Polypeptide hydrogel based on oxidized ganoderan and application

Abstract

The invention discloses polypeptide hydrogel based on oxidized ganoderan and application. The polypeptide hydrogel is simple in preparation method, rapid in gelling, good in stability and capable of resisting oxidation activity and inhibiting tyrosinase activity. The polypeptide hydrogel provides a physical barrier for the bioactive peptide, so that the influence of the gastrointestinal tract environment is overcome, 71% of polypeptide is released in the intestinal tract, the bioavailability of the peptide is improved, and the problem that the use condition of the polypeptide is limited is effectively solved.

Description

(I) Technical field

[0001] The invention relates to a polypeptide hydrogel based on oxidized ganoderma lucidum polysaccharide and application thereof. (II) Background technology

[0002] Pearl peptides are peptides extracted from pearls with multiple biological activities (antibacterial, antioxidant, anti-tyrosinase, etc.) (Jingying Pei, et al. Extraction, purification, bioactivities and application of matrix proteins from pearl powder and nacre powder: A review. Frontiers in Bioengineering and Biotechnology, 2021). However, proteins and peptides are very sensitive to the environment. Changes in pH and environmental ion concentrations will denature proteins and peptides and inactivate them (Jun Ye, Targeted release of soybean peptide from CMC / PVA hydrogels in simulated dintestinal fluid and their pharmacokinetics, Carbohydrate Polymers, 2023). The drastic pH changes and high ionic strength in the gastrointestinal tract make the bioavailability of peptides absorbed orally extremely low.

[0003] Hydrogel is a soft material with a three-dimensional network structure, and polysaccharide-based hydrogel has excellent biocompatibility, biodegradability, and water retention. Polysaccharides are easily available and have low toxicity, making them ideal carriers for oral small molecule active peptides (Arshied Manzoor, Recent insights into polysaccharide-based hydrogels and their potential applications in food sector: A review, International Journal of Biological Macromolecules, 2022). Active peptides can be encapsulated in hydrogels to avoid the effects of gastric acid, enzymes, etc. on peptides during gastrointestinal digestion.

[0004] Ganoderma lucidum is an edible fungus that can be used as both medicine and food. It has anti-cancer, anti-inflammatory, and antioxidant activities. Ganoderma lucidum polysaccharide is one of the main active ingredients of Ganoderma lucidum (Li Fang Zhu, Engineering of Ganoderma lucidum polysaccharide loaded polyvinyl alcohol nanofibers for biopharmaceutical delivery, Journal of Drug Delivery Science and Technology, 2019). However, the current research on Ganoderma lucidum polysaccharide mainly focuses on extraction and purification, activity verification, etc., while there are very few studies on the use of Ganoderma lucidum polysaccharide to prepare hydrogels and apply them to transport nutrients.

[0005] Sui Xiaoyu et al.'s patent "A method for preparing a ganoderma lucidum polysaccharide hydrogel and its application in diabetic wound repair" (CN117752851 A) shows that the ganoderma lucidum polysaccharide hydrogel prepared with ganoderma lucidum polysaccharide and carboxymethyl chitosan as raw materials, sodium alginate and cross-linking agent has a good effect in repairing wound damage. Animal experimental results show that the wound healing rate of the ganoderma lucidum polysaccharide hydrogel group reached 98.94±0.77% on the 21st day, which is higher than that of the CMC / SA group and the saline group. However, the hydrogel prepared by this method contains a cross-linking agent, which is highly toxic.

[0006] The patent "A galactosylated sodium alginate derivative gel microsphere and its preparation method and application" (CN202310710208) by Zhang Liming et al. shows that galactosylated sodium alginate derivative gel microsphere can be used as a carrier material to embed bovine serum albumin. When 5mg / ml bovine serum albumin solution is added, the embedding rate reaches 64.24%. In deionized water, the maximum cumulative release is reached on the 12th day, almost reaching 100%. However, the gelation of the gel takes 5-15 minutes, which is a long time. (III) Summary of the invention

[0007] The purpose of the present invention is to provide a polypeptide hydrogel based on oxidized Ganoderma lucidum polysaccharide and its application. The polypeptide hydrogel prepared by the method of the present invention has a simple preparation method and rapid gelation. The prepared hydrogel has certain antioxidant and anti-tyrosinase activities and shows good inhibitory effects on Escherichia coli and Staphylococcus aureus. At the same time, the hydrogel is targetedly released in the intestine to improve the bioavailability of oral peptides, effectively solving the problem that the polypeptides are easily inactivated by the gastrointestinal environment during the oral administration of bioactive peptides.

[0008] The technical solution adopted by the present invention is:

[0009] The present invention provides a polypeptide hydrogel based on oxidized ganoderma lucidum polysaccharide, and the polypeptide hydrogel is prepared according to the following method:

[0010] (1) Preparation of Oxidized Ganoderma Lucidum Polysaccharide

[0011] Add distilled water to ganoderma lucidum polysaccharide, stir until completely dissolved, add sodium periodate under light-proof conditions, react at room temperature in the dark for 5-10 hours (preferably 6 hours), add ethylene glycol and stir to terminate the reaction; put the reaction mixture into a dialysis bag and dialyze in pure water for 1-3 days (preferably 2 days), take the intercepted liquid and freeze-dry (preferably freeze-dry at -80°C for 48 hours) to obtain oxidized ganoderma lucidum polysaccharide;

[0012] (2) Preparation of polypeptide hydrogel

[0013] A polypeptide is added to a 2-10% carboxymethyl cellulose (CMCS) aqueous solution to obtain a polypeptide concentration of 0.05-1% to obtain a polypeptide solution; a 1-5% oxidized ganoderma lucidum polysaccharide aqueous solution is mixed with the polypeptide solution at a volume ratio of 1:0.5-2, and the mixture is allowed to stand at room temperature to form a gel to obtain a polypeptide hydrogel.

[0014] Furthermore, the ganoderma lucidum polysaccharide in step (1) is obtained by removing protein from ganoderma lucidum extract, and the polysaccharide mass content is 55-65%.

[0015] Furthermore, the step (1) of preparing the ganoderma polysaccharide is as follows: adding Sevage reagent to the ganoderma extract aqueous solution, mixing and shaking to remove the lower water layer and the denatured protein at the interface, repeating the process for multiple times until no denatured protein settles, and collecting the clarified polysaccharide solution; then adding anhydrous ethanol with a final volume concentration of 80%, standing at room temperature overnight, centrifuging, and collecting the precipitate; re-dissolving the precipitate with water and removing the organic solvent by rotary evaporation, and freeze-drying at -80°C for 48 hours to obtain the ganoderma polysaccharide; the Sevage reagent is a solution of chloroform and n-butanol in a volume ratio of 4:1, and the volume amount of the Sevage reagent is 1 / 3 of the volume of the ganoderma extract aqueous solution; the volume amount of distilled water in the ganoderma extract aqueous solution is 10 mL / g based on the mass of the ganoderma extract.

[0016] Furthermore, the volume amount of distilled water used in step (1) is 50-150 mL / g (preferably 100 mL / g) based on the mass of Ganoderma polysaccharide; the molar ratio of Ganoderma polysaccharide to sodium periodate is 1:0.1-1 (preferably 1:0.6); and the volume amount of ethylene glycol used is 1-10 mL / g (preferably 5 mL / g) based on the mass of Ganoderma polysaccharide.

[0017] Furthermore, the molecular weight cut-off of the dialysis bag in step (1) is 3.5 kDa.

[0018] Furthermore, in step (2), the mass concentration of the carboxymethyl cellulose (CMCS) aqueous solution is 5%, the mass concentration of the polypeptide is 0.1%, the mass concentration of the oxidized Ganoderma lucidum polysaccharide aqueous solution is 3%, and the oxidized Ganoderma lucidum polysaccharide aqueous solution and the polypeptide solution are mixed in a volume ratio of 1:1.

[0019] Furthermore, the polypeptide in step (2) includes pearl polypeptide, and the pearl polypeptide is prepared according to the method of patent application CN112390848A.

[0020] The present invention also provides an application of the polypeptide hydrogel in the preparation of a polypeptide intestinal sustained-release drug, wherein the drug can release 71% of the polypeptide in the intestinal tract, the drug dosage form includes a solid dosage form, and the administration method includes oral administration.

[0021] The present invention also provides an application of the polypeptide hydrogel in the preparation of antibacterial drugs, wherein the polypeptide in the hydrogel is a pearl polypeptide, and the antibacterial drug is a drug that inhibits the activity of Escherichia coli or Staphylococcus aureus.

[0022] Compared with the existing methods, the beneficial effects of the present invention are mainly reflected in:

[0023] (1) The preparation method of the polypeptide hydrogel of the present invention is simple and the gelation is rapid. The present invention increases the gelation speed by compounding oxidized Ganoderma lucidum polysaccharide with carboxymethyl chitosan.

[0024] (2) The polypeptide hydrogel of the present invention has good stability. The polypeptide hydrogel has a porous structure and has good mechanical properties, thermal stability and swelling characteristics.

[0025] (3) The polypeptide hydrogel of the present invention has antioxidant activity and inhibits tyrosinase activity; the scavenging rates of 1 mg / mL of Ganoderma lucidum polysaccharide hydrogel extract on DPPH, ABTS and hydroxyl free radicals are 25%, 30% and 48%, respectively, and the addition of pearl polypeptide further enhances its antioxidant capacity to 27%, 33% and 54%, respectively.

[0026] The 1 mg / mL pearl polypeptide hydrogel extract has an inhibition rate of 19% on tyrosinase, more than 99% on Escherichia coli, and more than 98% on Staphylococcus aureus.

[0027] (4) The polypeptide hydrogel of the present invention provides a physical barrier for the bioactive peptides, enabling them to overcome the influence of the gastrointestinal environment, allowing 71% of the polypeptides to be released in the intestine, thereby improving the bioavailability of the peptides and effectively solving the problems of restrictions on the use of polypeptides. (IV) Description of the drawings

[0028] Figure 1 , Photos of Ganoderma lucidum polysaccharide hydrogels with different oxidation degrees.

[0029] Figure 2 , antioxidant activity of Ganoderma lucidum polysaccharides with different oxidation degrees.

[0030] Figure 3, scanning electron micrographs of Ganoderma lucidum polysaccharide hydrogel and pearl polypeptide hydrogel with different volume ratios.

[0031] Figure 4 , infrared spectra of Ganoderma lucidum polysaccharide and oxidized Ganoderma lucidum polysaccharide.

[0032] Figure 5 , infrared spectra of Ganoderma lucidum polysaccharide hydrogel, pearl polypeptide hydrogel and CMCS.

[0033] Figure 6 , G'-G" curves of Ganoderma lucidum polysaccharide hydrogel and pearl polypeptide hydrogel.

[0034] Figure 7 , the swelling rate of Ganoderma lucidum polysaccharide hydrogel and pearl polypeptide hydrogel.

[0035] Figure 8 , release of pearl polypeptides from pearl polypeptide hydrogels by simulating gastrointestinal digestion in vitro.

[0036] Fig. 9 , antioxidant activity and anti-tyrosinase activity of Ganoderma lucidum polysaccharide hydrogel and pearl polypeptide hydrogel extracts.

[0037] Fig.10 , antibacterial activity of Ganoderma lucidum polysaccharide hydrogel and pearl polypeptide hydrogel. (V) Specific implementation methods

[0038] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0039] The ganoderma lucidum extract used in the present embodiment was purchased from Zhejiang Huihe Health Technology Co., Ltd., and the polysaccharide content was ≥ 10%. Pearl polypeptide was prepared according to the method of Example 3 of patent application CN112390848A.

[0040] The room temperature of the present invention is 25-30°C.

[0041] Example 1: Preparation and performance testing of Ganoderma lucidum polysaccharide hydrogel

[0042] 1. Preparation of Ganoderma lucidum polysaccharide hydrogel

[0043] (1) Preparation of Ganoderma lucidum polysaccharide

[0044] To a Ganoderma lucidum extract aqueous solution prepared by adding 40 g of Ganoderma lucidum extract to 400 mL of distilled water, add Sevage reagent (chloroform and n-butanol solution in a volume ratio of 4:1) in an amount of 1 / 3 of the volume of the Ganoderma lucidum extract aqueous solution, mix and shake, remove the lower water layer and the denatured protein at the junction, repeat several times until no denatured protein precipitates, and collect the clarified polysaccharide solution; then add anhydrous ethanol in a final concentration of 80% by volume, let stand at room temperature overnight, centrifuge, and collect the precipitate; after re-dissolving the precipitate with water, remove the organic solvent by rotary evaporation, and freeze-dry at -80°C for 48 hours to obtain 15 g of Ganoderma lucidum polysaccharide, which is recorded as GLP.

[0045] (2) Preparation of Oxidized Ganoderma Lucidum Polysaccharide

[0046] Weigh 1 g (0.0056 mol) of the Ganoderma lucidum polysaccharide sample prepared in step (1) and add 100 mL of distilled water, stirring until completely dissolved. Add 0.7 g (0.0032 mol) of sodium periodate under light-proof conditions, react at room temperature in the dark for 6 h, add 5 mL of ethylene glycol and stir for 30 min to terminate the reaction. The reaction mixture is placed in a dialysis bag (3.5 kDa) and dialyzed with pure water for 2 days. The intercepted liquid is freeze-dried at -80°C for 48 h to obtain 0.7 g of oxidized Ganoderma lucidum polysaccharide, recorded as OGLP.

[0047] (3) Preparation of Ganoderma lucidum polysaccharide hydrogel

[0048] Take oxidized Ganoderma lucidum polysaccharide and add water to prepare 3% oxidized Ganoderma lucidum polysaccharide aqueous solution, mix it with 5% carboxymethyl cellulose (CMCS) aqueous solution at a volume ratio of 1:1, and let it stand at room temperature to form a gel to obtain Ganoderma lucidum polysaccharide hydrogel, which is recorded as G-GLP2. Figure 1 .

[0049] 2. Performance test of Ganoderma lucidum polysaccharide hydrogel

[0050] (1) Gelation time

[0051] The gelation time was determined by the vial inversion method (Wang Bulei. Preparation and characterization of Bletilla striata polysaccharide hydrogel, 2021), see Table 1.

[0052] (2) Degree of oxidation

[0053] The oxidation degree of Ganoderma lucidum polysaccharide was determined by titration (Wang Kuntang. Preparation, performance study and application of Bletilla striata polysaccharide-based hydrogel in wound repair, 2022), see Table 1.

[0054] 3. Evaluate the antioxidant activity of oxidized Ganoderma lucidum polysaccharide.

[0055] (1) DPPH clearance rate

[0056] Take 50 μL of 0.1-5 mg / mL (0.1, 0.2, 0.5, 1, 2, 5 mg / mL) OGLP aqueous solution prepared in step 1 and add it to a 96-well plate, then add 150 μL of 0.2 mg / mL DPPH ethanol solution to form the sample group; 50 μL of OGLP aqueous solution (0.1, 0.2, 0.5, 1, 2, 5 mg / mL) mixed with 150 μL of anhydrous ethanol solution to form the blank control group; 150 μL of 0.2 mmol / L DPPH ethanol solution mixed with 50 μL of distilled water to form the substrate group. Let stand at room temperature for 30 minutes and measure the absorbance (517 nm). Under the same conditions, replace OGLP with V C , GLP, as a control. The results are shown in Figure 2 .

[0057]

[0058] Where: A0 is the absorbance of the blank control group; A i is the absorbance of the sample group; A j is the absorbance of the substrate group.

[0059] (2) ABTS clearance rate

[0060] Take 40 mg of ABTS (2,2-azino-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt) and 6.88 mg of potassium persulfate and dissolve them in 10 ml of distilled water. Keep the mixture away from light at room temperature for 12 hours to obtain ABTS mother solution. Add PBS buffer (pH 7.4) to the ABTS mother solution until its absorbance at 734 nm is 0.70±0.02 to obtain ABTS working solution.

[0061] Take 50 μL of 0.1-5 mg / mL (0.1, 0.2, 0.5, 1, 2, 5 mg / mL) OGLP aqueous solution prepared in step 1 and add it to a 96-well plate, then add 150 μL of ABTS working solution as the sample group; 50 μL of OGLP aqueous solution (0.1, 0.2, 0.5, 1, 2, 5 mg / mL) mixed with 150 μL PBS buffer as the blank control group; 50 μL of distilled water mixed with 150 μL ABTS working solution as the substrate group. After mixing, react at room temperature for 5 minutes, and measure its absorbance at a wavelength of 734 nm. Under the same conditions, replace OGLP with V C , GLP, as a control. The results are shown in Figure 2 .

[0062]

[0063] Where: A t is the absorbance of the blank control group; A b is the absorbance of the sample group; A sis the absorbance of the substrate group.

[0064] (3) Hydroxyl radical scavenging rate

[0065] Take 300μL 9mmol / L FeSO4 aqueous solution, 300μL 9mmol / L salicylic acid ethanol solution and add them to a test tube, add 100μL 0.1-5mg / mL (0.1, 0.2, 0.5, 1, 2, 5mg / mL) OGLP aqueous solution prepared in step 1, and then add 300μL 8.8mmol / L H2O2 aqueous solution as the sample group, use distilled water instead of H2O2 as the substrate group; use distilled water instead of OGLP aqueous solution as the blank control group. React in a 37℃ water bath for 30min, and then measure its absorbance at 510nm. Under the same conditions, replace OGLP with V C , GLP, as a control. Figure 2 .

[0066]

[0067] Where: A k is the absorbance of the blank control group; A q is the absorbance of the sample group; A h is the absorbance of the substrate group.

[0068] Comparative Example 1:

[0069] The amount of sodium periodate added in step (1) of Example 1 was changed to 20% of the molar amount of Ganoderma lucidum polysaccharide, and the remaining operations were the same. The gelation time and oxidation degree are shown in Table 1. The photo of Ganoderma lucidum polysaccharide hydrogel is shown in Figure 1 .

[0070] Comparative Example 2:

[0071] The amount of sodium periodate added in step (1) of Example 1 was changed to 40% of the molar amount of Ganoderma lucidum polysaccharide, and the remaining operations were the same. The gelation time and oxidation degree are shown in Table 1. The photo of Ganoderma lucidum polysaccharide hydrogel is shown in Figure 1 .

[0072] Comparative Example 3:

[0073] The amount of sodium periodate added in step (1) of Example 1 was changed to 80% of the molar amount of Ganoderma lucidum polysaccharide, and the remaining operations were the same. The gelation time and oxidation degree are shown in Table 1. The photo of the anti-Ganoderma lucidum polysaccharide hydrogel is shown in Figure 1 , antioxidant activity see Figure 2 .

[0074] Comparative Example 4:

[0075] The amount of sodium periodate added in step (1) of Example 1 was changed to 100% of the molar amount of Ganoderma lucidum polysaccharide, and the remaining operations were the same. The gelation time and oxidation degree are shown in Table 1, and the photo of Ganoderma lucidum polysaccharide hydrogel is shown in Figure 1 , antioxidant activity see Figure 2 .

[0076] Table 1 Gelation time of each hydrogel

[0077]

[0078] The results in Table 1 show that the Ganoderma lucidum polysaccharide hydrogel prepared by the molar ratio of Ganoderma lucidum polysaccharide to sodium periodate of 1:0.6 as described in Example 1 has a more ideal characterization result.

[0079] Figure 2 The results showed that the aqueous solution of Ganoderma lucidum polysaccharide had good antioxidant activity. Comparing the antioxidant activity of GLP and OGLP at different concentrations, the antioxidant activity of OGLP with an oxidation degree of 25%, 26% OGLP, and 29% OGLP was the best, but it was slightly lower than that of GLP. Therefore, the oxidized Ganoderma lucidum polysaccharide with an oxidation degree of 25% prepared by the molar ratio of Ganoderma lucidum polysaccharide to sodium periodate of 1:0.6 in Example 1 was selected as the raw material of the hydrogel.

[0080] Example 2: Preparation of pearl polypeptide hydrogel

[0081] (1) Preparation of Oxidized Ganoderma Lucidum Polysaccharide

[0082] Weigh 1 g (0.0056 mol) of the Ganoderma lucidum polysaccharide sample prepared by the method of Example 1, add 100 mL of distilled water, and stir until completely dissolved. Add 0.7 g (0.0032 mol) of sodium periodate under light-proof conditions, react at room temperature in the dark for 6 h, add 5 mL of ethylene glycol and stir for 30 min to terminate the reaction. The reaction mixture is placed in a dialysis bag (3.5 kDa) and dialyzed with pure water for 2 days. The intercepted liquid is freeze-dried at -80°C for 48 h to obtain 0.7 g of oxidized Ganoderma lucidum polysaccharide.

[0083] (2) Preparation of pearl polypeptide hydrogel

[0084] Take oxidized Ganoderma lucidum polysaccharide and add a certain amount of water to prepare an oxidized Ganoderma lucidum polysaccharide aqueous solution with a mass concentration of 3%.

[0085] Pearl polypeptide was added to a 5% mass concentration carboxymethyl cellulose (CMCS) aqueous solution to obtain a pearl polypeptide solution.

[0086] The oxidized Ganoderma lucidum polysaccharide aqueous solution and the pearl polypeptide solution were mixed in a volume ratio of 1:1 and allowed to stand at room temperature to form a gel to obtain the pearl polypeptide hydrogel, which was recorded as G-GLP-PP.

[0087] Comparative Example 5:

[0088] The volume ratio of the oxidized Ganoderma lucidum polysaccharide solution to the 5% carboxymethyl cellulose (CMCS) aqueous solution in step 1 (3) of Example 1 was changed to 1:2, and the remaining operations were the same to obtain a Ganoderma lucidum polysaccharide hydrogel, which was recorded as G-GLP1.

[0089] Comparative Example 6:

[0090] The volume ratio of the oxidized Ganoderma lucidum polysaccharide solution to the 5% carboxymethyl cellulose (CMCS) aqueous solution in step 1 (3) of Example 1 was changed to 1:0.5, and the remaining operations were the same to obtain a Ganoderma lucidum polysaccharide hydrogel, which was recorded as G-GLP3.

[0091] Example 3: Detection of different gel properties

[0092] 1. Scanning electron microscope detection

[0093] The gels prepared in Examples 1 and 2 and Comparative Examples 5 and 6 were examined by scanning electron microscopy. Figure 3 , the Ganoderma lucidum polysaccharide-based hydrogels all showed irregular porous network structures. In Example 1, Comparative Examples 5 and 6, as the proportion of Ganoderma lucidum polysaccharide increased, the pore size of the G-GLP hydrogel gradually decreased, and the dispersion gradually became uniform. However, the addition of pearl polypeptide in Example 2 did not destroy the three-dimensional network structure of the hydrogel.

[0094] 2. Fourier infrared spectroscopy detection

[0095] The oxidized Ganoderma lucidum polysaccharide (OGLP) and Ganoderma lucidum polysaccharide (GLP) prepared in Example 1 were detected by Fourier transform infrared spectroscopy. Figure 4 Compared with the spectrum of Ganoderma lucidum polysaccharide, the OGLP spectrum has a strong peak at 1702 cm due to the stretching vibration of -CHO. -1 A new absorption peak appeared at , which confirmed the presence of -CHO on OGLP.

[0096] Fourier transform infrared spectroscopy was performed on carboxymethyl cellulose (CMCS), the ganoderma lucidum polysaccharide hydrogel (G-GLP2) prepared in Example 1, and the pearl polypeptide hydrogel (G-GLP-PP) prepared in Example 2. The results are shown in Figure 5 , 1702cm in G-GLP and G-GLP-PP -1 The stretching vibration related to the aldehyde C=O at 1640 cm -1 The left and right sides show characteristic absorption peaks of imine bonds, indicating that a Schiff base reaction has occurred.

[0097] 3. Rheological test

[0098] A parallel plate with a gap of 1.0 mm and a diameter of 25 mm was used at 25°C. 0.6 mL of the gel samples G-GLP1, G-GLP-PP, G-GLP2, and G-GLP 3 prepared in Examples 1 and 2 and Comparative Examples 5 and 6 were fixed on the sample stage of a rheometer (HR20, TA Instruments Waters, USA) for dynamic frequency scanning and stress-strain scanning, and the storage modulus (G') and loss modulus (G") of the samples under different scanning modes were recorded. The frequency scanning range was 0.1-100 Hz, stress 1%; the stress scanning range was 0.1-1000%, frequency 1 Hz.

[0099] Results Figure 6 , the oscillatory strain scanning spectrum shows that all three hydrogels have a long linear viscoelastic region with solid and stable mechanical properties. As the proportion of oxidized Ganoderma lucidum polysaccharide increases, the aldehyde groups provided gradually increase, the cross-linking of the hydrogel becomes tighter, and the collapse point gradually moves backward. Among them, G-GLP1 intersects G' and G" at a strain of 332%, while the collapse points of G-GLP2 and 3 are 403% and 440%, which indicates that the increase in cross-linking strength can enhance the mechanical properties of the hydrogel. The results of the frequency scan show that the hydrogels of the three hydrogels remain stable in the range of 0.1-10Hz. After the addition of the polypeptide, the collapse point of the hydrogel shifted slightly backward (425%), indicating that the addition of the polypeptide makes the hydrogel have more stable mechanical properties.

[0100] 4. Swelling test

[0101] 1 g of the gels G-GLP1, G-GLP-PP, G-GLP2, and G-GLP3 prepared in Examples 1 and 2 and Comparative Examples 5 and 6 were immersed in PBS buffer (pH 7.4) at room temperature. After swelling, the hydrogel samples were taken out of the buffer at different times, and the water on the surface of the samples was wiped off with filter paper and weighed until the weight of the hydrogel no longer increased, which was recorded as W. S The weight of the sample before immersion was recorded as W0. The swelling data of each hydrogel were tested in parallel for 3 samples and the average value was taken.

[0102]

[0103] The results are as follows Figure 7As shown in the figure, after the hydrogel was immersed in PBS at room temperature for 21 hours, G-GLP 1, G-GLP 2, and G-GLP 3 hydrogels all reached swelling equilibrium, and their swelling rates were 71.69±2.0%, 57.84±4.5%, and 34.11±1.6%, respectively. As the proportion of oxidized Ganoderma lucidum polysaccharide increased, the aldehyde content increased, and the swelling rate of the hydrogel gradually decreased. This is related to the cross-linking density of the hydrogel. As the aldehyde content increases, the cross-linking density increases, and the structural stability of the hydrogel network increases, resulting in a decrease in the ability of the hydrogel to absorb liquid. After the addition of the peptide, the swelling rate of the hydrogel decreased, which may be due to the peptide strengthening the structural stability of the hydrogel.

[0104] 5. In vitro simulated gastrointestinal digestion experiment

[0105] Gastric juice: Add 2.0 g NaCl to 70 mL of 1.0 N HCl and make up to 1 L. Adjust the pH to 2.0 ± 0.1 with 0.2 N HCl or 0.2 N NaOH. After high-pressure steam sterilization, add 3.2 g pure pepsin to obtain simulated gastric fluid (SGF).

[0106] Intestinal fluid: Add 6.8 g potassium dihydrogen phosphate to 250 mL distilled water, then add 77 mL 0.2 N NaOH to make up to 1 L. Adjust the pH to 6.8 ± 0.1 with 0.2 N NaOH or 0.2 N HCl, then sterilize the solution. Add 10 g trypsin (porcine pancreas) and 1% bile salt to obtain simulated intestinal fluid (SIF).

[0107] 0.5 g of the G-GLP-PP hydrogel prepared by the method of Example 2 was placed in 5 mL of simulated gastric fluid and placed in a shaker at 37 ° C and 100 rpm for 2 hours. After that, the hydrogel was moved into 5 mL of simulated intestinal fluid and shaken for 6 hours. 1 mL of simulated gastric fluid or simulated intestinal fluid was taken every 20 minutes, and an equal volume of release medium (simulated gastric fluid or simulated intestinal fluid) was immediately added. The protein concentration in the sample solution was detected by the BCA method, and the polypeptide release rate was calculated.

[0108]

[0109] Where: n i is the protein content of the sample solution at the i-th time point; n0 is the protein content of simulated gastric fluid or simulated intestinal fluid; w is the polypeptide content in the hydrogel.

[0110] Results Figure 8During the 120-min incubation time of the G-GLP-PP hydrogel in SGF, only 29% of the peptides were slowly released. In contrast, when the hydrogel was transferred to SIF, the release rate of the peptides increased significantly, and the remaining peptides (PP, 71%) were released within 240 min. This indicates that the G-GLP-PP hydrogel effectively maintains the activity of PP after passing through SGF, reaches the intestine as a whole, and gradually releases PP.

[0111] 6. In vitro antioxidant and anti-tyrosinase experiments

[0112] 0.1 g of G-GLP2 and G-GLP-PP were soaked in 100 mL of pure water for 24 h, and 1 mg / mL of hydrogel extract was obtained by filtration. The hydrogel extract, 1 mg / mL CMCS aqueous solution, 1 mg / mL OGLP aqueous solution, and 1 mg / mL PP aqueous solution were used as the test sample solutions to detect antioxidant activity and tyrosinase inhibitory activity. The antioxidant activity detection method refers to the antioxidant activity determination method in Example 1.

[0113] 30 μL of mushroom tyrosinase (800 U / mL) and 30 μL of the sample solution to be tested were added to a 96-well plate, incubated at room temperature for 10 min, then 50 μL of L-DOPA aqueous solution (1 mM) was added, and 90 μL of phosphate buffer (0.2 M, pH = 6.8) was added, and the absorbance was measured at 475 nm, which was recorded as A; the absorbance value of replacing the sample solution with 30 μL of phosphate buffer was recorded as B; the absorbance value of replacing mushroom tyrosinase with 30 μL of phosphate buffer was recorded as A0; the absorbance value of 150 μL of phosphate buffer mixed with 50 μL of L-DOPA aqueous solution was recorded as B0. Kojic acid was used as a positive control.

[0114]

[0115] The results are as follows Fig. 9 As shown in the figure, compared with G-GLP2 hydrogel, the antioxidant activity of the hydrogel with PP was improved, among which the scavenging activity of ABTS and hydroxyl radicals was significantly improved, but the DPPH scavenging activity did not change significantly compared with G-GLP2, which may be due to the low DPPH scavenging activity of PP. The tyrosinase inhibition rate of CMCS and OGLP was low, so the tyrosinase inhibition rate of G-GLP was also low, but after adding PP, the tyrosinase inhibition rate of the hydrogel was significantly improved.

[0116] 7. Antibacterial experiment

[0117] The antibacterial properties of the hydrogels were studied using Escherichia coli (CMCC 44102) and Staphylococcus aureus (CMCC 26003). 600 μL of the pre-gel mixture of Examples 1 and 2 was prepared in a 24-well plate, and the hydrogels G-GLP2 and G-GLP-PP were formed at room temperature and sterilized under ultraviolet irradiation for 24 h. The bacterial suspension (10 μL, 1×10 6 CFU / mL) were evenly added to the hydrogel surface, and only bacteria were added as the control group. After incubation at 37°C for 4 hours, 1 mL of sterile PBS was added, ultrasonicated for 5 minutes, and 50 μL of bacterial suspension was taken and coated on LB agar plates. After inverted culture at 37°C for 18 hours, photos were taken and colonies were counted. The results are shown in Fig.10 As shown, G-GLP2 hydrogel exhibited antibacterial activity against Escherichia coli and Staphylococcus aureus. Compared with G-GLP2 hydrogel, the addition of PP further improved the antibacterial effect. There was almost no colony formation in the G-GLP-PP hydrogel group plate, and the inhibition rate of the two test bacteria was significantly improved. Moreover, only G-GLP2 hydrogel caused more than 96% bacterial death. The antibacterial performance of G-GLP-PP hydrogel was significantly better than that of hydrogel without PP, with an inhibition rate of more than 99% against Escherichia coli and more than 98% against Staphylococcus aureus.

[0118] The results show that the Ganoderma lucidum polysaccharide hydrogel has good mechanical stability and swelling properties, and hydrogels with different properties can be obtained by adjusting the proportion of oxidized Ganoderma lucidum polysaccharide in the hydrogel. The addition of pearl polypeptide improves the mechanical stability of the hydrogel. In addition, in vitro digestion experiments have shown that the Ganoderma lucidum polysaccharide hydrogel can provide a physical barrier for the pearl polypeptide, allowing it to be released in the intestine and improving its bioavailability.

Claims

1. A polypeptide hydrogel based on oxidized Ganoderma lucidum polysaccharide, It is characterized in that The polypeptide hydrogel is prepared as follows: (1) Preparation of Oxidized Ganoderma Lucidum Polysaccharide Add distilled water to the ganoderma lucidum polysaccharide and stir until it is completely dissolved. Add sodium periodate under light-proof conditions and react at room temperature in the dark for 5-10 hours. Add ethylene glycol and stir to terminate the reaction. Put the reaction mixture into a dialysis bag and dialyze it in pure water for 1-3 days. Take the intercepted liquid and freeze-dry it to obtain oxidized ganoderma lucidum polysaccharide. (2) Preparation of polypeptide hydrogel A polypeptide is added to a 2-10% carboxymethyl cellulose aqueous solution to obtain a polypeptide concentration of 0.05-1% to obtain a polypeptide solution; a 1-5% oxidized ganoderma lucidum polysaccharide aqueous solution and the polypeptide solution are mixed at a volume ratio of 1:0.5-2, and allowed to stand at room temperature to form a gel to obtain a polypeptide hydrogel.

2. The polypeptide hydrogel according to claim 1, It is characterized in that Step (1) The ganoderma lucidum polysaccharide is obtained by removing protein from ganoderma lucidum extract, and the mass content of the polysaccharide is 55-65%.

3. The polypeptide hydrogel according to claim 1, It is characterized in that Step (1) Ganoderma lucidum polysaccharide is prepared as follows: Sevage reagent is added to the aqueous solution of Ganoderma lucidum extract, and the lower water layer and the denatured protein at the interface are removed after mixing and shaking, and the process is repeated for multiple times until no denatured protein is precipitated, and the clarified polysaccharide solution is collected; Then add anhydrous ethanol with a final volume concentration of 80%, stand at room temperature overnight, centrifuge, and collect the precipitate; after the precipitate is re-dissolved with water, the organic solvent is removed by rotary evaporation, and freeze-dried at -80°C for 48 hours to obtain Ganoderma lucidum polysaccharide; the Sevage reagent is a chloroform and n-butanol solution with a volume ratio of 4:1, and the volume amount of the Sevage reagent is 1 / 3 of the volume of the Ganoderma lucidum extract aqueous solution; the volume amount of distilled water in the Ganoderma lucidum extract aqueous solution is 10mL / g based on the mass of the Ganoderma lucidum extract.

4. The polypeptide hydrogel according to claim 1, It is characterized in that In step (1), the volume amount of distilled water is 50-150 mL / g based on the mass of Ganoderma lucidum polysaccharide; the molar ratio of Ganoderma lucidum polysaccharide to sodium periodate is 1:0.1-1; and the volume amount of ethylene glycol is 1-10 mL / g based on the mass of Ganoderma lucidum polysaccharide.

5. The polypeptide hydrogel according to claim 1, It is characterized in that The molecular weight cut-off of the dialysis bag in step (1) is 3.5 kDa.

6. The polypeptide hydrogel according to claim 1, It is characterized in that Step (2) The carboxymethyl cellulose aqueous solution has a mass concentration of 5%, and the polypeptide has a mass concentration of 0.1%; the oxidized Ganoderma polysaccharide aqueous solution has a mass concentration of 3%; the oxidized Ganoderma polysaccharide aqueous solution and the polypeptide solution are mixed in a volume ratio of 1:

1.

7. The polypeptide hydrogel according to claim 1, It is characterized in that The polypeptide in step (2) includes pearl polypeptide.

8. Use of the polypeptide hydrogel according to claim 1 in preparing polypeptide intestinal sustained-release drugs.

9. Use of the polypeptide hydrogel according to claim 1 in the preparation of antibacterial drugs.

10. The use according to claim 9, It is characterized in that The antibacterial drug is a drug that inhibits the activity of Escherichia coli or Staphylococcus aureus.

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