Short peptide molecule and application thereof, oleanolic acid hydrogel and preparation method and application thereof

Abstract

The invention relates to the field of antibacterial hydrogel, and discloses an oligopeptide molecule and application thereof, oleanolic acid hydrogel as well as a preparation method and application thereof. The structural formula of the oligopeptide molecule is shown in the formula (I). The oligopeptide molecule provided by the invention has an excellent application prospect in preparation of a hydrogel material. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the technical field of antibacterial hydrogels, and in particular to a short peptide molecule and application thereof, an oleanolic acid hydrogel and a preparation method and application thereof. Background Art

[0002] As an emerging antimicrobial material, antimicrobial hydrogels have attracted considerable attention due to their superior properties. These hydrogels not only provide a near-natural moist environment under physiological conditions, accelerating wound healing, but also possess antimicrobial activity, effectively inhibiting bacterial growth. Consequently, they are widely used in wound care and the treatment of skin infections.

[0003] Antibacterial hydrogels can form a protective barrier on the wound surface, maintain appropriate moisture, promote cell regeneration and reduce the risk of infection.

[0004] However, many current antimicrobial hydrogel developments rely on supramolecular materials such as synthetic polymers, metal nanoparticles, or peptides.

[0005] To achieve stable properties, such as self-support and self-healing properties, the synthesis of such materials often requires complex chemical reactions and purification processes. During this process, organic solvents, catalysts, and toxic chemicals may be introduced into the hydrogel, thus affecting the quality of the final product.

[0006] Chemical residues may not only reduce the antimicrobial activity of hydrogels, but also affect their biocompatibility and may even induce immune responses and local inflammation. In addition, some synthetic antimicrobial hydrogels are highly toxic to mammalian cells, potentially posing a threat to human health.

[0007] Oleanolic acid is a natural pentacyclic triterpenoid small molecule compound, primarily extracted from the whole herb of Glechoma longituba or the fruit of Ligustrum lucidum. It is widely found in a variety of plants, such as ginger, nettle, and olive, and has a high distribution frequency in the plant kingdom.

[0008] Studies have shown that oleanolic acid can bind to bacterial cell membranes and disrupt their integrity. This increases cell membrane permeability, allowing substances inside the cell to leak out, leading to bacterial death. Oleanolic acid also possesses antioxidant activity, which can reduce oxidative damage to cells by scavenging free radicals, thereby indirectly enhancing its antibacterial effects. Oxidative stress often increases bacterial pathogenicity, and inhibiting oxidative damage can help reduce the severity of infection.

[0009] As research deepens, oleanolic acid is expected to become a new antibacterial drug, especially in the context of increasingly serious bacterial resistance, its application potential will be more prominent.

[0010] However, due to the highly hydrophobic pentacyclic structure of the oleanolic acid molecule, oleanolic acid is difficult to dissolve in aqueous solutions, resulting in low solubility and poor absorption. In addition, oral administration results in a significant first-pass effect in the liver (which occurs when a drug, after gastrointestinal absorption, first enters the liver for metabolism, causing some drug components to be rapidly metabolized or inactivated, thus affecting its effectiveness). Furthermore, poor hydrophilicity can lead to excessively high local concentrations, which is detrimental to cell growth and reduces its biocompatibility.

[0011] The above factors result in low bioavailability of oleanolic acid, which seriously affects its clinical therapeutic effect.

[0012] Currently, reports on the preparation of oleanolic acid into stable hydrogels are rare and are mostly limited to specific organic solvents such as ethanol, tetrahydrofuran, chlorinated alkane solvents and benzene solvents, which are highly toxic to cells and cannot be used in vivo.

[0013] In order to solve the above problems, it is necessary to develop more biocompatible and effective oleanolic acid antibacterial hydrogels. Summary of the Invention

[0014] The purpose of the present invention is to overcome the problems of poor hydrophilicity and low biocompatibility of oleanolic acid drugs in the prior art.

[0015] In order to achieve the above object, the first aspect of the present invention provides a short peptide molecule, the structural formula of the short peptide molecule is shown in formula (I):

[0016]

[0017] The second aspect of the present invention provides the use of the short peptide molecule described in the first aspect in preparing a hydrogel material.

[0018] A third aspect of the present invention provides a method for preparing oleanolic acid hydrogel, the method comprising:

[0019] (1) contacting and mixing a solution I containing oleanolic acid and a short peptide with an acid solution to obtain a mixed solution;

[0020] (2) subjecting the mixed solution to gel treatment to obtain oleanolic acid hydrogel;

[0021] The mass ratio of oleanolic acid to short peptide in the solution I is 1:(10-50);

[0022] By controlling the amount of the acid solution, the pH of the mixed solution is made to be 5.0-7.5;

[0023] The short peptide is the short peptide molecule described in the first aspect above.

[0024] The fourth aspect of the present invention provides an oleanolic acid hydrogel prepared by the method described in the third aspect.

[0025] The fifth aspect of the present invention provides use of the oleanolic acid hydrogel described in the fourth aspect in biomedical materials.

[0026] The short peptide molecules provided by the present invention have good application prospects in the preparation of hydrogel materials.

[0027] The oleanolic acid hydrogel prepared by the method provided by the present invention not only has good biocompatibility, but also has excellent self-repairing properties and biological antibacterial activity. It has great potential application value in the field of biomaterials and provides a research basis for the clinical application of oleanolic acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is the nuclear magnetic resonance hydrogen spectrum of the short peptide molecule provided by the present invention.

[0029] Figure 2 These are the biocompatibility results of the oleanolic acid hydrogel prepared in Example 1 of the present invention at 24 hours, 48 hours, and 72 hours, respectively.

[0030] Figure 3 This is the rheological property result of the oleanolic acid hydrogel prepared in Example 1 of the present invention.

[0031] Figure 4 This is the self-repairing performance result of the oleanolic acid hydrogel prepared in Example 1 of the present invention.

[0032] Figure 5 This is a diagram showing the antibacterial effect of the oleanolic acid hydrogel prepared in Example 1 of the present invention on Escherichia coli and Staphylococcus. DETAILED DESCRIPTION

[0033] The endpoints of the ranges and any values disclosed herein are not limited to the precise ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0034] As mentioned above, the first aspect of the present invention provides a short peptide molecule, the structural formula of the short peptide molecule is shown in formula (I):

[0035]

[0036] According to a preferred embodiment, the short peptide molecule is prepared by a method comprising the following steps:

[0037] S1: In the presence of N,N'-dimethylformamide, piperidine and Rink amide MBHA resin are subjected to a first contact reaction to obtain intermediate I;

[0038] S2: conducting a first coupling reaction of N-methylpyrrolidone, N-(9-fluorenylmethoxycarbonyl)-N'-methyltrityl-L-lysine, O-benzotriazole-tetramethyluronium hexafluorophosphate, N,N-diisopropylethylamine and the intermediate I to obtain intermediate II;

[0039] S3: subjecting N-Fmoc-L-phenylalanine, Fmoc-L-glycine and the intermediate II to a second coupling reaction to obtain intermediate III;

[0040] S4: In the presence of dichloromethane, trifluoroacetic acid, triisopropylsilane and the intermediate III are subjected to a second contact reaction to obtain the short peptide molecule.

[0041] Preferably, in step S1, the mass ratio of the N,N'-dimethylformamide, the piperidine and the Rink amide MBHA resin is 1:1-2.5:1.5-2.0.

[0042] Preferably, before the first contact reaction, the Rink amide MBHA resin is washed with dichloromethane (I), and then the washed Rink amide MBHA resin is swelled in a mixed solution (I) containing dichloromethane and N,N'-dimethylformamide.

[0043] Preferably, the volume ratio of dichloromethane to N,N'-dimethylformamide in the mixed solution I is 1:0.8-1.2.

[0044] Preferably, the swelling treatment is carried out in a shaking table.

[0045] Preferably, the swelling treatment conditions include: a time of 30-40 minutes and a rotation speed of 20-50 rpm.

[0046] Preferably, in step S1, the mass fraction of piperidine is 15-25 wt%.

[0047] Preferably, the conditions of the first contact reaction include: temperature of 25-30° C. and time of 3-5 min.

[0048] Preferably, after the first contact reaction, the material after the first contact reaction is washed II with the mixed solution I to obtain the intermediate I.

[0049] There are no special requirements for the conditions of washing II of the present invention, as long as the material after the first contact reaction is washed cleanly. For example, it is washed with deionized water 3-5 times.

[0050] Preferably, in step S2, the mass ratio of the N-methylpyrrolidone, N-(9-fluorenylmethoxycarbonyl)-N'-methyltrityl-L-lysine, the O-benzotriazole-tetramethyluronium hexafluorophosphate, the N,N-diisopropylethylamine and the intermediate I is 1:1.5-2.0:1.5-2.0:2.5-3.0:2.0-2.5.

[0051] Preferably, in step S3, the mass ratio of the N-Fmoc-L-phenylalanine, the Fmoc-L-glycine and the intermediate II is 1:1.0-1.5:2.5-3.0.

[0052] Preferably, in step S4, the mass ratio of the trifluoroacetic acid, triisopropylsilane and the intermediate III is 1:1.0-1.5:2.5-3.0.

[0053] Preferably, in step S4, the amount of dichloromethane used is 4-6 g per 1 g of trifluoroacetic acid.

[0054] Preferably, in step S2, the conditions of the first coupling reaction include: temperature of 25-30° C., and time of 10-20 min.

[0055] Preferably, in step S3, the conditions of the second coupling reaction include: temperature of 25-30° C., and time of 10-20 min.

[0056] Preferably, in step S4, the conditions of the second contact reaction include: temperature of 25-30° C., and time of 20-40 min.

[0057] Preferably, in step S4, after the second contact reaction, the material after the second contact reaction is purified by HPLC and dried to obtain the short peptide molecule.

[0058] There are no particular requirements for the drying conditions of the present invention, and those skilled in the art can select them as needed. For example, freeze drying is performed in a freeze drying chamber for 24-48 hours.

[0059] As mentioned above, the second aspect of the present invention provides the use of the short peptide molecules described in the first aspect in preparing hydrogel materials.

[0060] As mentioned above, the third aspect of the present invention provides a method for preparing oleanolic acid hydrogel, the method comprising:

[0061] (1) contacting and mixing a solution I containing oleanolic acid and a short peptide with an acid solution to obtain a mixed solution;

[0062] (2) subjecting the mixed solution to gel treatment to obtain oleanolic acid hydrogel;

[0063] The mass ratio of oleanolic acid to short peptide in the solution I is 1:(10-50);

[0064] By controlling the amount of the acid solution, the pH of the mixed solution is made to be 5.0-7.5;

[0065] The short peptide is the short peptide molecule described in the first aspect above.

[0066] Preferably, in step (1), the mass ratio of oleanolic acid to short peptide in the solution I is 1:(20-40). The inventors of the present invention have found that under this preferred condition, the obtained oleanolic acid hydrogel has more excellent mechanical properties.

[0067] Preferably, in step (1), the solvent in the solution I is selected from at least one of a carbonate solution, a bicarbonate solution, and an acetate solution.

[0068] Preferably, in step (1), the acid solution is acetic acid and / or citric acid.

[0069] Preferably, in step (1), the contact mixing conditions include: ultrasonic power of 100-400W, and time of 5-10 minutes.

[0070] Preferably, in step (2), the gel treatment conditions include: a temperature of 25-50° C. and a time of 30-60 min. The inventors of the present invention have found that under this preferred condition, the obtained oleanolic acid hydrogel has better stability.

[0071] As mentioned above, the fourth aspect of the present invention provides an oleanolic acid hydrogel prepared by the method described in the second aspect.

[0072] As mentioned above, the fifth aspect of the present invention provides the use of the oleanolic acid hydrogel described in the fourth aspect in biomedical materials.

[0073] The present invention will be described in detail below through examples. In the following examples, unless otherwise specified, the instruments, reagents, materials, etc. involved are all conventional instruments, reagents, materials, etc., which can be obtained through regular commercial channels. In particular, unless otherwise specified, the reagents used are all commercially available analytical grade products.

[0074] Rink amide MBHA resin: purchased from Shanghai MacLean Biochemical Technology Co., Ltd., brand R916084.

[0075] Mixed solution I: the volume ratio of dichloromethane and N,N'-dimethylformamide is 1:1.

[0076] Piperidine: mass fraction is 20 wt%.

[0077] N-(9-Fluorenylmethyloxycarbonyl)-N'-methyltrityl-L-lysine: purchased from Shanghai MacLean Biochemical Technology Co., Ltd., brand F809911.

[0078] O-Benzotriazole-tetramethyluronium hexafluorophosphate: purchased from Aladdin Biochemical Technology Co., Ltd., brand H106174.

[0079] N-Fmoc-L-phenylalanine: purchased from Shanghai MacLean Biochemical Technology Co., Ltd., brand number F809550.

[0080] Fmoc-L-glycine was purchased from Aladdin Biochemical Technology Co., Ltd. with the brand number F103019.

[0081] Sodium carbonate solution: pH = 10.5.

[0082] Acid solution: 30% acetic acid by mass.

[0083] Preparation Example 1:

[0084] This preparation example is used to illustrate the preparation of a short peptide molecule represented by formula (I) according to the following steps:

[0085] S1: washing the Rink amide MBHA resin with dichloromethane I, and then swelling the washed Rink amide MBHA resin with the mixed solution I on a shaker;

[0086] S2: In the presence of N,N'-dimethylformamide (1.0 g), piperidine (1.0 g) and the swollen Rink amide MBHA resin (1.5 g) were subjected to a first contact reaction, and the material after the first contact reaction was washed with the mixed solution I (washed with deionized water 5 times) to obtain intermediate I;

[0087] S3: N-methylpyrrolidone (1.0 g), N-(9-fluorenylmethoxycarbonyl)-N'-methyltrityl-L-lysine (1.5 g), O-benzotriazole-tetramethyluronium hexafluorophosphate (1.5 g), N,N-diisopropylethylamine (2.5 g) and the intermediate I (2.0 g) were subjected to a first coupling reaction to obtain intermediate II;

[0088] S4: N-Fmoc-L-phenylalanine (1.0 g), Fmoc-L-glycine (1.0 g) and the intermediate II (2.5 g) were subjected to a second coupling reaction to obtain intermediate III;

[0089] S5: In the presence of dichloromethane (5.0 g), trifluoroacetic acid (1.0 g), triisopropylsilane (1.0 g) and the intermediate III (2.5 g) are subjected to a second contact reaction. The material after the second contact reaction is purified by HPLC and dried (freeze-dried in a freeze drying oven for 24 h) to obtain the short peptide molecule;

[0090] Swelling treatment: time 30 min, speed 30 rpm;

[0091] First contact reaction: temperature 25°C, time 3 minutes;

[0092] First coupling reaction: temperature 25°C, time 15 min;

[0093] Second coupling reaction: temperature 25°C, time 20 min;

[0094] Second contact reaction: temperature is 25°C, time is 30 minutes.

[0095] The short peptide molecules prepared in this preparation example were characterized by nuclear magnetic resonance, and the results were as follows Figure 1 As shown:

[0096] Through Figure 1 Analysis of the peak areas in the ions showed that: the peaks of the two hydrogen atoms with a chemical shift range of 3.5-4.5 ppm correspond to the α-carbon (-CH2) on the glycine main chain; the peak with a chemical shift range of 7.5-8.5 ppm corresponds to the amino (-NH2) hydrogen atom; the peak with a chemical shift range of 6.5-8.5 ppm corresponds to the benzene ring hydrogen atoms of phenylalanine, and the α-amino (-CH2) hydrogen of phenylalanine appears around 4.0-4.5 ppm; the peak with a chemical shift range of 1.5-3.5 ppm corresponds to the hydrogen atoms in the lysine side chain (-CH2-CH2-CH2-NH2), especially the β-hydrogen (-CH2) appears between 1.5-2.5 ppm.

[0097] The above analysis results show that the short peptide molecule prepared in this preparation example is the short peptide molecule represented by formula (I).

[0098] Example 1

[0099] This example is used to illustrate the preparation of oleanolic acid hydrogel according to the following steps with reference to the formulation in Table 1:

[0100] (1) contacting and mixing the solution I containing oleanolic acid and the short peptide with the acid solution (ultrasonic power of 300 W, time of 5 min) to obtain a mixed solution;

[0101] (2) The mixed solution is subjected to gel treatment to obtain oleanolic acid hydrogel.

[0102] Unless otherwise specified, Examples 2 to 4 were all carried out using a method similar to Example 1, with the differences being the formulation and process, to prepare oleanolic acid hydrogels, as shown in Table 1.

[0103] Table 1

[0104]

[0105] Example 5

[0106] This example is carried out in a similar manner to Example 1, except that the amount of the acid solution is controlled so that the pH of the mixed solution is 4.0.

[0107] The unlisted parts were the same as in Example 1 to prepare oleanolic acid hydrogel.

[0108] Comparative Example 1

[0109] This comparative example was carried out using a method similar to that of Example 1, except that the short peptide molecule in step (1) was adjusted to glycine-DL-phenylalanine (CAS No.: 721-66-4). The unlisted parts were the same as those of Example 1, and no oleanolic acid hydrogel could be prepared.

[0110] Test Case

[0111] 1. The cytotoxicity (L929 cells) of the oleanolic acid hydrogel prepared in Example 1 was tested using the CCK8 method (reference: Properties of Cell-Compatible Poly (vinylalcohol) Hydrogels Cross-Linked with Hydrophobic Luteolin. ACS Applied Polymer Materials 2021, 3 (6), 3019-3027). The results are as follows: Figure 2As shown in:

[0112] pass Figure 2 As can be seen, the survival rate of L929 cells decreased significantly when pure oleanolic acid was present. However, the survival rate of L929 cells in the combination of the short peptide and oleanolic acid hydrogel was significantly higher. This indicates that the prepared hydrogel has no significant toxicity to normal cells and that the short peptide can form a hydrogel with oleanolic acid that is highly hydrophilic and biocompatible.

[0113] 2. The rheological properties of the oleanolic acid hydrogel prepared in Example 1 were tested (the oleanolic acid hydrogel was placed on a rheometer, the temperature was set to 25°C, the parallel plate diameter was 50 mm, the gap was set to 0.1 mm, and the dynamic strain scan was performed: strain 0.1% to 100%, frequency 6.28 rad / s.). The results are as follows: Figure 3 As shown in:

[0114] pass Figure 3 It can be seen that: within the strain scanning range, G′ is much larger than G″ at low strain, indicating that the oleanolic acid hydrogel is in a gel state; when the strain increases to 24.5%, G′ begins to be smaller than G″, and as the strain increases, the difference becomes larger and larger, and it is in a solution state at this time.

[0115] 3. The self-repairing performance of the oleanolic acid hydrogel prepared in Example 1 was tested. The results are as follows: Figure 4 As shown in:

[0116] pass Figure 4 It can be seen from (A) (at 25°C) that when the strain is higher than 40%, the oleanolic acid hydrogel undergoes an obvious gel-sol transition. At a low strain of 0.1%, it lasts for 2 minutes and G′ is greater than G″, indicating a gel state. When the strain increases by 40%, it lasts for 1 minute and G′ and G″ are opposite in size, indicating a transition from the gel state to the sol state. After the strain is reduced to 0.1% again, the G′ value is higher than the G″ value again after about 5 seconds, and the value is basically consistent with the original state. This reversible gel-sol transition can be cycled at least 5 times, and the gel state can still be instantly restored after multiple high strain stimulations.

[0117] pass Figure 4 (B) (oscillation experiment) shows that when the oleanolic acid hydrogel is vigorously shaken, it will transform into a sol and return to a gel state after standing. It can be seen that the hydrogel has strong self-healing properties and is expected to be developed into a locally injectable biomedical material.

[0118] 4. The mechanical properties of the oleanolic acid hydrogels prepared in Examples 1 to 6 were analyzed using rheological testing methods. The results are shown in Table 2:

[0119] Table 2

[0120] Storage modulus / Pa Loss modulus / Pa Example 1 2688.9 557.8 Example 2 2232.3 620.1 Example 3 1484.0 676.4 Example 4 1127.0 658.6 Example 5 816.7 606.6

[0121] 5. The antibacterial properties of the oleanolic acid hydrogel prepared in Example 1 were tested with reference to the literature (Multifunctional Oxidized Dextran–Metformin as a Tissue-Adhesive Hydrogel to Prevent Postoperative Peritoneal Adhesions in Patients with Metabolic Syndrome. Advance Sciences 2023, 2303767). Specifically, the oleanolic acid hydrogel was cultured with Escherichia coli and Staphylococcus aureus for 48 hours to evaluate its antibacterial ability, and the results were as follows: Figure 5 As shown:

[0122] pass Figure 5 As can be seen in (A), compared with the blank group (no treatment), there was no obvious growth of E. coli on the agar plate treated with oleanolic acid hydrogel, while a certain amount of colonies appeared on the agar plate treated with short peptide molecules;

[0123] pass Figure 5 As can be seen in (B), compared with the blank group (no treatment), there was no obvious growth of Staphylococcus aureus on the agar plate treated with oleanolic acid hydrogel, while a certain amount of colonies appeared on the agar plate treated with short peptide molecules;

[0124] The above results show that oleanolic acid hydrogel treatment has outstanding antibacterial effects on Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus).

[0125] The above results indicate that the short peptide molecules provided by the present invention have promising application prospects in the preparation of hydrogel materials. The oleanolic acid hydrogel prepared using the method provided by the present invention not only exhibits good biocompatibility but also possesses excellent self-repairing properties and bioantibacterial activity. This has great potential application value in the field of biomaterials and provides a research foundation for the clinical application of oleanolic acid.

[0126] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, various simple variations of the technical solution of the present invention may be made, including combining the various technical features in any other appropriate manner. These simple variations and combinations should also be regarded as disclosed in the present invention and fall within the scope of protection of the present invention.

Claims

1. A short peptide molecule, characterized in that The structural formula of the short peptide molecule is shown in formula (I):

2. Use of the short peptide molecule according to claim 1 in the preparation of hydrogel materials.

3. A method for preparing oleanolic acid hydrogel, characterized in that: The method includes: (1) contacting and mixing a solution I containing oleanolic acid and a short peptide with an acid solution to obtain a mixed solution; (2) subjecting the mixed solution to gel treatment to obtain oleanolic acid hydrogel; The mass ratio of oleanolic acid to short peptide in the solution I is 1:(10-50); By controlling the amount of the acid solution, the pH of the mixed solution is made to be 5.0-7.5; The short peptide is the short peptide molecule described in claim 1.

4. The method according to claim 3, characterized in that In step (1), the mass ratio of oleanolic acid to short peptide in the solution I is 1:(20-40).

5. The method according to claim 3 or 4, characterized in that In step (1), the solvent in the solution I is selected from at least one of a carbonate solution, a bicarbonate solution, and an acetate solution.

6. The method according to any one of claims 3 to 5, characterized in that In step (1), the acid solution is acetic acid and / or citric acid.

7. The method according to any one of claims 3 to 6, characterized in that In step (1), the contact mixing conditions include: ultrasonic power of 100-400W, and time of 5-10 minutes.

8. The method according to any one of claims 3 to 7, characterized in that In step (2), the gel treatment conditions include: temperature of 25-50° C. and time of 30-60 min.

9. Oleanolic acid hydrogel prepared by the method according to any one of claims 3 to 8.

10. Use of the oleanolic acid hydrogel according to claim 9 in biomedical materials.

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