Glycosylated giant salamander skin collagen curcumin hydrogel bead as well as preparation method and application thereof
Curcumin hydrogel beads were prepared by glycosylated giant salamander skin collagen, and the gel network technology was used to improve the load rate and biological allowable rate of curcumin, which solved the low solubility, poor stability and absorption of curcumin in actual applications. The problem of low rate achieves the orientation, stable release and anti-inflammatory effects of curcumin in the intestine.
Patent Information
- Application Number
- CN202411563257.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-05
- Publication Date
- 2025-02-07
AI Technical Summary
In actual applications, curcumin has problems such as low solubility, poor chemical stability and low absorption, which leads to its low bioavailability, limiting its wide application in the food and pharmaceutical industries.
Curcumin hydrogel beads were prepared by glycosylation of modified giant salamander skin collagen, and glycosylated proteins and sodium alginate-chitosan form a gel network to increase the load rate and biological allowable rate of curcumin. and release it in the intestine direction and stably.
It achieves high loading and stability of curcumin, improves its biological allowable rate, promotes the directional release and anti-inflammatory effect of curcumin in the intestine, and has potential application value for the treatment of colitis.
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Figure CN119385923A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of hydrogel preparation, and in particular relates to glycosylated giant salamander skin collagen curcumin hydrogel beads and a preparation method and application thereof. Background Art
[0002] The giant salamander, commonly known as the baby fish, is called the "living ginseng in the water" by aquatic experts. It is a tailed amphibian with rich nutritional value. A total of 17 amino acids were detected in the skin of the giant salamander, with a total amino acid content of 21.86%, of which 7 essential amino acids accounted for 5.91%. The essential amino acid index (EAAI) was 81.65, with a rich amino acid composition and high nutritional and functional value.
[0003] Glycosylation, as an important post-protein modification method, plays a vital role in regulating the physicochemical properties and biological functions of proteins, and can improve the structural stability and biological activity of proteins.
[0004] Curcumin, as a naturally occurring active substance, is known for its significant anti-inflammatory effects. However, in actual applications, curcumin has exposed some limitations, including low solubility, poor chemical stability, and low absorption rate in the body. These defects together lead to the low bioavailability of curcumin, which to some extent restricts its widespread application in the food and pharmaceutical industries. Summary of the invention
[0005] In view of this, the purpose of the present invention is to provide a glycosylated giant salamander skin collagen curcumin hydrogel beads and a preparation method and application thereof, prepare the hydrogel by glycosylation-modified collagen, use glycosylation as a means to improve the physicochemical properties of the protein, and then improve the gel network structure, improve the shortcomings of curcumin such as low solubility, poor stability, and low absorption rate, increase the loading rate and bioavailability of curcumin, so that it can be released in a directed and stable manner in the intestine, and curcumin and glycosylated protein work together to reduce the level of inflammation and promote the recovery of colitis.
[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:
[0007] The present invention provides a method for preparing glycosylated giant salamander skin collagen curcumin hydrogel beads, comprising the following steps:
[0008] The glycosylated giant salamander skin collagen solution was adjusted to pH 11-13, and then curcumin was added, and the pH was adjusted to neutral, and the supernatant was collected by centrifugation;
[0009] The supernatant and the sodium alginate solution are mixed to obtain a mixed solution, and the mixed solution is dropped into a chitosan solution and soaked for 1 to 3 hours to obtain glycosylated giant salamander skin collagen curcumin hydrogel beads.
[0010] Preferably, the concentration of the glycosylated giant salamander skin collagen solution is 0.005-0.015 g / mL, and the concentration of curcumin in the glycosylated giant salamander skin collagen solution is 0.005-0.015 mg / mL.
[0011] Preferably, the sodium alginate solution consists of the following ingredients: 20-30 g / L sodium alginate, 15-25 g / L polyvinyl alcohol, 5-15 g / L glycerol and the balance water.
[0012] Preferably, the volume ratio of the supernatant to the sodium alginate solution is 1:1-3.
[0013] Preferably, the chitosan solution comprises acetic acid solution, calcium chloride and chitosan, the volume mass ratio of the acetic acid solution, calcium chloride and chitosan is 80-120 mL:1-3 g:0.5-1.5 g, and the volume fraction of the acetic acid solution is 0.5-1.5%.
[0014] Preferably, the glycosylated giant salamander skin collagen solution is prepared from glycosylated giant salamander skin collagen, and the preparation method of the glycosylated giant salamander skin collagen comprises the following steps:
[0015] D-xylose and choline chloride are mixed to obtain a natural low eutectic solvent, and the giant salamander skin collagen, the natural low eutectic solvent and water are mixed and ultrasonically treated in a water bath, and then dialyzed and dried to obtain glycosylated giant salamander skin collagen.
[0016] Preferably, the molar ratio of D-xylose to choline chloride is 0.5-1.5:0.5-1.5;
[0017] The mass ratio of the giant salamander skin collagen, the natural low eutectic solvent and water is 1g:45-55g:10g.
[0018] Preferably, the temperature of the ultrasonic water bath is 60-90° C., the power of the ultrasonic water bath is 1000W-1500W, and the time of the ultrasonic water bath is 50-90 min.
[0019] The present invention also provides glycosylated giant salamander skin collagen curcumin hydrogel beads prepared by the preparation method.
[0020] The present invention also provides the glycosylated giant salamander skin collagen curcumin hydrogel beads prepared by the preparation method or the use of the glycosylated giant salamander skin collagen curcumin hydrogel beads in the preparation of drugs for treating colitis.
[0021] Compared with the prior art, the present invention has the following beneficial effects:
[0022] The invention provides a method for preparing glycosylated giant salamander skin collagen curcumin hydrogel beads. The prepared hydrogel beads can achieve a high loading amount of curcumin and maintain high stability.
[0023] The present invention utilizes glycosylated protein in combination with sodium alginate-chitosan to form a gel network, effectively improving the bioavailability, enabling its directed and stable release in the intestine, effectively reducing the level of inflammation, and providing certain technical support for the treatment and rehabilitation of colitis.
[0024] The invention can regulate the glycosylation level of giant salamander skin collagen protein and change the structural system of the protein, and can synergize with curcumin to regulate the relevant inflammatory response in the body and jointly promote the recovery of colitis.
[0025] The preparation method of the invention is simple, stable, and has a mature process, is suitable for industrial mass production, and has great industrial value. BRIEF DESCRIPTION OF THE DRAWINGS
[0026] Figure 1 It is an X-ray diffraction spectrum analysis diagram of the glycosylated giant salamander skin collagen curcumin hydrogel beads and curcumin (Cur) prepared in Example 1 and Comparative Examples 1 to 3;
[0027] Figure 2 It is a fluorescence microscopic analysis diagram of the glycosylated giant salamander skin collagen curcumin hydrogel beads prepared in Example 1 and Comparative Example 3;
[0028] Figure 3 is a graph showing the loading rate analysis results of the glycosylated giant salamander skin collagen curcumin hydrogel beads prepared in Example 1 and Comparative Example 3;
[0029] Figure 4 It is a result diagram of the bioavailability of glycosylated giant salamander skin collagen curcumin hydrogel beads and curcumin (Cur) prepared in Example 1 and Comparative Examples 1 to 3;
[0030] Figure 5 This is a graph showing the colon length results of mice with acute colitis induced by dextran sulfate sodium (DSS) after being treated with glycosylated giant salamander skin collagen curcumin hydrogel beads prepared in Example 1 and Comparative Examples 1 to 3;
[0031] Figure 6 This is a graph showing the results of treating IL-1β levels in mice with acute colitis induced by dextran sulfate sodium (DSS) using glycosylated giant salamander skin collagen curcumin hydrogel beads prepared in Example 1 and Comparative Examples 1 to 3;
[0032] Figure 7 This is a graph showing the results of treating IL-6 levels in mice with acute colitis induced by dextran sulfate sodium (DSS) using glycosylated giant salamander skin collagen curcumin hydrogel beads prepared in Example 1 and Comparative Examples 1 to 3;
[0033] Figure 8 This is a graph showing the results of treating TNF-α levels in mice with acute colitis induced by dextran sulfate sodium (DSS) using glycosylated giant salamander skin collagen curcumin hydrogel beads prepared in Example 1 and Comparative Examples 1 to 3;
[0034] Fig. 9 This is a graph showing the results of the treatment of mice with acute colitis induced by dextran sulfate sodium (DSS) using glycosylated giant salamander skin collagen curcumin hydrogel beads prepared in Example 1 and Comparative Examples 1 to 3;
[0035] Fig.10 This is a graph showing the results of treating the acute colitis mice induced by dextran sulfate sodium (DSS) using glycosylated giant salamander skin collagen curcumin hydrogel beads prepared in Example 1 and Comparative Examples 1 to 3;
[0036] Fig.11 This is a graph showing the results of treating mice with acute colitis induced by dextran sulfate sodium (DSS) using glycosylated giant salamander skin collagen curcumin hydrogel beads prepared in Example 1 and Comparative Examples 1 to 3;
[0037] Fig.12 This is a graph showing the results of treating mice with acute colitis induced by dextran sulfate sodium (DSS) using glycosylated giant salamander skin collagen curcumin hydrogel beads prepared in Example 1 and Comparative Examples 1 to 3;
[0038] Fig.13 This is a graph showing the results of the treatment of mice with acute colitis induced by dextran sulfate sodium (DSS) using glycosylated giant salamander skin collagen curcumin hydrogel beads prepared in Example 1 and Comparative Examples 1 to 3;
[0039] Fig.14 This is a graph showing the results of treating CAT content in mice with acute colitis induced by dextran sodium sulfate (DSS) using glycosylated giant salamander skin collagen curcumin hydrogel beads prepared in Example 1 and Comparative Examples 1 to 3. DETAILED DESCRIPTION
[0040] The present invention provides a method for preparing glycosylated giant salamander skin collagen curcumin hydrogel beads, comprising the following steps:
[0041] The glycosylated giant salamander skin collagen solution was adjusted to pH 11-13, and then curcumin was added, and the pH was adjusted to neutral, and the supernatant was collected by centrifugation;
[0042] The supernatant and the sodium alginate solution are mixed to obtain a mixed solution, and the mixed solution is dropped into a chitosan solution and soaked for 1 to 3 hours to obtain glycosylated giant salamander skin collagen curcumin hydrogel beads.
[0043] In the present invention, the glycosylated giant salamander skin collagen solution is prepared from glycosylated giant salamander skin collagen, and the preparation method of the glycosylated giant salamander skin collagen comprises the following steps:
[0044] D-xylose and choline chloride are mixed to obtain a natural low eutectic solvent, and the giant salamander skin collagen, the natural low eutectic solvent and water are mixed and ultrasonically treated in a water bath, and then dialyzed and dried to obtain glycosylated giant salamander skin collagen.
[0045] In the present invention, the molar ratio of D-xylose to choline chloride is preferably 0.5-1.5:0.5-1.5, and more preferably 0.8-1.2:0.8-1.2; the D-xylose and choline chloride are stirred when mixed, and the stirring speed is preferably 150-250rpm, and more preferably 180-220rpm; the temperature during stirring is preferably 85-95°C, and more preferably 88-92°C; the stirring time is preferably 2.5-5.5h, and more preferably 3-5h. The mass ratio of the giant salamander skin collagen, the natural low eutectic solvent and water is preferably 1g:45-55g:10g, and more preferably 1g:48-52g:10g; the temperature of the ultrasonic water bath is preferably 60-90°C, and more preferably 70-80°C; the power of the ultrasonic water bath is preferably 1000W-1500W, and more preferably 1100W-1400W; the time of the ultrasonic water bath is preferably 50-90min, and more preferably 60-80min; the dialysis is preferably carried out using a dialysis bag, and the molecular weight cutoff of the dialysis bag is preferably 250-350Da, and more preferably 280-320Da; the dialysis time is preferably 36-60h, and more preferably 42-5 2h; the dialysis is followed by centrifugation to obtain a supernatant, and the supernatant is dried to obtain glycosylated giant salamander skin collagen; the centrifugation temperature is preferably 3-5°C, and more preferably 4°C; the centrifugation speed is preferably 10000-14000rpm, and more preferably 11000-13000rpm; the centrifugation time is preferably 5-15min, and more preferably 8-12min; the drying method is preferably vacuum freeze drying, and the vacuum freeze drying is preferably pre-frozen at -90--70°C for 1-3h, and then dried at 0kPa, 15-25°C for 42-54h, and more preferably pre-frozen at -85--75°C for 1.5-2.5h, and then dried at 0kPa, 18-22°C for 45-50h. By regulating the glycosylation level of giant salamander skin collagen protein and changing the protein structure system, the inflammatory response in the intestine can be regulated, and it can work synergistically with curcumin to regulate the level of pro-inflammatory or anti-inflammatory factors in the body, thereby regulating the degree and persistence of inflammation, and improving or alleviating the inflammatory response. In addition, compared with other proteins, pepsin will not destroy its triple helix structure, but the molecular weight will be reduced, giving it better biological activity in the stomach, which is why it can work synergistically with curcumin to regulate the level of inflammation compared to other proteins.
[0046] In the present invention, the method for preparing giant salamander skin collagen comprises the following steps:
[0047] Oxalic acid and choline chloride are mixed to obtain a natural low eutectic solvent. Giant salamander skin and the natural low eutectic solvent are mixed and then placed in a water bath. The mixture is dialyzed and dried to obtain giant salamander skin collagen.
[0048] In the present invention, the molar ratio of oxalic acid to choline chloride is preferably 0.5-1.5:0.5-1.5, more preferably 0.8-1.2:0.8-1.2; the oxalic acid and choline chloride are stirred when mixed, and the stirring speed is preferably 150-250rpm, more preferably 180-220rpm; the temperature during stirring is preferably 85-95°C, more preferably 88-92°C; the stirring time is preferably 2.5-5.5h, more preferably 3-5h. The giant salamander skin is soaked in NaCl solution and isopropanol solution in sequence before mixing with the natural low eutectic solvent, pulped after soaking, and freeze-dried; the concentration of the NaCl solution is preferably 0.05-0.15mol / mL; the mass concentration of the isopropanol solution is 5-15%; the soaking time of the NaCl solution and the isopropanol solution is preferably 18-30h, more preferably 22-28h; the freeze-drying is preferably vacuum freeze-drying, and the vacuum freeze-drying is preferably first at -90~ -70 ℃ pre-freeze 1 ~ 3h, then 0kPa, 15 ~ 25 ℃ drying 42 ~ 54h, more preferably -85 ~ -75 ℃ pre-freeze 1.5 ~ 2.5h, then 0kPa, 18 ~ 22 ℃ drying 45 ~ 50h; the mass ratio of the giant salamander skin and the natural low eutectic solvent is preferably 1g: 15 ~ 35g, more preferably 1g: 20 ~ 30g; the temperature of the water bath is preferably 50 ~ 80 ℃, more preferably 55 ~ 75 ℃; the water bath time is preferably 3h ~ 7 h, more preferably 4 to 6 h; the dialysis is preferably performed using a dialysis bag, the molecular weight cutoff of the dialysis bag is preferably 250 to 350 Da, more preferably 280 to 320 Da; the dialysis time is preferably 36 to 60 h, more preferably 42 to 52 h; the dialysis is centrifuged to obtain a supernatant, and the supernatant is dried to obtain giant salamander skin collagen; the centrifugal speed is preferably 10000 to 14000 rpm, more preferably 11000 to 13000 rpm ; The centrifugation temperature is preferably 3-5°C, more preferably 4°C; the centrifugation time is preferably 5-15 min, more preferably 8-12 min; the drying method is preferably vacuum freeze drying, and the vacuum freeze drying is preferably pre-frozen at -90-70°C for 1-3 h, and then dried at 0 kPa, 15-25°C for 42-54 h, and more preferably pre-frozen at -85-75°C for 1.5-2.5 h, and then dried at 0 kPa, 18-22°C for 45-50 h.
[0049] In the present invention, after adjusting the pH of the glycosylated giant salamander skin collagen solution to 11-13, curcumin is added, the pH is adjusted to neutral, and the supernatant is taken by centrifugation. The concentration of the glycosylated giant salamander skin collagen solution is preferably 0.005-0.015 g / mL, and more preferably 0.008-0.012 g / mL; the concentration of curcumin in the glycosylated giant salamander skin collagen solution is preferably 0.005-0.015 mg / mL, and more preferably 0.008-0.012 mg / mL; the centrifugal speed is preferably 10000-14000 rpm, and more preferably 11000-13000 rpm; the centrifugal temperature is preferably 3-5°C, and more preferably 4°C; the centrifugal time is preferably 5-15 min, and more preferably 8-12 min.
[0050] In the present invention, the supernatant and the sodium alginate solution are mixed to obtain a mixed solution, and the mixed solution is dropped into a chitosan solution and soaked for 1 to 3 hours to obtain glycosylated giant salamander skin collagen curcumin hydrogel beads. The sodium alginate solution is composed of the following ingredients: 20-30 g / L of sodium alginate, 15-25 g / L of polyvinyl alcohol, 5-15 g / L of glycerol and the balance of water; the concentration of the sodium alginate is preferably 22-28 g / L; the concentration of the polyvinyl alcohol is preferably 18-22 g / L; the concentration of the glycerol is preferably 8-12 g / L; the volume ratio of the supernatant to the sodium alginate solution is preferably 1:1-3, and more preferably 1:2; the chitosan solution comprises acetic acid solution, calcium chloride and chitosan, and the volume mass ratio of the acetic acid solution, calcium chloride and chitosan is preferably 80-120 mL: 1-3 g: 0.5-1.5 g, and more preferably 90-110 mL: 1.5-2.5 g: 0.8-1.2 g; the volume fraction of the acetic acid solution is preferably 0.5-1.5%, and more preferably 0.8-1.2%; the soaking time is preferably 1.5-2.5 h.
[0051] The present invention also provides glycosylated giant salamander skin collagen curcumin hydrogel beads prepared by the preparation method.
[0052] The present invention also provides the glycosylated giant salamander skin collagen curcumin hydrogel beads prepared by the preparation method or the use of the glycosylated giant salamander skin collagen curcumin hydrogel beads in the preparation of a drug for treating colitis. In the present invention, the glycosylated giant salamander skin collagen curcumin hydrogel beads reduce the level of pro-inflammatory factors and increase the level of anti-inflammatory factors, thereby promoting the recovery of colitis.
[0053] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.
[0054] Example 1
[0055] The skin of giant salamander was soaked in 0.1 mol / mL NaCl aqueous solution for 24 h and in isopropanol (1:10) (w / v) for 24 h, washed after each soaking, and finally passed through a pulper to make a homogenate and vacuum freeze-dried (pre-frozen at -80°C for 2 h, then dried at 0 kPa, 20°C for 48 h).
[0056] Anhydrous oxalic acid and choline chloride in a molar ratio of 1:1 were heated at 90°C, mixed and stirred at 200 rpm for 5 hours to obtain uniform and transparent NADES (natural low eutectic solvent), the fish skin freeze-dried powder was immersed in NADES (natural low eutectic solvent), heated in a water bath at 60°C for 5 hours, dialyzed with a dialysis bag with a molecular weight of 300 Da for 48 hours, centrifuged at 12000 rpm and 4°C for 10 minutes, and the supernatant was vacuum freeze-dried (pre-frozen at -80°C for 2 hours, and then dried at 0 kPa, 20°C for 48 hours) to obtain giant salamander skin collagen freeze-dried powder.
[0057] D-xylose and choline chloride in a molar ratio of 1:1 were placed in a beaker, the beaker was placed in a 90° C. water bath, and stirred at a speed of 200 rpm for 5 hours to obtain a uniform and transparent NADES (natural deep eutectic solvent).
[0058] The prepared giant salamander skin collagen, natural low eutectic solvent (NADES) and water were mixed evenly in a mass ratio of 1g:50g:10g, and heated at 1200w, 80℃, for glycosylation reaction for 70min. After the reaction, the giant salamander skin collagen glycosylation product solution was obtained, and dialyzed for 48h with a 300Da molecular weight dialysis bag at room temperature to remove excess glycosyl donors. The dialyzate was then vacuum freeze-dried (pre-frozen at -80℃ for 2h, and then dried at 0kPa, 20℃ for 48h) to obtain glycosylated giant salamander skin collagen freeze-dried powder.
[0059] The pH of the 0.01 g / ml protein solution was adjusted to 12 with NaOH solution and HCL solution, and curcumin was added and stirred to completely dissolve it. The concentration was 0.01 mg / ml, and the pH was adjusted to 7. Then, the solution was centrifuged at 12000 rpm and 4°C for 10 min, and the supernatant was taken to obtain a curcumin-protein solution.
[0060] Sodium alginate solution was prepared by adding 2.5 g of sodium alginate, 2 g of polyvinyl alcohol and 1 g of glycerol per 100 mL of water to obtain a sodium alginate mixed solution, which was then evenly mixed with the protein solution in a volume ratio of 2:1.
[0061] Add 1.5 g of CaCl2 and 1 g of chitosan to every 100 mL of 1% acetic acid solution to obtain an immersion solution. Use a syringe to drop the sodium alginate mixed solution into the immersion solution and soak for 2 hours to obtain glycosylated giant salamander skin collagen curcumin hydrogel beads. Wash the glycosylated giant salamander skin collagen curcumin hydrogel beads with distilled water and store them at 4°C away from light.
[0062] Example 2
[0063] The skin of giant salamander was soaked in 0.05 mol / mL NaCl aqueous solution for 18 h and in isopropanol (1:20) (w / v) for 18 h, washed after each soaking, and finally passed through a pulper to make a homogenate, and vacuum freeze-dried (pre-frozen at -90°C for 1 h, then dried at 0 kPa, 15°C for 42 h).
[0064] Anhydrous oxalic acid and choline chloride in a molar ratio of 0.5:1.5 were heated to 85°C, mixed and stirred at a speed of 150 rpm for 2.5 hours to obtain uniform and transparent NADES (natural low eutectic solvent), the fish skin freeze-dried powder was immersed in NADES (natural low eutectic solvent), heated in a water bath at 50°C for 7 hours, dialyzed with a dialysis bag with a molecular weight of 250 Da for 36 hours, centrifuged at 10000 rpm and 3°C for 5 minutes, and the supernatant was vacuum freeze-dried (pre-frozen at -90°C for 1 hour, and then dried at 0 kPa, 15°C for 42 hours) to obtain giant salamander skin collagen freeze-dried powder.
[0065] D-xylose and choline chloride in a molar ratio of 0.5:1.5 were placed in a beaker, the beaker was placed in a water bath at 85° C., and stirred at a speed of 150 rpm for 2.5 h to obtain a uniform and transparent NADES (natural deep eutectic solvent).
[0066] The prepared giant salamander skin collagen, natural low eutectic solvent (NADES) and water were mixed evenly in a mass ratio of 1g:45g:10g, and heated at 1000w, 60℃, for glycosylation reaction for 50min. After the reaction, the giant salamander skin collagen glycosylation product solution was obtained, and dialyzed for 48h with a 250Da molecular weight dialysis bag at room temperature to remove excess glycosyl donors. The dialyzate was then vacuum freeze-dried (pre-frozen at -90℃ for 1h, and then dried at 0kPa, 15℃ for 42h) to obtain glycosylated giant salamander skin collagen freeze-dried powder.
[0067] The pH of the 0.005 g / ml protein solution was adjusted to 11 with NaOH solution and HCL solution, and curcumin was added and stirred to completely dissolve it to a concentration of 0.005 mg / ml. The pH was adjusted to 7, and then centrifuged at 10000 rpm and 3°C for 5 min. The supernatant was taken to obtain a curcumin-protein solution.
[0068] Sodium alginate solution was prepared by adding 2 g of sodium alginate, 1.5 g of polyvinyl alcohol and 0.5 g of glycerol per 100 mL of water to obtain a sodium alginate mixed solution, which was then evenly mixed with the protein solution in a volume ratio of 1:1.
[0069] Add 1 g of CaCl2 and 0.5 g of chitosan to every 80 mL of 0.5% acetic acid solution to obtain an immersion solution. Use a syringe to drop the sodium alginate mixed solution into the immersion solution and soak for 1 hour to obtain glycosylated giant salamander skin collagen curcumin hydrogel beads. Wash the glycosylated giant salamander skin collagen curcumin hydrogel beads with distilled water and store them at 4°C away from light.
[0070] Example 3
[0071] The skin of giant salamander was soaked in 0.15 mol / mL NaCl aqueous solution for 30 h and in isopropanol (3:20) (w / v) for 30 h, washed after each soaking, and finally passed through a pulper to make a homogenate, and then vacuum freeze-dried (pre-frozen at -85°C for 3 h, and then dried at 0 kPa, 25°C for 54 h).
[0072] Anhydrous oxalic acid and choline chloride in a molar ratio of 1.5:0.5 were heated at 95°C, mixed and stirred at a speed of 250 rpm for 5.5 hours to obtain uniform and transparent NADES (natural low eutectic solvent), the fish skin freeze-dried powder was immersed in NADES (natural low eutectic solvent), heated in a water bath at 80°C for 3 hours, dialyzed with a dialysis bag with a molecular weight of 350 Da for 60 hours, centrifuged at 14000 rpm and a temperature of 5°C for 15 minutes, and the supernatant was vacuum freeze-dried (pre-frozen at -85°C for 3 hours, and then dried at 0 kPa, 25°C for 54 hours) to obtain giant salamander skin collagen freeze-dried powder.
[0073] D-xylose and choline chloride in a molar ratio of 1.5:0.5 were placed in a beaker, the beaker was placed in a 95° C. water bath, and stirred at 250 rpm for 5.5 h to obtain a uniform and transparent NADES (natural deep eutectic solvent).
[0074] The prepared giant salamander skin collagen, natural low eutectic solvent (NADES) and water were mixed evenly in a mass ratio of 1g:55g:10g, and heated at 1500w, 90℃, for glycosylation reaction for 90min. After the reaction, the giant salamander skin collagen glycosylation product solution was obtained, and dialyzed for 48h with a 350Da molecular weight dialysis bag at room temperature to remove excess glycosyl donors. The dialyzate was then vacuum freeze-dried (pre-frozen at -85℃ for 3h, and then dried at 0kPa, 25℃ for 54h) to obtain glycosylated giant salamander skin collagen freeze-dried powder.
[0075] The pH of the 0.015 g / ml protein solution was adjusted to 13 with NaOH solution and HCL solution, and curcumin was added and stirred to completely dissolve it. The concentration was 0.015 mg / ml, and the pH was adjusted to 7. Then, the solution was centrifuged at 14000 rpm and 5°C for 15 min, and the supernatant was taken to obtain a curcumin-protein solution.
[0076] Sodium alginate solution was prepared by adding 3 g of sodium alginate, 2.5 g of polyvinyl alcohol and 1.5 g of glycerol per 100 mL of water to obtain a sodium alginate mixed solution, which was then evenly mixed with the protein solution in a volume ratio of 3:1.
[0077] Add 1.5 g of CaCl2 and 1 g of chitosan to every 100 mL of 1.5% acetic acid solution to obtain an immersion solution. Use a syringe to drop the sodium alginate mixed solution into the immersion solution and soak for 3 hours to obtain glycosylated giant salamander skin collagen curcumin hydrogel beads. Wash the glycosylated giant salamander skin collagen curcumin hydrogel beads with distilled water and store them at 4°C away from light.
[0078] Comparative Example 1
[0079] The difference from Example 1 is that there is no glycosylation treatment and no process of adding curcumin.
[0080] Comparative Example 2
[0081] The difference from Example 1 is that there is no process of adding curcumin.
[0082] Comparative Example 3
[0083] The difference from Example 1 is that there is no glycosylation process.
[0084] Experimental Example 1
[0085] X-ray diffraction energy spectrum analysis (XRD) was performed on the glycosylated giant salamander skin collagen curcumin hydrogel beads and curcumin (Cur) prepared in Example 1 and Comparative Examples 1 to 3.
[0086] Experimental results: Figure 1 As shown. Figure 1 It can be seen that neither Example 1 nor Comparative Group 3 has the characteristic peak of curcumin, indicating that curcumin is completely embedded in the gel.
[0087] Experimental Example 2
[0088] The glycosylated giant salamander skin collagen curcumin hydrogel beads prepared in Example 1 and Comparative Example 3 were subjected to fluorescence microscopy analysis and loading rate analysis.
[0089] Experimental results: Figure 2 , 3 As shown, the curcumin loading rate of Example 1 is significantly higher.
[0090] The glycosylated giant salamander skin collagen curcumin hydrogel beads prepared in Example 1 and Comparative Examples 1 to 3 were subjected to in vitro simulated digestion with curcumin (Cur), and the bioavailability of each group at each time period was analyzed.
[0091] Experimental results: Figure 4 As shown, as time goes by, the bioavailability of Example 1 is higher.
[0092] from Figures 2 to 4 It can be seen that compared with Comparative Example 3, the curcumin loading rate of Example 1 is higher and the bioavailability is high. The glycosylated giant salamander skin collagen curcumin hydrogel beads prepared in Example 1 can release more curcumin into the intestine in a targeted manner.
[0093] Experimental Example 3
[0094] The glycosylated giant salamander skin collagen curcumin hydrogel beads prepared in Example 1 and Comparative Examples 1 to 3 were used to treat the acute colitis mouse model induced by dextran sulfate sodium (DSS). The acute colitis mouse model was established by allowing mice (BALB / c, male, 7 weeks old, purchased from Jiangsu Jicui Pharmaceutical Co., Ltd.) to freely drink 3% DSS solution for 7 consecutive days, and the colon length, IL-1β level, IL-6 level, TNF-α level, and antioxidant enzyme activities such as DAO, MPO, MDA, GSH-Px, SOD, and CAT were evaluated.
[0095] Experimental results: Figures 5 to 14 shown.
[0096] from Figure 5 It can be seen that the colon length can reflect the severity of colitis, and the colon length of Example 1 is longer than that of Comparative Examples 1 to 3, and is close to that of the blank group.
[0097] from Figures 6 to 14 It can be seen that compared with comparative examples 1 to 3, the negative control and the positive control, Example 1 has lower IL-1β level, IL-6 level, TNF-α level, DAO, MPO and MDA level, which can effectively reduce the level of pro-inflammatory factors, and the higher levels of GSH-Px, SOD and CAT increase the level of anti-inflammatory factors and promote the recovery of colitis.
[0098] In particular, Comparative Example 2 of the present invention has a better promoting effect on the recovery of colitis compared with Comparative Example 1, and Example 1 has a better promoting effect on the recovery of colitis compared with Comparative Example 3. That is to say, the glycosylated fish skin collagen of the present invention can promote the recovery of colitis.
[0099] It can be seen from the above embodiments and experimental examples that the glycosylated giant salamander skin collagen curcumin hydrogel beads prepared by the present invention can achieve a high loading amount of curcumin, maintain a high stability, and have a high bioavailability, and can release more curcumin into the intestine in a targeted manner, effectively reducing the level of pro-inflammatory factors, increasing the level of anti-inflammatory factors, and promoting the recovery of colitis.
[0100] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.
Claims
1. A method for preparing glycosylated giant salamander skin collagen curcumin hydrogel beads, characterized in that: The following steps are involved: The glycosylated giant salamander skin collagen solution was adjusted to pH 11-13, and then curcumin was added, and the pH was adjusted to neutral, and the supernatant was collected by centrifugation; The supernatant and the sodium alginate solution are mixed to obtain a mixed solution, and the mixed solution is dropped into a chitosan solution and soaked for 1 to 3 hours to obtain glycosylated giant salamander skin collagen curcumin hydrogel beads.
2. The preparation method according to claim 1, characterized in that: The concentration of the glycosylated giant salamander skin collagen solution is 0.005-0.015 g / mL, and the concentration of the curcumin in the glycosylated giant salamander skin collagen solution is 0.005-0.015 mg / mL.
3. The preparation method according to claim 1, characterized in that: The sodium alginate solution consists of the following ingredients: 20-30 g / L of sodium alginate, 15-25 g / L of polyvinyl alcohol, 5-15 g / L of glycerol and the balance of water.
4. The preparation method according to claim 1, characterized in that: The volume ratio of the supernatant to the sodium alginate solution is 1:1-3.
5. The preparation method according to claim 1, characterized in that: The chitosan solution comprises acetic acid solution, calcium chloride and chitosan, wherein the volume mass ratio of the acetic acid solution, calcium chloride and chitosan is 80-120 mL: 1-3 g: 0.5-1.5 g, and the volume fraction of the acetic acid solution is 0.5-1.5%.
6. The preparation method according to claim 1, characterized in that: The glycosylated giant salamander skin collagen solution is prepared from glycosylated giant salamander skin collagen, and the preparation method of the glycosylated giant salamander skin collagen comprises the following steps: D-xylose and choline chloride are mixed to obtain a natural low eutectic solvent, and the giant salamander skin collagen, the natural low eutectic solvent and water are mixed and ultrasonically treated in a water bath, and then dialyzed and dried to obtain glycosylated giant salamander skin collagen.
7. The preparation method according to claim 6, characterized in that: The molar ratio of D-xylose to choline chloride is 0.5-1.5:0.5-1.5; The mass ratio of the giant salamander skin collagen, the natural low eutectic solvent and water is 1g:45-55g:10g.
8. The preparation method according to claim 6, characterized in that: The temperature of the ultrasonic water bath is 60-90° C., the power of the ultrasonic water bath is 1000W-1500W, and the time of the ultrasonic water bath is 50-90 minutes.
9. Glycosylated giant salamander skin collagen curcumin hydrogel beads prepared by the preparation method according to any one of claims 1 to 8.
10. Use of the glycosylated giant salamander skin collagen curcumin hydrogel beads prepared by the preparation method according to any one of claims 1 to 8 or the glycosylated giant salamander skin collagen curcumin hydrogel beads according to claim 9 in the preparation of drugs for treating colitis.