Bone-derived collagen peptide with joint protection effect and preparation method thereof

Abstract

The invention discloses a bone-derived collagen peptide with a joint protection effect and a preparation method thereof, and belongs to the technical field of collagen peptide preparation, the preparation method comprises the following steps: S1, cleaning and crushing fresh fish bones, and performing impurity protein removal treatment on fish bone powder by using a 0.1 M NaOH solution to obtain pretreated bone powder; s2, mixing the pretreated bone meal with 0.5-1.5 mol / L hydrochloric acid according to a solid-to-liquid ratio of 1: 5, and carrying out a decalcification reaction for 2-4 hours to obtain decalcified bone collagen; s3, performing sequential enzymolysis on the decalcified ossein by adopting pepsin and trypsin to obtain enzymatic hydrolysate; and carrying out inactivation, purification, vacuum concentration and freeze drying on the enzymatic hydrolysate to obtain the collagen peptide. According to the collagen peptide and the preparation method, the characteristic amino acid content of the prepared collagen peptide is increased by 40% or above compared with that of a traditional process, it is ensured that the collagen peptide is combined with a cartilage cell receptor in a targeted mode and inhibits secretion of inflammatory factors, and the inhibition rate reaches 58-68%; at a concentration of 500 [mu] g / mL, there is no toxicity to HaCaT cells (IC50gt; and the traditional Chinese medicine composition has no toxic or side effect and no skin irritation.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of collagen peptide preparation, and particularly relates to a collagen peptide with joint protection effect from bone and a preparation method. BACKGROUND

[0002] Type II collagen is the main component of articular cartilage, and its hydrolysate, collagen peptide, has been confirmed to relieve osteoarthritis symptoms by inhibiting inflammatory factors (such as IL-1β and TNF-α) and promoting chondrocyte proliferation. Traditional type II collagen peptides are mostly derived from pig and bovine cartilage, while a large amount of fish bones (such as silver carp, grass carp and salmon) in aquatic product processing are not effectively utilized as waste.

[0003] In addition, the existing collagen peptide extraction process has obvious technical bottlenecks. For example, when high-concentration hydrochloric acid or strong alkali is used for decalcification or degreasing, the triple helix structure of collagen protein is easily denatured irreversibly, the key active sites (such as Gly-X-Y repeat sequence) are destroyed, and the immunoregulatory activity of the final product is significantly reduced. At the same time, although high-temperature treatment (> 60℃) can accelerate decalcification or sterilization, it further accelerates the degradation of collagen protein, resulting in a disordered molecular weight distribution (500-10000 Da) of the product, which is difficult to meet the standard of small molecular peptides (<3000 Da) required for joint repair.

[0004] In the enzymatic process, the existing technology mostly relies on a single protease to cut collagen protein, but the targeting recognition ability for cross-linked structure is insufficient, resulting in a large amount of macromolecular fragments in the enzymatic product, and the bioavailability is less than 30%. In addition, the extensive control of enzymatic parameters further reduces the process efficiency. For example, when pepsin is used under non-optimal conditions, the enzyme activity loss is more than 50%, the reaction time is forced to be prolonged to more than 12 hours, and the production cost is significantly increased.

[0005] In the decalcification link, a large amount of hydrochloric acid is consumed, and the treatment cost of the acid-containing waste liquid accounts for more than 15% of the total cost. Incomplete decalcification will hinder the subsequent enzymatic hydrolysis, resulting in a sharp decrease in the extraction rate of collagen protein to less than 40%. The backwardness of the purification technology exacerbates the problem of insufficient product purity. Conventional centrifugation or microfiltration cannot effectively remove small molecular impurities, and the purity of type II collagen peptide in the final product is only 60-70%, and the thermal aggregation of peptide chains caused by high temperature (> 200℃) in the spray drying process further reduces the chondrocyte proliferation activity of collagen peptide by 40%. SUMMARY

[0006] The application aims to provide a collagen peptide with joint protection effect from bone sources and a preparation method, so as to solve the low utilization rate of fish bones in the prior art, and improve the preparation method of the collagen peptide from fish bones, and solve the technical problems of high cost and low activity in the existing preparation process.

[0007] To achieve the above-mentioned object, the application provides a preparation method of a collagen peptide with joint protection effect from bone sources, comprising the following steps:

[0008] S1, washing and crushing fresh fish bones to obtain fish bone powder, and removing impure proteins from the fish bone powder by using 0.1M NaOH solution to obtain pretreated bone powder;

[0009] S2, mixing the pretreated bone powder obtained in S1 with 0.5-1.5mol / L hydrochloric acid at a solid-liquid ratio of 1:5, and carrying out decalcification reaction for 2-4 hours to obtain decalcified bone collagen;

[0010] S3, sequentially carrying out enzymolysis of the decalcified bone collagen obtained in S2 by using pepsin and trypsin to obtain an enzymolysis solution;

[0011] S4, inactivating, purifying, vacuum concentrating and freeze-drying the enzymolysis solution obtained in S3 to obtain collagen peptide powder.

[0012] Preferably, in S1, the fish bone powder is mixed with 0.1M NaOH solution at a solid-liquid ratio of 1:10, and stirred at 200rpm in a 25℃ constant temperature water bath for 1.5 hours, and then washed by centrifugation with deionized water at 5000xg until the pH of the filtrate is 7.0±0.2, and the filtrate is filtered to obtain the pretreated bone powder.

[0013] Preferably, in S2, the pretreated bone powder is mixed with 1mol / L hydrochloric acid at a solid-liquid ratio of 1:5, and the mixture is placed in a 30℃ constant temperature shaker for decalcification reaction at 120rpm for 3 hours to obtain the decalcified bone collagen.

[0014] Preferably, the enzymolysis reaction of S3 specifically comprises the following steps:

[0015] S31, dispersing the decalcified bone collagen in 0.1M acetic acid buffer with pH 2.5 to prepare a suspension with a solid-liquid ratio of 1:15, adding pepsin to the suspension, controlling the enzyme-substrate ratio to be 2% w / w, maintaining the pH of the system at 2.5±0.1, and continuously stirring at 37℃ and 400rpm for 4 hours;

[0016] S32, after the stirring in S31 is completed, the pH is adjusted to 7.5 by using 1M NaOH, trypsin is added, the enzyme-substrate ratio is controlled to be 1% w / w, and the enzymolysis is carried out at 37℃ for 2 hours.

[0017] Preferably, in S4, the enzyme solution is rapidly heated to 95℃ and maintained for 10 minutes to inactivate the enzyme, and then immediately cooled to 4℃ in an ice bath. The un-dissociated particles are removed by plate and frame filtration to obtain a clear enzyme solution.

[0018] Preferably, in S4, the clear enzyme solution is sequentially subjected to 8000xg centrifugation, 0.1μm ceramic membrane microfiltration, and 3kDa ultrafiltration membrane ultrafiltration to purify the enzyme solution. The active peptide segments with a molecular weight of <3000Da are collected to obtain an ultrafiltration solution.

[0019] The ultrafiltration solution is vacuum concentrated to 30% solid content, pre-frozen at -80℃ for 24 hours, and dried in a freeze dryer for 48 hours to obtain a collagen peptide powder.

[0020] Preferably, the cold trap of the freeze dryer is -55℃, and the vacuum degree is 10Pa.

[0021] A collagen peptide prepared by the above preparation method.

[0022] Use of the above collagen peptide in the preparation of a joint protection drug.

[0023] Therefore, the bone-derived collagen peptide with joint protection effect and the preparation method have the characteristics that the content of characteristic amino acids is increased by more than 40% compared with traditional processes, the collagen peptide can ensure targeted binding to cartilage cell receptors, inhibit the secretion of inflammatory factors, and the inhibition rate is 58-68%; the collagen peptide has no toxicity (IC 50 >500μg / mL) to HaCaT cells at a concentration of 500μg / mL, has no toxic side effects, and has no skin irritation.

[0024] The technical solutions of the present application will be further described in detail below with the aid of the accompanying drawings and examples. BRIEF DESCRIPTION OF DRAWINGS

[0025] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed in the description of the embodiments of the present application will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0026] Figure 1 To test the cell survival rate of primary human chondrocytes in Example 1;

[0027] Figure 2 To test the inhibition rate of TNF-α and IL-6 detected by ELISA in Example 1;

[0028] Figure 3 To test the reduction rate of joint swelling in Example 2. DETAILED DESCRIPTION

[0029] The technical solutions of the present application are further described below by means of the accompanying drawings and examples.

[0030] In order to make the purpose, technical solutions and advantages of the present application more clear, thorough and complete, the technical solutions of the present application are described clearly and completely below by means of the accompanying drawings and examples. The following detailed description is the description of examples, which aims to provide further detailed description of the present application. Unless otherwise specified, all technical terms used in the present application have the same meaning as generally understood by those skilled in the art to which the present application belongs.

[0031] The instrument equipment and reagent materials used in the examples are obtained through commercial channels.

[0032] Example 1

[0033] A preparation method of a collagen peptide of bone origin, comprising the following steps:

[0034] S1, raw material pretreatment.

[0035] Select fresh silver carp bones 1 kg, rinse to remove surface residual meat and impurities.

[0036] The washed silver carp bones were pre-frozen at -20℃ for 2 hours, then crushed to 150 mesh by a low-temperature crusher at 15000 rpm and a temperature ≤10℃.

[0037] Mix the silver carp bone powder with 0.1M NaOH solution at a solid-liquid ratio of 1:10, stir at a low speed of 200 rpm in a constant temperature water bath at 25℃ for 1.5 hours, then centrifuge with deionized water at 5000×g until the filtrate pH = 7.0±0.2.

[0038] S2, decalcification treatment

[0039] Divide the pretreated bone powder into three groups, 300g each, and mix with 0.5, 1.0, 1.5 mol / L hydrochloric acid solution at a solid-liquid ratio of 1:5, respectively, and place in a constant temperature shaker (shaking frequency 120 rpm) at 30℃ for decalcification reaction, with reaction time of 2, 3, 4 hours respectively.

[0040] S3, enzymatic hydrolysis: disperse the decalcified bone collagen in 0.05M pH 2.5 acetic acid buffer, prepare a suspension with a solid-liquid ratio of 1:15. Add pepsin (enzyme activity 2500U / mg), control the enzyme substrate ratio to be 2% (w / w).

[0041] Stir at 300 rpm in a precision constant temperature bath at 37℃ for 6 hours, and maintain the system pH at 2.5±0.1 by automatic pH titrator.

[0042] The enzymatic solution was rapidly warmed to 90℃ and maintained for 10 minutes to inactivate the enzyme, immediately cooled to 4℃ in an ice bath, and filtered through a plate and frame filter (pore size 5 μm) to remove undissociated particles, obtaining a clear enzymatic solution.

[0043] The enzymatic solution was ultrafiltered with a molecular weight cutoff of 5000 Da, and the permeate was collected. The permeate was concentrated to a solid content of 25% at 60℃ and -0.09 MPa using a rotary evaporator, and finally dried using a spray dryer to obtain a white powder of collagen peptide product type II. The drying conditions were: inlet air temperature 180℃, outlet air temperature 80℃, and atomization pressure 0.2 MPa.

[0044] The molecular weight distribution of the collagen peptide type II in each group was as follows: the group with 1.0 mol / L hydrochloric acid decalcification for 3 hours had a molecular weight concentrated in the range of 1000-3000 Da (accounting for 85%), which was better than the group with 0.5 mol / L (2000-5000 Da, accounting for 60%) and the group with 1.5 mol / L (<1000 Da with too many fragments).

[0045] Example Two

[0046] A method for preparing a collagen peptide of bone origin, comprising the following steps:

[0047] S1, raw material pretreatment

[0048] Fresh silver carp bones 1 kg were selected and rinsed to remove surface residual meat and impurities.

[0049] The washed silver carp bones were pre-frozen at -20℃ for 2 hours, and then crushed to 150 mesh by a low-temperature crusher at 15000 rpm and a temperature ≤10℃.

[0050] The silver carp bone powder was mixed with 0.1 M NaOH solution at a solid-liquid ratio of 1:10, and stirred at a low speed of 200 rpm in a constant temperature water bath at 25℃ for 1.5 hours. Then, the filtrate was washed by centrifugation at 5000 x g until the pH of the filtrate was 7.0±0.2, and the filtrate was filtered to obtain the pretreated bone powder. The silver carp bone powder was mixed with a low concentration of NaOH solution to remove impurities.

[0051] S2, decalcification treatment

[0052] The optimal conditions selected in S1 were used for decalcification treatment: the pretreated bone powder was mixed with 1.0 mol / L hydrochloric acid at a solid-liquid ratio of 1:5, and the mixture was placed in a constant temperature shaker (shaking frequency 120 rpm) at 30℃ for decalcification reaction, with a reaction time of 3 hours.

[0053] S3, enzymatic hydrolysis:

[0054] Group A used sequential enzymatic hydrolysis with pepsin and trypsin.

[0055] First stage (acidic enzymatic hydrolysis): The decalcified bone collagen was dispersed in 0.1M acetic acid buffer with pH 2.5, and a suspension with solid-liquid ratio of 1:15 was prepared. Pepsin (enzyme activity 3500 U / mg) was added, and the enzyme-substrate ratio was controlled at 2% (w / w). The mixture was stirred at 400 rpm and 37°C for 4 hours.

[0056] Second stage (neutral enzymatic hydrolysis): The pH was adjusted to 7.5 (1M NaOH), and trypsin (enzyme activity 2500 U / mg, added amount 1% w / w) was added. The enzymatic hydrolysis was continued at 37°C for 2 hours.

[0057] Group B used single pepsin enzymatic hydrolysis treatment.

[0058] The decalcified bone collagen was dispersed in 0.1M acetic acid buffer with pH 2.5, and a suspension with solid-liquid ratio of 1:15 was prepared. Pepsin (enzyme activity 3500 U / mg) was added, and the enzyme-substrate ratio was controlled at 2% (w / w). The mixture was stirred at 400 rpm and 37°C for 4 hours.

[0059] S4, Inactivation and purification.

[0060] The enzyme solution was quickly heated to 95°C and maintained for 10 minutes to inactivate the enzyme, and then immediately cooled to 4°C in an ice bath. The un-dissociated particles were removed by plate and frame filtration (pore size 5μm) to obtain a clear enzyme solution.

[0061] The enzyme solution was sequentially purified by centrifugation at 8000 x g, microfiltration with a 0.1μm ceramic membrane, and ultrafiltration with a 3kDa ultrafiltration membrane. The active peptide fragments with a molecular weight <3000 Da were collected.

[0062] After vacuum concentration of the ultrafiltrate to 30% solids, pre-freezing at -80°C for 24 hours, and drying in a freeze dryer for 48 hours, a white porous peptide powder was obtained. The cold trap of the freeze dryer was -55°C, and the vacuum degree was 10 Pa.

[0063] Amino acid composition analysis (HPLC-UV): Group A: Glycine Gly, 32.1%; Proline Pro, 12.8%; Hydroxyproline Hyp; 9.5%, total collagen characteristic amino acid ratio 54.4%; Group B: Gly, 28.3%; Pro, 10.2%; Hyp, 7.1%; total ratio 45.6%. The Hyp retention rate of Group A was increased by 34% compared to Group B, indicating that compound enzymatic hydrolysis reduces the loss of hydroxyproline oxidation.

[0064] The decalcified waste acid solution was neutralized to form food-grade calcium phosphate (purity ≥95%), and the COD value of the waste liquid was reduced to 1200-1500 mg / L.

[0065] Comparative Example One

[0066] The traditional collagen peptide process from mammalian sources has the following preparation method:

[0067] S1, select fresh pig cartilage 2 kg, remove attached muscle and fascia, cut into 5 cm x 5 cm blocks, rinse to remove surface impurities. After draining, coarse grinding to a particle size of about 1 cm through a meat grinder, and then fine grinding to a slurry (particle size ≤2 mm) through a colloid mill.

[0068] S2, mix the cartilage slurry with 2.0 mol / L hydrochloric acid solution at a solid-liquid ratio of 1:10, place it in a 45°C constant temperature shaker, and shake at 100 rpm for 6 hours. Centrifuge at 5000 x g for 15 min, discard the supernatant, and rinse the solid residue with deionized water until it is neutral. Freeze-dry for later use.

[0069] S3, disperse the decalcified cartilage in 0.1 M phosphate buffer (pH 7.5) at a solid-liquid ratio of 1:20, add trypsin (enzyme activity 2000 U / mg), control the enzyme substrate ratio to 2% (w / w), and continuously stir at 200 rpm at 37°C for 12 hours.

[0070] Inactivate by raising the temperature to 85°C for 15 minutes, centrifuge at 8000 x g for 20 min to remove undissociated residues, and collect the supernatant.

[0071] The COD value of the decalcification waste liquid reaches 8000 mg / L, which is significantly higher than that of Example 2.

[0072] S4, add ammonium sulfate to the supernatant to a saturation degree of 80%, stand at 4°C overnight, centrifuge at 10000 x g for 30 min to collect the precipitate, and dialyze for 48 hours to remove salt.

[0073] The dialysate is spray dried (inlet temperature 200°C, outlet temperature 90°C) to obtain a light yellow powder, which is sealed and stored.

[0074] SDS-PAGE shows that the main band is dispersed (500-8000 Da), and the proportion of active peptide segments (<3000 Da) is only 40%.

[0075] Comparative Example 2

[0076] A high-acid short-time decalcification process is used to prepare collagen peptide, which includes the following steps:

[0077] S1, take 2 kg of silver carp bones, directly mechanically crush to 50 mesh (particle size about 300 μm), without defatting or removing impure protein treatment.

[0078] S2, high-acid rapid decalcification:

[0079] Mix the silver carp bone powder with 2.0 mol / L hydrochloric acid at a solid-liquid ratio of 1:10, stand at 25°C (without stirring) for 1 hour. After centrifugation at 5000 x g for 10 min, wash with water to pH 4.0-5.0.

[0080] S3, the decalcified bone powder was directly suspended in deionized water (initial pH ≈ 3.5), pepsin (enzyme activity 2500 U / mg) was added, the enzyme substrate ratio was controlled at 1% (w / w), and the enzyme hydrolysis was carried out at 37°C for 6 hours. The enzyme was inactivated by heating at 90°C for 10 minutes, centrifuged at 6000 x g for 15 min, and the supernatant was taken. The supernatant was dried in an oven at 80°C for 24 hours, and the brown powder was crushed. The fishy smell was obvious.

[0081] SDS-PAGE showed that the main band was >5000 Da, and ultrafiltration analysis showed that the proportion of active peptides (<3000 Da) was ≤25%. HPLC detected a large amount of free fatty acids and impurities, and the purity of the product was ≤55%; it was inferred that this was due to the lack of defatting and alkali treatment.

[0082] Test Example One

[0083] An inflammation model of human chondrocytes was constructed. Primary human chondrocytes (purchased from ScienCell, No. #4650) were cultured in DMEM / F12 medium (containing 10% FBS and 1% double-antibiotic).

[0084] The experimental groups were treated as follows:

[0085] Blank control group: normal culture medium was used;

[0086] Inflammatory model group: stimulated with IL-1β (10 ng / mL) for 24 hours;

[0087] Intervention group: after IL-1β stimulation, the collagen peptides of Example One, Example Two (Group A), and Comparative Example One and Comparative Example Two were added (concentration was 100 μg / mL), respectively, and cultured for another 48 hours.

[0088] The cell survival rate was determined by CCK-8 method. Specifically, 10 μL of CCK-8 reagent (Dojindo) was added to each well, and incubated at 37°C for 2 hours. Then the absorbance was detected using a microplate reader (450 nm wavelength), and the survival rate (%) was calculated according to the formula:

[0089] Survival rate (%) = (A 干预组 / A 空白组 ) x 100%.

[0090] The concentration of inflammatory factors was determined by ELISA kit. The cell supernatant was collected, and TNF-α / IL-6 ELISA kit (R&D Systems) was used to operate according to the instructions. The concentration was calculated by standard curve method, and the inhibition rate (%) was calculated by the formula:

[0091] Inhibition rate (%) = (model group concentration - intervention group concentration) / model group concentration x 100%.

[0092] Cell survival rate results are shown in Table 1. Figure 1 As shown in Table 1, when the chondrocytes were induced by IL-1β (10 ng / mL) to cause inflammation, and then the products of Example 1 and Example 2 Group A (100 μg / mL) were added for intervention for 48 hours, the cell survival rates reached 92% and 95%, respectively, which were significantly higher than those of Comparative Example 1 (65%) and Comparative Example 2 (58%), indicating that the optimized process significantly reduced the cytotoxicity in the inflammatory environment.

[0093] The inhibition rates of TNF-α and IL-6 detected by ELISA are shown in Table 2. Figure 2 As shown in Table 2, the inhibition rate of TNF-α of Example 1 was 58%, and that of Example 2 Group A was further improved to 68%, which was much higher than those of Comparative Example 1 (35%) and Comparative Example 2 (22%). In terms of IL-6 inhibition effect, Example 1 and Example 2 achieved inhibition rates of 53% and 61%, respectively, while the inhibition rates of the comparative examples were less than 30%.

[0094] Test Example 2

[0095] Evaluation of in vivo cartilage repair ability.

[0096] Healthy SPF male SD rats weighing 200-250 g and 8-10 weeks old were selected and single-caged in a constant temperature (22±2°C), humidity (50±10%), 12-hour day-night cycle environment. They were adaptively fed for 7 days with free access to food and water.

[0097] SD rats were injected with sodium monoiodoacetate (MIA) at a concentration of 3 mg / 50 μL normal saline into the right knee joint cavity to construct a rat osteoarthritis model.

[0098] The experimental groups and treatment methods are as follows:

[0099] Blank group: only normal saline was injected;

[0100] Model group: after MIA induction, normal saline was given by gavage;

[0101] Intervention group: after MIA induction, collagen peptide of Example 1, Example 2 (Group A), and Comparative Example 1 and Comparative Example 2 (dose of 100 mg / kg / day, for 4 weeks) were given by gavage.

[0102] The swelling degree reduction rate was measured at 7 days, 14 days, and 28 days after MIA induction. The right knee joint diameter was measured using a vernier caliper at the midpoint level of the patella. The swelling degree reduction rate (%) was calculated according to the following formula:

[0103] Swelling degree reduction rate (%) = (diameter of model group - diameter of intervention group) / (diameter of model group - diameter of blank group) x 100%.

[0104] Histopathological analysis of rats was performed as follows: Twenty-eight days after MIA induction, rats were euthanized under overdose anesthesia, and the right knee articular cartilage was completely isolated and fixed in 4% paraformaldehyde for 48 hours. Decalcification was carried out in 10% EDTA solution (pH 7.4) for 4 weeks, with the decalcification solution changed daily. The articular cartilage was paraffin-embedded and sectioned (sagittal sections 5 μm thick), and stained with H&E and Safranin O-Fixed Green. The Osteoarthritis Research Society International (OARSI) scoring system (0-6 points) was used for scoring. The improvement rate (%) was calculated using the following formula:

[0105]

[0106] The rate of reduction in joint swelling is as follows Figure 3 As shown, the joint swelling in Example 1 and Example 2 groups decreased by 45% and 52% respectively, which was significantly higher than that in Comparative Example 1 (25%) and Comparative Example 2 (18%).

[0107] The average OARSI score and OARSI score improvement rate for each group are shown in Table 1 below:

[0108] Table 1. Average OARSI score and OARSI score improvement rate

[0109] Group Average OARSI score Improvement rate (%) Blank group 0.2 - Model group 4.2 - Example 1 1.8 60％ Example 2 1.5 67.5％ Comparative Example 1 3.0 30％ Comparative Example 2 3.5 17.5％

[0110] Histopathological analysis (H&E and Safranin O staining) further confirmed that the improvement rates of OARSI scores in Example 1 and Example 2 groups were 57% and 64%, respectively, while the improvement rates in Comparative Example 1 and Comparative Example 2 were less than 30%.

[0111] The expression level and distribution of type II collagen in articular cartilage tissue were detected by immunohistochemistry to verify the promoting effect of the product of the example on cartilage repair.

[0112] The paraffin-embedded section of SD rat knee joint cartilage with a thickness of 5 pm was sequentially immersed in xylene I and II for 10 minutes each, gradient ethanol (100%, 95%, 80%) for 5 minutes each, and finally washed with PBS. It was again immersed in preheated sodium citrate buffer (pH 6.0) and placed in a 95°C water bath for 20 minutes, and then naturally cooled to room temperature. 3% H2O2 was added dropwise to cover the tissue, and incubated at room temperature for 10 minutes in the dark, and then washed with PBS for 3 times for 5 minutes each time. 5% BSA blocking solution was added dropwise, and incubated at room temperature for 1 hour to reduce non-specific binding. The diluted primary antibody (rabbit anti-type II collagen polyclonal antibody (Abeam ab34712, 1:200 dilution)) was added dropwise to cover the tissue, and incubated at 4°C in a wet box overnight (16-18 hours). PBS was washed for 3 times for 5 minutes each time, and the HRP-labeled secondary antibody (HRP-labeled goat anti-rabbit IgG, CST#7074, 1:500 dilution) was added dropwise, and incubated at room temperature for 1 hour. DAB was used for color development (the color development time was controlled under a microscope, about 1-3 minutes), and then the reaction was terminated by flowing water. The nuclei were stained with hematoxylin for 1 minute, and then differentiated with hydrochloric acid ethanol, and then dehydrated, transparentized, and mounted with neutral resin after returning to blue. The positive control was a tissue section known to express the target protein, and the negative control was a tissue section in which the primary antibody was omitted.

[0113] The scoring criteria are as follows:

[0114] Staining intensity score: 0 points: no staining; 1 point: weak staining (light yellow); 2 points: moderate staining (brownish yellow); 3 points: strong staining (dark brown).

[0115] Positive area ratio: 0 points: 0%; 1 point: 1-25%; 2 points: 26-50%; 3 points: 51-75%; 4 points: 76-100%.

[0116] Total score = Staining intensity score x Positive area ratio score (range 0-12 points).

[0117] The positive area and integral optical density (IOD) were analyzed using ImageJ, and the relative expression amount was calculated.

[0118] The scores and relative expression amounts are shown in Table 2:

[0119] Table 2: Staining intensity score, positive area ratio, total score, and IOD value of each group

[0120]

[0121] Note: * indicates p<0.05 compared with the model group, and ** indicates p<0.01 compared with the model group.

[0122] The immunohistochemical results confirmed that Example One and Example Two significantly increased the expression of type II collagen in cartilage tissue (IOD value increased by 3-4 times compared with the model group, p<0.01), and the positive signal was uniformly distributed in the extracellular matrix, indicating that it played a repair role by promoting cartilage matrix synthesis.

[0123] Test Example Three

[0124] The biological safety of type II collagen peptide was evaluated by cytotoxicity experiment and acute toxicity test.

[0125] (1) Cytotoxicity experiment: human immortalized epidermal cells (HaCaT) were used, and the cells were inoculated in 96-well plates (density 1×10 4 cells / well), and cultured for 24 hours until adherent, then different concentrations of collagen peptide (0-500 μg / mL) were added, and cultured for another 24 hours. The cell survival rate was detected by CCK-8 reagent: 10 μL CCK-8 solution was added to each well, incubated at 37°C for 2 hours, and the absorbance (A) at 450 nm was measured by a microplate reader to calculate the survival rate. A positive control group (1% Triton X-100) was set up to verify the sensitivity of the experiment.

[0126]

[0127] The survival rate results of each group are shown in Table 3:

[0128] Table 3 Cytotoxicity experiment results

[0129] Group Blank group Example 1 Example 2 Comparative Example 1 Comparative Example 2 50 μg / mL survival rate (%) 100±0 98±2 99±1 85±5* 80±6* 100 μg / mL survival rate (%) 100±0 97±3 98±2 78±4* 70±5* 200 μg / mL survival rate (%) 100±0 95±4 96±3 72±5* 65±4* 500 μg / mL survival rate (%) 100±0 92±3 95±2 65±6 58±6 IC 50 (μg / mL) - >500** >500** 320±30* 280±25*

[0130] Note: * indicates p<0.05 compared with the blank group, and ** indicates p<0.01 compared with the blank group.

[0131] In the concentration range of 0-500 μg / mL, the cell survival rates of Example One and Example Two groups were both >95% (IC 50 >500 μg / mL), indicating that they had no significant cytotoxicity. The survival rates of Comparative Example One and Comparative Example Two groups decreased significantly with increasing concentration, with IC 50 values of 320 μg / mL and 280 μg / mL, respectively, indicating that the traditional process products had a risk of toxicity.

[0132] (2) Acute toxicity test: healthy ICR mice (half male and half female, body weight 18-22 g) were selected and randomly divided into 4 groups (n=10): blank control group, Example One group, Example Two group, and Comparative Example Two group (Comparative Example One was not included due to ethical restrictions). A single gavage of collagen peptide solution (2000 mg / kg) was given, and the survival rate, body weight change, organ coefficient, and histopathological changes were observed for 14 days.

[0133] The activity, food and water intake, hair condition and abnormal behavior were observed daily. The body weight was measured every 3 days, and the body weight gain rate was calculated. The serum was collected on day 15, and the liver and kidney function indexes such as ALT, AST, BUN and Cr were detected. The heart, liver, spleen, lung and kidney were taken, fixed with 4% paraformaldehyde, embedded in paraffin with a thickness of 4 μm, and observed under a microscope after HE staining.

[0134] The survival rate of mice in all groups was 100% for 14 days, and no abnormal reactions such as acute death or convulsions occurred.

[0135] The body weight changes of mice in each group are shown in Table 4 below:

[0136] Table 4 Body weight changes of mice

[0137] Group Initial body weight (g) Body weight on day 14 (g) Body weight gain rate (%) Blank control group 20.5±1.2 23.2±1.5 13.2±2.1 Example 1 group 20.8±1.3 23.4±1.6 12.5±1.8 Example 2 group 20.6±1.1 23.3±1.4 13.1±1.9 Comparative Example 2 group 20.7±1.4 22.4±1.7 8.3±2.5*

[0138] The body weight gain rate of the example group had no significant difference (p>0.05) from the blank group, indicating that it had no metabolic inhibition or nutritional absorption disorder; the body weight gain of the comparative example two group was significantly slowed down (p<0.05), which may be related to the interference of residual fatty acids in the defatting process with energy metabolism.

[0139] The blood biochemical indexes of mice in each group are shown in Table 5 below:

[0140] Table 5 Blood biochemical indexes of mice

[0141] Index Blank control group Example 1 group Example 2 group Comparative Example 2 group ALT (U / L) 16.5±2.8 18.3±3.2 17.9±3.1 35.6±5.4 AST (U / L) 20.1±3.5 22.5±4.1 21.8±3.9 48.2±6.7 BUN (mmol / L) 6.5±1.0 6.8±1.2 6.7±1.1 7.2±1.5 Cr (μmol / L) 24.8±3.0 25.3±3.5 24.9±3.2 38.5±4.8

[0142] The ALT and AST of the comparative example two group were significantly increased (p<0.01), indicating liver cell damage, which may be related to oxidative stress induced by residual fatty acids or impurities; the blood creatinine (Cr) of the comparative example two group was increased (p<0.05), indicating impaired glomerular filtration function, while the indexes of the example group were within the normal range and had no statistical difference from the blank group.

[0143] The organ coefficients and pathological damage of mice in each group are shown in Table 6 below:

[0144] Table 6 Organ coefficients and pathological damage of mice

[0145]

[0146] The liver cells of the example group were arranged in order, and the glomerular structure was clear, without inflammatory infiltration or necrosis. The liver cells of the comparative example two group were vacuolar degeneration in part, and there was a small amount of lymphocyte infiltration in the portal area; the renal tubular epithelial cells were edematous, the lumen was narrow, and protein cast was occasionally seen.

[0147] In summary, Example 1 and 2 groups have 100% survival rate at the maximum tolerated dose (2000 mg / kg), no abnormality in body weight gain, blood biochemistry and organ pathology, which meets the actual non-toxic level (LD 50 >5000 mg / kg) standard. Example 2 group caused liver and kidney function damage due to process defects (not defatted, not completely decalcified), which confirmed that the traditional process has safety hazards. In addition, the free fatty acid residues in the product of Example 2 may release inflammatory factors (such as TNF-α, IL-6) by activating Kupffer cells, triggering hepatocyte apoptosis; hydroxyapatite residues may deposit in the renal tubules, interfere with filtration function, leading to elevated blood creatinine.

[0148] (3) Skin irritation test: After shaving the back of New Zealand rabbits (n = 6), apply Example 2 collagen peptide solution (10% concentration, 0.5 mL), once a day, for 7 consecutive days, observe the erythema, edema and other irritation reactions, and score according to the "Cosmetic Safety Technical Specifications" (0-4 points). All experimental rabbits had no erythema and edema (score 0), which confirmed that it had no skin irritation.

[0149] Therefore, the bone-derived collagen peptide with joint protection effect and the preparation method, the prepared collagen peptide has more than 40% higher characteristic amino acid content than the traditional process, which ensures its targeted combination with chondrocyte receptors, inhibits the secretion of inflammatory factors, and the inhibition rate reaches 58-68%; at a concentration of 500 μg / mL, it has no toxicity (IC 50 >500 μg / mL) to HaCaT cells, no toxic side effects, and no skin irritation.

[0150] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application and not to limit it, although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that: it can still modify or equivalently replace the technical solutions of the present application, and these modifications or equivalent replacements also cannot make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present application.

Claims

1. A method for preparing a bone-derived collagen peptide with joint-protective properties, characterized in that, Includes the following steps: S1. Wash and crush fresh fish bones to obtain fish bone powder. Treat the fish bone powder with 0.1M NaOH solution to remove impurities and proteins to obtain pretreated bone powder. S2. The pretreated bone powder obtained in S1 is mixed with 0.5-1.5 mol / L hydrochloric acid at a solid-liquid ratio of 1:5, and the decalcification reaction is carried out for 2-4 hours to obtain decalcified bone collagen. S3. The decalcified collagen obtained in S2 is sequentially hydrolyzed with pepsin and trypsin to obtain the hydrolysate. S4. The enzymatic hydrolysate obtained in S3 is inactivated, purified, vacuum concentrated, and freeze-dried to obtain collagen peptide powder.

2. The method for preparing a bone-derived collagen peptide with joint-protective function according to claim 1, characterized in that: In S1, fish bone powder and 0.1M NaOH solution are mixed at a solid-liquid ratio of 1:10 and stirred at 200 rpm for 1.5 hours in a constant temperature water bath at 25℃. The mixture is then washed with deionized water at 5000×g by centrifugation until the pH of the filtrate is 7.0±0.

2. The filtrate is filtered to obtain pretreated bone powder.

3. The method for preparing a bone-derived collagen peptide with joint-protective function according to claim 1, characterized in that: In S2, pretreated bone powder is mixed with 1 mol / L hydrochloric acid at a solid-liquid ratio of 1:

5. The mixture is placed in a constant temperature shaker at 30℃ and decalcified at 120 rpm for 3 hours to obtain decalcified bone collagen.

4. The method for preparing a bone-derived collagen peptide with joint-protective function according to claim 1, characterized in that, The enzymatic hydrolysis reaction of S3 specifically includes the following steps: S31. Disperse decalcified collagen in 0.1M pH 2.5 acetate buffer to prepare a solid-liquid ratio of 1:

15. Add pepsin to the suspension, control the enzyme-substrate ratio at 2% w / w, maintain the pH of the system at 2.5±0.1, and stir continuously at 37℃ and 400rpm for 4 hours. After stirring S32 and S31, adjust the pH to 7.5 with 1M NaOH, add trypsin, control the enzyme-substrate ratio to 1% w / w, and enzymatically hydrolyze at 37℃ for 2 hours.

5. The method for preparing a bone-derived collagen peptide with joint-protective function according to claim 1, characterized in that: In S4, the enzyme hydrolysate is rapidly heated to 95°C and maintained for 10 minutes to inactivate the enzyme. It is then immediately cooled to 4°C in an ice bath and filtered through a plate and frame filter to remove undissociated particles, yielding a clear enzyme hydrolysate.

6. A method for preparing a bone-derived collagen peptide with joint-protective effects according to claim 1 or 5, characterized in that: In S4, the clarified enzymatic hydrolysate was purified by centrifugation at 8000×g, microfiltration through a 0.1μm ceramic membrane, and ultrafiltration through a 3kDa ultrafiltration membrane. Active peptides with a molecular weight <3000Da were collected to obtain the ultrafiltrate. The ultrafiltrate was concentrated under vacuum to 30% solids, the solids were pre-frozen at -80°C for 24 hours, and then dried in a freeze dryer for 48 hours to obtain collagen peptide powder.

7. The method for preparing a bone-derived collagen peptide with joint-protective function according to claim 6, characterized in that: The freeze dryer has a cold trap temperature of -55°C and a vacuum level of 10Pa.

8. A collagen peptide prepared by the preparation method according to any one of claims 1 to 7.

9. The use of the collagen peptide as described in claim 8 in the preparation of a joint-protective medicament.

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