11. Use of cartilage extract, characterized in that: The cartilage extract according to any one of claims 7 to 10 is used to strengthen joints and cartilage structure, improve joints, improve joint health, and improve skin.

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