Yak collagen oligopeptide, bone-targeted complex, and preparation method and application thereof

Abstract

The yak collagen oligopeptide, bone-targeted complex and preparation method and application thereof belong to the field of biological oligopeptides. The technical key point is that the amino acid sequence is shown in SEQ ID NO.1, wherein the bone-targeted complex is a phytic acid conjugate of the oligopeptide included in cyclodextrin. The effect is that the oligopeptide of the present invention has the function of promoting osteoblast proliferation and inhibiting osteoclast function, and can be applied to drugs, foods or health products for treating osteoporosis.

Description

Technical Field

[0001] The invention belongs to the field of biological oligopeptides, and specifically relates to an oligopeptide derived from collagen in bovine bones and a bone-targeted composite using the oligopeptide, as well as a preparation method and application of the composite. Background Art

[0002] Osteoporosis (OP) is a systemic bone metabolic disease characterized by decreased bone mass, degeneration of bone microstructure, and increased bone brittleness. With the aging population, the prevalence of osteoporosis is also increasing, and the prevalence in women is much higher than that in men, especially in postmenopausal women. Due to estrogen deficiency, bone density decreases and bone loss accelerates. Among people aged 80-89, the prevalence in women is about three times that of men.

[0003] At present, the drugs used to treat osteoporosis mainly include bisphosphonates, estrogen replacement therapy, selective estrogen receptors, etc. Although these anti-osteoporosis drugs can effectively increase bone density and reduce fractures, they are prone to cause side effects such as nausea and vomiting, and there are certain risks in long-term use. In recent years, peptide substances extracted from animal bones have attracted more and more attention from researchers due to their high safety and good efficacy. Literature has reported that different types of bone peptide drugs have therapeutic effects in regulating bone metabolism, promoting osteoblast proliferation to form new bone, regulating calcium and phosphorus metabolism, and increasing bone calcium deposition.

[0004] Among them, bovine collagen peptides have received extensive attention due to their rich sources and simple acquisition, but lack the support of appropriate bone tissue targeted delivery and target location sustained release technology, and the role of bovine collagen peptides in maintaining bone health and relieving symptoms such as osteoporosis in vivo has not been fully utilized. The construction process of existing bone-targeted delivery carriers is mostly complicated and difficult to apply to industrial preparation. Therefore, further research on bovine collagen peptides and the development of their bone tissue targeted delivery system to expand their application in the prevention and treatment of osteoporosis are technical issues that need to be solved in this field. Summary of the invention

[0005] The present invention aims at the above-mentioned purpose, in the first aspect, provides a yak collagen oligopeptide having the function of promoting osteoblast proliferation and inhibiting osteoclast activity. In the second aspect, a bone-targeted complex and its preparation method can be further used for delivering oligopeptides extracted from bones to bones. In the third aspect, the application of the yak collagen oligopeptide and the bone-targeted complex is provided.

[0006] In a first aspect, according to the yak collagen oligopeptide in some embodiments of the present application, the amino acid sequence is shown as SEQ ID NO.1.

[0007] In a second aspect, according to the method for preparing the yak collagen oligopeptide in some embodiments of the present application, the method comprises:

[0008] S110. Add a composite protease to the beef bone cooking liquid for enzymatic hydrolysis, wherein the mass ratio of the composite protease is neutral protease: alkaline protease: papain = 2:1:3, the enzymatic hydrolysis temperature is 45-55°C, the enzyme reaction pH is 7.5-8.5, the enzymatic hydrolysis time is 3-4h, and the enzyme is inactivated after enzymatic hydrolysis to obtain beef bone enzymatic hydrolysate.

[0009] S120. The clear liquid of the bovine bone hydrolysate is subjected to membrane separation with a molecular weight cut-off of 3000Da.

[0010] S130. The membrane separation liquid is separated and purified by a gel chromatography column. The elution solvent is deionized water. The elution flow rate is 0.8 mL / min. The absorbance is detected at 280 nm, and the third elution peak is collected.

[0011] S140. Further purification is carried out using a chromatographic column, wherein mobile phase A is water containing 0.05% trifluoroacetic acid by volume, mobile phase B is acetonitrile, and the gradient elution conditions are: 0-5 min, 10% B, 5-15 min, 10%-15% B, 15-25 min, 15% B-25% B, 25-40 min, 25% B-35% B, the flow rate is 1.0 mL / min, and the elution peak with a retention time of 8.23-9.84 min is collected to obtain the yak bone collagen oligopeptide.

[0012] According to the preparation method of the yak collagen oligopeptide in some embodiments of the present application,

[0013] According to the preparation method of yak bone collagen oligopeptide in some embodiments of the present application, in step S110, enzymolysis is performed in an enzymolysis tank, and the preparation of the cow bone cooking liquid includes crushing the cow bones, adding 10 times the mass volume of water and cooking at high temperature. After cooking for 4 hours, cooling, centrifugation and filtration are performed to remove fat to obtain the cow bone cooking liquid.

[0014] According to the method for preparing yak collagen oligopeptide in some embodiments of the present application, in step S110, 0.3% of the composite protease by weight of bovine bone is added.

[0015] According to the method for preparing yak collagen oligopeptide in some embodiments of the present application, in step S110, the enzymatic hydrolysis temperature is 55° C., the enzyme reaction pH is 8.0, and the enzymatic hydrolysis time is 4 hours.

[0016] According to the method for preparing yak bone collagen oligopeptide in some embodiments of the present application, in step S110, after enzymatic hydrolysis, the temperature is raised to 80° C. to inactivate the enzyme for 15 minutes to obtain a bovine bone enzymatic hydrolysate.

[0017] According to the method for preparing yak bone collagen oligopeptide in some embodiments of the present application, in step S120, the bovine bone enzymatic hydrolysate is centrifuged to obtain the supernatant to obtain the clear liquid of the bovine bone enzymatic hydrolysate.

[0018] According to the method for preparing yak collagen oligopeptide in some embodiments of the present application, in step S120, the centrifugal speed is 8000 rev / min, and the centrifugation is 10 minutes.

[0019] According to the method for preparing yak collagen oligopeptide in some embodiments of the present application, in step S130, a 20 mm×100 mm Sephadex G10 gel chromatography column is used for separation and purification.

[0020] According to the method for preparing yak collagen oligopeptide in some embodiments of the present application, in step S140, a C18 chromatographic column is used for further purification.

[0021] According to the preparation method of the yak bone collagen oligopeptide in some embodiments of the present application, in step S140, the yak bone collagen oligopeptide is concentrated and then freeze-dried to obtain yak bone collagen oligopeptide powder.

[0022] In the first direction of the third aspect, according to some embodiments of the present application, the bone-targeted complex of the oligopeptide is a phytic acid conjugate of the oligopeptide encapsulated by cyclodextrin, wherein the oligopeptide is an oligopeptide extracted from bones, and the oligopeptide is the yak collagen oligopeptide as described in claim 1.

[0023] According to the bone-targeted oligopeptide complex in some embodiments of the present application, the preparation method of the bone-targeted oligopeptide complex is characterized by comprising:

[0024] S210. Mixing the aqueous solution of oligopeptide and the aqueous solution of phytic acid to obtain a mixed solution of oligopeptide and phytic acid.

[0025] S220. Add hydrochloric acid to the oligopeptide and phytic acid mixture and stir until dissolved to obtain a mixture containing hydrochloric acid, wherein the molar ratio of the oligopeptide, phytic acid and hydrochloric acid is 3-6:1:10.

[0026] S230. React the mixed solution containing hydrochloric acid in an oven at 110-120°C for 8-12 hours.

[0027] S240. Wash the reactants.

[0028] S250. Dropping the cyclodextrin solution into the aqueous solution of the reactant to obtain a bone-targeted complex of the oligopeptide, wherein the mass ratio of the reactant to the cyclodextrin is 1 to 3:1.

[0029] According to the bone-targeted complex of oligopeptides in some embodiments of the present application, the oligopeptide is an oligopeptide extracted from bones, and the oligopeptide is the yak collagen oligopeptide.

[0030] According to the bone-targeted oligopeptide complex in some embodiments of the present application, the molar concentration of the aqueous solution of the oligopeptide in step 210 is 20-40 mmoL, and the molar concentration of the aqueous solution of phytic acid is 10-20 mmoL.

[0031] According to the bone-targeted oligopeptide complex in some embodiments of the present application, in step 210, the aqueous solution of the oligopeptide is a deionized aqueous solution of the oligopeptide, and the aqueous solution of phytic acid is a deionized aqueous solution of phytic acid.

[0032] According to the bone-targeted complex of oligopeptides in some embodiments of the present application, the molar ratio of oligopeptide, phytic acid and hydrochloric acid in step S220 is 0.006:0.001:0.01.

[0033] According to the bone-targeted oligopeptide complex in some embodiments of the present application, in step S230, the mixed solution containing hydrochloric acid is reacted in an oven at 120° C. for 12 hours.

[0034] According to the bone-targeted oligopeptide complex in some embodiments of the present application, step S240 is to wash the reactants at least once using 0.1 M HCl, 0.1 M NaOH and water in sequence.

[0035] According to the bone-targeted oligopeptide complex in some embodiments of the present application, in step S250, the mass volume ratio of the aqueous solution of the reactants is 1-5%, and the mass volume ratio of the aqueous solution of cyclodextrin is 1-5%.

[0036] According to the bone-targeted complex of oligopeptides in some embodiments of the present application, in step S250, the aqueous solution of the reactants is a deionized aqueous solution of the reactants, and the aqueous solution of cyclodextrin is a deionized aqueous solution of cyclodextrin.

[0037] According to the bone-targeted oligopeptide complex in some embodiments of the present application, in step S250, the mass volume ratio of the aqueous solution of the reactants is 5%, and the mass volume ratio of the aqueous solution of cyclodextrin is 5%.

[0038] In the second direction of the third aspect, the bone-targeted complex of the oligopeptide according to some embodiments of the present application is prepared by any one of the methods.

[0039] In the fourth aspect, according to some embodiments of the present application, the yak collagen oligopeptide or the bone-targeted complex of the oligopeptide or the bone-targeted complex of the oligopeptide is used in the preparation of drugs, foods or health products that promote osteoclast proliferation, or in the preparation of drugs, foods or health products that inhibit osteoclasts, or in the preparation of drugs, foods or health products for treating osteoporosis.

[0040] Beneficial effects:

[0041] The present invention obtains an active peptide from bovine bones through separation and purification by gel exclusion chromatography and reversed-phase high performance liquid chromatography. The active peptide has good activity in promoting osteoblast proliferation and inhibiting osteoclasts. The amino acid sequence of the active peptide is: Gly-Pro-Phe-Gly-Leu-Asp-Lys-Tyr-Trp. After retrieval from the online databases BIOPEP and EROP-Moscow, the sequence is a new small molecule active peptide.

[0042] The present invention provides a bone-targeted oligopeptide complex and a preparation method thereof. The complex is a bone-targeted oligopeptide that can be used to deliver the oligopeptide extracted from the bone to the bone. The carboxyl end of the yak collagen oligopeptide is grafted with the hydroxyl group of the outer ring of the phytic acid structure by esterification cross-linking. Due to the chelation of the deprotonated hydroxyl group in the phytic acid phosphate group with Ca2+ on the hydroxyapatite in the bone, a strong affinity for the bone is achieved, thereby achieving the enrichment of the oligopeptide in the bone part. At the same time, the stability of the BBP-PA conjugate in the gastrointestinal fluid is improved by the inclusion of cyclodextrin. The hydrophobic end of the peptide segment of the BBP-PA conjugate is located in the hydrophobic inner cavity of the cyclodextrin, and the phosphate group with bone targeting is located on the outside, thereby avoiding the premature hydrolysis of the coupled ester bond and reducing the targeting efficiency. After the complex is enriched in the bone, the BBP-PA conjugate is released, and then the reversible esterification cross-linking is broken to allow the oligopeptide to be slowly released in the bone tissue, thereby improving the bioavailability of the active peptide in the bone tissue. This method is simple and easy to operate, suitable for industrial preparation scenarios, and has broad application prospects in the prevention and treatment of osteoporosis symptoms. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 . Proliferation rate of mouse preosteoblast cell line MC3T3-E1 cells (*P<0.05, **P<0.01).

[0044] Figure 2 .The number of osteoclasts stained by TRAP (*P<0.05, **P<0.01).

[0045] Figure 3 .BBP-PA-CD complex and BBP adsorption rate on hydroxyapatite.

[0046] Figure 4 .Comparison of bone density values ​​in mice. DETAILED DESCRIPTION

[0047] The present invention is further described below in conjunction with the accompanying drawings and embodiments. It should be noted that the embodiments do not limit the scope of protection claimed for the present invention.

[0048] In view of the above-mentioned purpose, the present invention provides an oligopeptide having the function of promoting osteoblast proliferation and inhibiting osteoclast activity. In some specific examples, the present invention uses the oligopeptide derived from bovine bones, especially collagen from yak (Bosgrunniens) bones. Therefore, in some examples, the present invention refers to it as yak bone collagen oligopeptide or bovine bone collagen oligopeptide.

[0049] In the present invention, the amino acid sequence of the oligopeptide is shown in SEQ ID NO: 1. SEQ ID NO. 1: Gly-Pro-Phe-Gly-Leu-Asp-Lys-Tyr-Trp.

[0050] The present invention also provides a method for preparing yak collagen oligopeptide, comprising:

[0051] S110. Add a composite protease to the yak bone cooking liquid for enzymatic hydrolysis, wherein the mass ratio of the composite protease is neutral protease: alkaline protease: papain = 2:1:3, the enzymatic hydrolysis temperature is 45-55°C, the enzyme reaction pH is 7.5-8.5, the enzymatic hydrolysis time is 3-4h, and the enzyme is inactivated after the enzymatic hydrolysis to obtain yak bone enzymatic hydrolysate.

[0052] S120. The supernatant of the yak bone enzymatic hydrolysate is subjected to membrane separation with a molecular weight cutoff of 3000Da.

[0053] S130. The membrane separation liquid is separated and purified by a gel chromatography column. The elution solvent is deionized water. The elution flow rate is 0.8 mL / min. The absorbance is detected at 280 nm, and the third elution peak is collected.

[0054] S140. Further purification is carried out using a chromatographic column, wherein mobile phase A is water containing 0.05% trifluoroacetic acid by volume, mobile phase B is acetonitrile, and the gradient elution conditions are: 0-5 min, 10% B, 5-15 min, 10%-15% B, 15-25 min, 15% B-25% B, 25-40 min, 25% B-35% B, the flow rate is 1.0 mL / min, and the elution peak with a retention time of 8.23-9.84 min is collected to obtain the yak bone collagen oligopeptide.

[0055] Preferably, in step S110, enzymolysis is performed in an enzymolysis tank, and the preparation of yak bone cooking liquid includes crushing the yak bones, adding 10 times the mass volume of water and cooking at high temperature, cooking for 4 hours, cooling, and centrifuging to remove fat to obtain yak bone cooking liquid.

[0056] Preferably, in step S110, 0.3% of the composite protease by weight of yak bone is added.

[0057] Preferably, in step S110, the enzymatic hydrolysis temperature is 55° C., the enzyme reaction pH is 8.0, and the enzymatic hydrolysis time is 4 h.

[0058] Preferably, in step S110, after enzymatic hydrolysis, the temperature is raised to 80° C. to inactivate the enzyme for 15 minutes to obtain yak bone enzymatic hydrolysate.

[0059] Preferably, in step S120, the yak bone enzymatic hydrolysate is centrifuged to obtain the supernatant to obtain the clear liquid of the yak bone enzymatic hydrolysate.

[0060] More preferably, in step S120, the centrifugal speed is 8000 rev / min, and the centrifugation is performed for 10 min.

[0061] Preferably, in step S130, a 20 mm×100 mm Sephadex G10 gel chromatography column is used for separation and purification.

[0062] Preferably, in step S140, a C18 chromatographic column is used for further purification.

[0063] Preferably, in step S140, the yak bone collagen oligopeptide is concentrated and then freeze-dried to obtain yak bone collagen oligopeptide powder.

[0064] The present invention also provides a method for preparing the bone-targeted composite of the yak collagen oligopeptide, which comprises the following process steps:

[0065] Step 1. Prepare aqueous solutions of yak bone collagen oligopeptide and phytic acid with deionized water, add hydrochloric acid and stir mechanically until they are completely dissolved after thorough mixing, and place in an oven at 110-120°C for reaction for 8-12 hours. Wash the obtained reaction product with 0.1M HCl, 0.1M NaOH and water respectively until the washing liquid is clear, and freeze-dry to obtain yak bone collagen oligopeptide-phytic acid conjugate (BBP-PA).

[0066] Furthermore, the molar concentration of the yak collagen oligopeptide solution in step 1 is 20-60 mmoL;

[0067] Furthermore, the molar concentration of the phytic acid solution in step 1 is 10-20 mmoL;

[0068] Furthermore, in step 1, the molar ratio of yak collagen oligopeptide, phytic acid and hydrochloric acid during mixing is 3-6:1:10.

[0069] Step 2. After dissolving the BBP-PA powder obtained in step 1 with deionized water, slowly drip it into the cyclodextrin solution and slowly stir it for a period of time. After freeze-drying, obtain the yak collagen oligopeptide-phytic acid-cyclodextrin bone-targeted complex (BBP-PA-CD).

[0070] Furthermore, the mass volume ratio of the BBP-PA solution in step 2 is 1-5%.

[0071] Furthermore, the mass volume ratio of the cyclodextrin solution in step 2 is 1-5%.

[0072] Furthermore, the mass ratio of BBP-PA to cyclodextrin in step 2 is 1 to 3:1.

[0073] Of course, the oligopeptide in the bone-targeted complex of the oligopeptide in the present invention can also be an oligopeptide extracted from other bones for delivery to the bone. As long as the carboxyl end of the oligopeptide is grafted with the hydroxyl group of the outer ring of the phytic acid structure through esterification cross-linking, a strong affinity for the bone can be achieved through the chelation of the deprotonated hydroxyl group in the phytic acid phosphate group with Ca2+ on the hydroxyapatite in the bone, thereby achieving the purpose of enriching the oligopeptide in the bone area.

[0074] Example 1. Preparation method of yak collagen oligopeptide:

[0075] 1000g of fresh frozen yak bones were soaked in cold water, cleaned and crushed with a bone crusher, added with 10 times the mass volume of water, steamed at high temperature for 4 hours, cooled, centrifuged to remove fat and added to an enzymatic hydrolysis tank, added with 0.3% of the raw material weight of composite protease, the composite protease ratio was neutral protease: alkaline protease: papain = 2:1:3, enzymatic hydrolysis was carried out at 55°C for 4 hours, the enzyme reaction pH was 8.0, the enzyme was inactivated at 80°C for 15 minutes, and the supernatant was centrifuged to obtain an enzymatic hydrolyzate; centrifuged at 8000 rpm for 10 minutes, and then separated by 3000Da ultrafiltration, and the membrane liquid was separated by SephadexG 10 (20mm×100mm) gel chromatography column for separation and purification, the elution solvent is deionized water, the elution flow rate is 0.8mL / min, the absorbance is detected at 280nm, and the third elution peak is collected; C18 chromatography column, mobile phase A is water containing 0.05% trifluoroacetic acid by volume, mobile phase B is acetonitrile, gradient elution conditions are: 0-5min, 10%B, 5-15min, 10%-15%B, 15-25min, 15%B-25%B, 25-40min, 25%B-35%B, the flow rate is 1.0mL / min, the elution peak with a retention time of 8.23-9.84min is collected, and the yak bone collagen oligopeptide powder is obtained by freeze-drying after concentration.

[0076] Example 2. Preparation of BBP-PA-CD

[0077] Step 1. Take 6g of yak collagen oligopeptide powder and 0.66g of phytic acid obtained in Example 1 and prepare 0.06M and 0.01M aqueous solutions with 100mL of deionized water, mix them thoroughly, add 0.83ml of concentrated hydrochloric acid and stir mechanically until they are completely dissolved, and place them in a 120°C oven to react for 12h. The obtained reaction product is washed with 0.1M HCl, 0.1M NaOH and water respectively until the washing liquid is clear, and freeze-dried to obtain 6.3g of yak collagen oligopeptide-phytic acid conjugate (BBP-PA).

[0078] Step 2. Take 5 g of the BBP-PA powder obtained in step 1, dissolve it in 100 ml of deionized water, slowly drip it into 100 ml of 5% cyclodextrin solution and slowly stir it for a while. After freeze-drying, 9.7 g of yak collagen oligopeptide-phytic acid-cyclodextrin bone targeting complex (BBP-PA-CD) was obtained.

[0079] Example 3. Cytotoxicity

[0080] MCF-7 cells in the logarithmic growth phase were inoculated into 96-well plates and incubated in a 37°C incubator for 24 hours. 100 μL of BBP-PA-CD aqueous solution samples (concentrations were 0.25, 0.5, 1, 5, and 10 μg / mL) were added to each well. After the cells were cultured at 37°C and 5% CO2 for 48 hours, the supernatant was discarded, and MTT solution was added. The cells were cultured for another 4 hours at 37°C and 5% CO2. The supernatant was discarded, 150 μL of DMSO was added to each well, and the cells were shaken lightly on a shaker for 1 minute in the dark. After the formazan was dissolved, the absorbance of each well was detected using an enzyme-labeled instrument. The relative activity of the cells was calculated according to the following formula:

[0081]

[0082] The results showed that when the concentration of PA-CD reached 10 μg / mL, the cell survival rate was also higher than 90%, indicating that the prepared BBP-PA-CD had low cytotoxicity.

[0083] Example 4. Promoting osteoblast proliferation and inhibiting osteoclasts

[0084] Mouse preosteoblast cell line MC3T3-E1 cells were seeded in a 96-well plate at 2×104 / mL and 100 μL per well. After constant temperature culture for 24 hours, the original culture medium was removed, and then 0.1, 0.5, 1.0, and 3.0 mg / mL BBP peptide solutions were prepared and added to the 96-well plate at 200 μL per well. The cells were cultured continuously for 48 hours in a constant temperature incubator at 37°C and 5% CO2 volume fraction. The blank group was added with an equal volume of culture medium, and the proliferation rate was determined by the CCK-8 method. Each group of experiments was performed in parallel 3 times to calculate the relative proliferation rate of preosteoblasts. The results are shown in Figure 1 As shown, the proliferation rate of mouse preosteoblast cell line MC3T3-E1 cells (*P<0.05, **P<0.01) showed that the BBP peptide solution group had a significant osteoblast proliferation effect starting from a mass concentration of 0.5 mg / L, and the relative proliferation rate of the 3.0 mg / mL group reached 131%.

[0085] Bone marrow mononuclear cells (BMMs) from C57BL / 6 mice were obtained and cultured in culture medium. After 24 hours, the non-adherent cells at the bottom of the bottle were collected and continued to be cultured. They were inoculated into 96-well plates (2×104 cells / well) as purified BMMs. After 24 hours, 100 ng / mL of RANKL was added to induce differentiation into osteoclasts. At the same time, different concentrations (0.1, 0.5, 1.0, 3.0 mg / mL) of BBP peptide solutions were added. A blank control group without RANKL was set up. Each group had 3 replicates, and the induction intervention lasted for 5-7 days. The cells induced to the endpoint were stained using the TRAP staining kit, and the number of osteoclasts in each group was calculated. The results are shown in Figure 2As shown in the figure, the number of osteoclasts stained by TRAP (*P<0.05, **P<0.01), no osteoclasts were generated in the blank group, a large number of osteoclasts were generated in the RANKL group, and the number of osteoclasts decreased significantly in a concentration-dependent manner after BBP treatment.

[0086] Example 5. Release rate of BBP-PA-CD complex

[0087] Take 5mL concentrated hydrochloric acid (37%), 5mL Tween 80, 2g Na Cl, 3.2g pepsin, add water to 1L, adjust the pH value to 1.2, and filter with a 0.22μm organic filter membrane before use as simulated gastric fluid. Take 6.8g potassium dihydrogen phosphate, 10g trypsin, 5mL Tween 80, add water to 1L, ultrasonically dissolve them completely, adjust the pH value to 6.8 with dilute sodium hydroxide solution, and filter with a 0.22μm organic filter membrane before use as simulated intestinal fluid. 4 mg of the BBP-PA-CD powder obtained in Example 2 was placed in a dialysis bag (MWCO 3500), immersed in 200 mL of artificial gastric juice (or intestinal juice), and stirred in a constant temperature magnetic stirrer at a system temperature of 37° C. The rotation speed was 100 rpm / min, and 1 mL was sampled at 30 min and 1, 6, 12, and 24 h, respectively. After sampling, 1 mL of blank artificial gastric juice (or intestinal juice) of the same matrix was added. The sample was centrifuged and the supernatant was taken. After filtering with a 0.22 μm organic filter membrane, the content of the BBP-PA conjugate was detected by high performance liquid chromatography.

[0088] The results showed that the release rate of BBP-PA in artificial gastrointestinal fluid gradually increased with time, reaching 10.54% in artificial gastric fluid and 15.72% in artificial intestinal fluid at 12 hours when the release rate tended to stabilize. The encapsulation effect of cyclodextrin on the esterification coupling of BBP-PA improved the stability, indicating that the BBP-PA-CD system can exist stably in the artificial gastrointestinal fluid environment, which protects the esterification coupling of BBP-PA, delays the hydrolysis of the ester bond, and facilitates its absorption by the intestine into the blood circulation.

[0089] Experimental Example 6. Hydroxyapatite affinity of BBP-PA-CD

[0090] Take 10 ml of 1 mg / mL BBP-PA-CD solution and BBP solution, put them into clean test tubes, add 50 mg of hydroxyapatite, shake at 37°C for 2 hours, centrifuge at 8000 r / min for 10 minutes, remove the supernatant, dissolve the precipitate with 10 ml of methanol, ultrasonically vibrate for 5 minutes, centrifuge at 12000 r / min for 10 minutes, take the supernatant and filter through a 0.22 μm filter membrane, and determine the content of BBP-PA-CD and BBP in the sample by LC-MS / MS. The adsorption rate is calculated according to the following formula: adsorption rate (%) = mass of BBP-PA-CD and BBP adsorbed by hydroxyapatite (mg) / mass of BBP-PA-CD and BBP in the original sample solution (mg) × 100%.

[0091] The results are as follows Figure 3 As shown in the figure, the BBP-PA-CD complex and BBP adsorption rate on hydroxyapatite, the adsorption amount of BBP-PA-CD complex on hydroxyapatite reached 84.52% after 2h of incubation, while the adsorption amount of BBP as the control group on hydroxyapatite was only 18.47%. After PA grafting, the adsorption amount of the complex on hydroxyapatite was 4.58 times the original adsorption amount.

[0092] Example 7. Osteoporosis mouse model

[0093] Except for the sham operation group, SD rats were adaptively fed for 1 week, and then the rat osteoporosis model was established by ovariectomy: after intraperitoneal anesthesia with 1% sodium pentobarbital (4 ml / kg), the abdominal skin was prepared and disinfected, and then an incision was made along the midline. The abdominal cavity was explored and the fat mass at the end of the uterine horn was pulled out of the abdominal cavity. The ovary was ligated and removed with hemostatic forceps, and then the tissue was restored. The other ovary was removed in the same way, and finally the wounds were sutured in sequence. In the sham operation group, only the fat tissue of the same size next to the ovary was removed. Drug administration began 4 weeks after surgery, and the castrated rats were randomly divided into 4 groups, with 10 rats in each group, and the sham operation group and model group were used as controls. The sham operation group and the model control group were given equal doses of normal saline; the BBP group and the BBP-PA-CD group were given at 200 mg / kg·d. At the end of the 8th week of drug administration, the mice were anesthetized with 1% sodium pentobarbital solution, and the changes in the distal femoral bone density of each group of mice were measured using a dual-energy X-ray absorptiometry, and the images formed by the X-ray were analyzed. The results are shown in Figure 4 As shown in the figure, the bone density values ​​of mice in each group were found to be significantly decreased compared with the sham operation group (P < 0.01), while the bone density values ​​of the BBP-PA-CD group and the BBP group were increased compared with the model group, and the improvement effect of oral BBP-PA-CD was significantly better than that of BBP (P < 0.01). BBP-PA-CD intervention can increase the whole-body bone density of mice and reduce the loss of whole-body bone mass.

[0094] Combined with the effects of the above embodiments, it can be seen that the yak collagen oligopeptide provided by the present invention has good effects of promoting osteoblast proliferation and inhibiting osteoclast activity. At the same time, the bone-targeted composite preparation method provided can make the composite have good bone tissue targeting through phytic acid grafting, so that it can slowly release active peptide components in bone tissue, improve the bioavailability of yak collagen oligopeptide, and thus improve the phenomenon of osteoporosis.

[0095] The present invention discloses a bovine collagen peptide and a method for preparing a bone-targeted complex thereof, and belongs to the field of active peptide application. An oligopeptide that promotes osteoblast proliferation and inhibits osteoclast activity has an amino acid sequence as shown in SEQ ID NO:1. Also disclosed is a method for preparing a bone-targeted active peptide conjugate, which first esterifies and couples the yak collagen oligopeptide with phytic acid to form a bone-targeted conjugate, and then uses cyclodextrin to load the conjugate to form a bone-targeted complex. On the one hand, phytic acid is used to bind to bone hydroxyphospholipids.

[0096] The targeting of the graystone is used to deliver the yak collagen oligopeptide. On the other hand, cyclodextrin is used to protect the conjugate in the gastrointestinal fluid, and finally the active peptide is slowly released in the bone tissue, thereby improving the bioavailability of the active peptide. The yak collagen oligopeptide of the present invention is derived from cattle bones, has good safety, has a significant effect of promoting osteoblast proliferation, promoting osteoblast differentiation, and inhibiting bone resorption. The bone-targeted complex is better enriched in the bone tissue in vivo, alleviating osteoporosis. The conjugate can be industrially produced and has good application prospects in the fields of food and medicine.

[0097] Finally, it should be noted that the above examples are only some specific embodiments of the present invention, and all derivatives that can be directly derived or associated with the contents disclosed by a person skilled in the art should be considered as the protection scope of the present invention.

Claims

1. A yak collagen oligopeptide, characterized in that: The amino acid sequence is shown in SEQ ID NO.

1.

2. A method for preparing the yak collagen oligopeptide according to claim 1, characterized in that: include S110. Add 0.3% of the mass of the composite protease of the cow bone to the cow bone cooking liquid for enzymolysis, and perform enzymolysis in an enzymolysis tank, wherein the mass ratio of the composite protease is neutral protease: alkaline protease: papain = 2:1:3, the enzymolysis temperature is 55°C, the enzyme reaction pH is 8.0, the enzymolysis time is 4h, and the enzyme is inactivated after the enzymolysis to obtain a cow bone enzymolysis solution; The preparation of the beef bone cooking liquid comprises crushing the beef bones, adding 10 times the mass volume of water and cooking at high temperature; cooling after cooking for 4 hours, and removing fat by centrifugation to obtain the beef bone cooking liquid; S120. The bovine bone hydrolysate was centrifuged to obtain the supernatant of the bovine bone hydrolysate, and the bovine bone hydrolysate was subjected to membrane separation with a molecular weight cutoff of 3000Da; S130. The membrane separation liquid was separated and purified by passing through a 20 mm × 100 mm Sephadex G10 gel chromatography column, the elution solvent was deionized water, the elution flow rate was 0.8 mL / min, the absorbance was detected at 280 nm, and the third elution peak was collected; S140. Further purification was performed using a C18 chromatographic column, wherein mobile phase A was water containing 0.05% trifluoroacetic acid by volume, mobile phase B was acetonitrile, and gradient elution conditions were: 0-5 min, 10% B, 5-15 min, 10%-15% B, 15-25 min, 15% B-25% B, 25-40 min, 25% B-35% B, the flow rate was 1.0 mL / min, and the elution peaks with retention times of 8.23-9.84 min were collected to obtain the yak bone collagen oligopeptide.

3. The preparation method according to claim 2, characterized in that: In step S110, after enzymatic hydrolysis, the temperature is raised to 80°C to inactivate the enzyme for 15 minutes to obtain a bovine bone enzymatic hydrolysate.

4. The preparation method according to claim 2, characterized in that: In step S120, the centrifugal speed is 8000 rev / min, and the centrifugation is performed for 10 minutes.

5. The preparation method according to claim 2, characterized in that: In step S140, the yak bone collagen oligopeptide is concentrated and then freeze-dried to obtain yak bone collagen oligopeptide powder.

6. A bone-targeting oligopeptide complex, characterized in that: It is a phytic acid conjugate of a cyclodextrin-enclosed oligopeptide, wherein the oligopeptide is the yak collagen oligopeptide according to claim 1.

7. A method for preparing the bone-targeting oligopeptide complex according to claim 6, characterized in that: include S210. The aqueous solution of the oligopeptide and the aqueous solution of phytic acid are mixed to obtain a mixture of oligopeptide and phytic acid; S220. adding hydrochloric acid to the oligopeptide and phytic acid mixture and stirring until dissolved to obtain a mixture containing hydrochloric acid, wherein the molar ratio of the oligopeptide, phytic acid and hydrochloric acid is 3-6:1:10; S230. The mixture containing hydrochloric acid is reacted in an oven at 110 to 120° C. for 8 to 12 hours; S240. Cleaning the reactants; S250. Dropping the aqueous solution of cyclodextrin into the aqueous solution of the reactant to obtain a bone-targeted complex of the oligopeptide, wherein the mass ratio of the reactant to the cyclodextrin is 1 to 3:

1.

8. The method for preparing the bone-targeted oligopeptide complex according to claim 7, characterized in that: The molar concentration of the aqueous solution of the oligopeptide in step 210 is 20-40 mmoL, and the molar concentration of the aqueous solution of phytic acid is 10-20 mmoL.

9. The method for preparing the bone-targeted oligopeptide complex according to claim 8, characterized in that: In step 210, the aqueous solution of oligopeptides is a deionized aqueous solution of oligopeptides, and the aqueous solution of phytic acid is a deionized aqueous solution of phytic acid.

10. The method for preparing the bone-targeted oligopeptide complex according to claim 7, characterized in that: In step S220, the molar ratio of the oligopeptide, phytic acid and hydrochloric acid is 0.006:0.001:0.

01.

11. The method for preparing the bone-targeted oligopeptide complex according to claim 7, characterized in that: In step S230, the mixed solution containing hydrochloric acid is reacted in an oven at 120° C. for 12 hours.

12. The method for preparing the bone-targeted oligopeptide complex according to claim 7, characterized in that: Step S240: The reactant is washed at least once using 0.1 M HCl, 0.1 M NaOH and water in sequence.

13. The method for preparing the bone-targeted oligopeptide complex according to claim 7, characterized in that: In step S250, the mass volume ratio of the aqueous solution of the reactants is 1-5%, and the mass volume ratio of the aqueous solution of cyclodextrin is 1-5%.

14. The method for preparing the bone-targeted oligopeptide complex according to claim 13, characterized in that: In step S250, the aqueous solution of the reactants is a deionized aqueous solution of the reactants, and the aqueous solution of cyclodextrin is a deionized aqueous solution of cyclodextrin.

15. The method for preparing the bone-targeted oligopeptide complex according to claim 13, characterized in that: In step S250, the mass volume ratio of the aqueous solution of the reactants is 5%, and the mass volume ratio of the aqueous solution of cyclodextrin is 5%.

16. A bone-targeting oligopeptide complex, characterized in that: Prepared by the method according to any one of claims 7 to 15.

17. Use of the yak collagen oligopeptide according to claim 1, the bone-targeted complex of the oligopeptide according to claim 6, or the bone-targeted complex of the oligopeptide according to claim 16 in the preparation of a drug for promoting osteocyte proliferation.

18. Use of the yak collagen oligopeptide according to claim 1, the bone-targeted complex of the oligopeptide according to claim 6, or the bone-targeted complex of the oligopeptide according to claim 16 in the preparation of a drug for inhibiting osteoclasts.

19. Use of the yak collagen oligopeptide according to claim 1, the bone-targeted complex of the oligopeptide according to claim 6, or the bone-targeted complex of the oligopeptide according to claim 16 in the preparation of a drug for treating osteoporosis.

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