Stomatitis composition based on micro-crosslinked polyethylene glycol lysozyme and preparation method and application of preparation of stomatitis composition

Abstract

The invention relates to a stomatitis composition based on micro-crosslinked polyethylene glycol lysozyme and a preparation method and application of a preparation of the stomatitis composition, and the stomatitis composition comprises the following components in parts by mass: polyethylene glycol carboxyl activated ester, polyethylene glycol, chitosan and lysozyme. According to the invention, by virtue of a microstructure formed by micro-crosslinking of polyethylene glycol carboxyl activated ester and lysozyme, the retention time of the lysozyme is prolonged, and the defect that single lysozyme is easy to lose is overcome. On one hand, after part of sites of polyethylene glycol carboxyl activated ester are crosslinked with lysozyme, the rest non-crosslinked activated ester sites continue to be crosslinked with protein in oral mucosa, so that a polyethylene glycol lysozyme complex is fixed on tissues, and the retention efficiency of lysozyme is enhanced; on the other hand, by means of the entrapment effect of the polymer microstructure, the risk that the lysozyme is scoured due to direct contact with saliva can be reduced, and the retention time of the lysozyme is remarkably prolonged.

Description

Technical Field

[0001] The invention relates to the technical field of animal oral health products, and in particular to a stomatitis composition based on micro-cross-linked polyethylene glycol lysozyme and a preparation method and application of the preparation thereof. Background Art

[0002] Stomatitis is a common oral disease in pets, which may be caused by a variety of reasons, including bacterial infection, food residue, dental calculus, etc. Stomatitis can cause pain and discomfort in the pet's mouth, and even affect eating and swallowing. Suppressing stomatitis can relieve the pain of pets. In addition, stomatitis is often accompanied by bacterial infection. If it is not suppressed in time, the infection may spread to other parts and even affect the overall health of the pet. Therefore, suppressing stomatitis can improve the quality of life of pets and make them live healthier and happier.

[0003] Lysozyme plays an important role in the treatment of stomatitis. First, lysozyme can destroy bacterial cell walls, leading to bacterial death, which helps to reduce inflammation caused by bacterial infection in the oral cavity. Secondly, lysozyme has an anti-inflammatory effect, which relieves the discomfort caused by stomatitis by reducing the inflammatory response of the oral mucosa. Finally, stomatitis is often accompanied by oral mucosal damage. Lysozyme can promote wound healing, enhance the body's immunity, and accelerate the repair process of the oral mucosa. For traditional stomatitis sprays, lysozyme is directly used by dissolving in a solution. However, lysozyme is easily affected by factors such as saliva secretion and chewing in the oral cavity, and has a short retention time, so it needs to be used multiple times in a short period of time, which seriously limits its clinical usability and efficacy.

[0004] Therefore, how to improve the retention time of lysozyme has become a problem that needs to be solved urgently. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a preparation method and application of a stomatitis composition based on micro-crosslinked polyethylene glycol lysozyme and its preparation. In the present invention, polyethylene glycol carboxyl activated ester and lysozyme are used to prepare a composite spray. The microstructure formed by micro-crosslinking with lysozyme increases the retention time of lysozyme, thereby overcoming the defect that a single lysozyme is easily lost. The principle is that, on the one hand, after the polyethylene glycol carboxyl activated ester partial sites are cross-linked with lysozyme, the remaining uncross-linked activated ester sites continue to cross-link with the protein in the oral mucosa, thereby fixing the polyethylene glycol lysozyme complex on the tissue, and on the other hand, the encapsulation effect of the polymer microstructure can reduce the risk of lysozyme being washed away due to direct contact with saliva, and significantly enhance the retention efficiency of lysozyme.

[0006] To achieve this object, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a stomatitis composition based on micro-cross-linked polyethylene glycol lysozyme, wherein the stomatitis composition comprises polyethylene glycol carboxyl activated ester, polyethylene glycol, chitosan and lysozyme.

[0008] In the present invention, a composite spray composition is prepared by using polyethylene glycol carboxyl activated ester and lysozyme, and the microstructure formed by micro-crosslinking with lysozyme is used to enhance the retention efficiency of lysozyme by utilizing the size effect, overcome the defect that a single lysozyme is easily lost, and significantly improve the retention time of lysozyme. The principle is that the polyethylene glycol carboxyl activated ester is crosslinked with lysozyme on the one hand, and crosslinked with the protein in the oral mucosa on the other hand, thereby fixing the polyethylene glycol lysozyme complex on the tissue, further improving the retention efficiency. Increase the retention time of lysozyme, and overcome the defect that a single lysozyme is easily lost. The principle is that, on the one hand, after the polyethylene glycol carboxyl activated ester partial sites are crosslinked with lysozyme, the remaining uncrosslinked activated ester sites continue to crosslink with the protein in the oral mucosa, thereby fixing the polyethylene glycol lysozyme complex on the tissue, prolonging the retention time of lysozyme; on the other hand, the encapsulation effect of the polymer microstructure can reduce the risk of lysozyme being washed away due to direct contact with saliva, and significantly enhance the retention efficiency of lysozyme.

[0009] Preferably, the stomatitis composition comprises, by mass, 0.001-20 parts of polyethylene glycol carboxyl activated ester, 0.1-10 parts of polyethylene glycol, 0.01-25 parts of chitosan, and 0.0001-20 parts of lysozyme.

[0010] The 0.001-20 parts can be, for example, 0.001 parts, 0.01 parts, 0.1 parts, 1 parts, 2 parts, 3 parts, 4 parts, 5 parts, 6 parts, 7 parts, 8 parts, 9 parts, 10 parts, 11 parts, 12 parts, 13 parts, 14 parts, 15 parts, 16 parts, 17 parts, 18 parts, 19 parts or 20 parts, etc.

[0011] The 0.1-10 parts may be, for example, 0.01 parts, 0.1 parts, 1 parts, 2 parts, 3 parts, 4 parts, 5 parts, 6 parts, 7 parts, 8 parts, 9 parts or 10 parts.

[0012] The 0.01-25 parts of chitosan can be, for example, 0.01 parts, 0.1 parts, 1 parts, 2 parts, 3 parts, 4 parts, 5 parts, 6 parts, 7 parts, 8 parts, 9 parts, 10 parts, 11 parts, 12 parts, 13 parts, 14 parts, 15 parts, 16 parts, 17 parts, 18 parts, 19 parts, 20 parts, 21 parts, 22 parts, 23 parts, 24 parts or 25 parts, etc.

[0013] The 0.0001-20 parts can be, for example, 0.0001 parts, 0.001 parts, 0.01 parts, 0.1 parts, 1 parts, 2 parts, 3 parts, 4 parts, 5 parts, 6 parts, 7 parts, 8 parts, 9 parts, 10 parts, 11 parts, 12 parts, 13 parts, 14 parts, 15 parts, 16 parts, 17 parts, 18 parts, 19 parts or 20 parts, etc.

[0014] Preferably, the polyethylene glycol carboxyl activated ester may be any one of Formula I, Formula II, Formula III and Formula IV:

[0015]

[0016] Wherein, in each formula, Z is a natural number between 20 and 300, and may be 29 to 228, 29, 34, 38, 45, 57, 136, 181 or 228; the group R is formula A or formula B, in which M represents a nitrogen atom, an oxygen atom, a sulfur atom or a phosphorus atom, preferably an oxygen atom, a nitrogen atom or a sulfur atom, connected to the end of the polyethylene glycol, and X or Y is an integer between 1 and 5, such as 0, 2 or 3; the group R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 , R 14 , R 15 and R 16 Each of them is independently a hydrogen atom, a hydroxyl group, an alkyl group having 1 to 10 carbon atoms (preferably a methyl group), a phenyl group or a sulfonic acid group.

[0017] Preferably, the polyethylene glycol carboxyl activated ester may be any one of the following 1)-8):

[0018] 1) As shown in formula I, wherein R is formula A, in which X is 2 to 3, M is an oxygen atom, Z is 136, and R 1 , R 2 , R 3 and R 4 All are hydrogen atoms;

[0019] 2) As shown in formula I, wherein R is formula B, in which Y is 1 to 2, M is an oxygen atom, Z is 228, and R 1 , R 2 , R 3 and R 4 All are hydrogen atoms;

[0020] 3) As shown in formula II, wherein R is formula A, in which X is 2 to 3, M is an oxygen atom, Z is 57, and R 1 , R 2 , R 3 and R 4 are all hydrogen atoms, R 5 , R 6 , R 7 and R 8 All are hydroxyl groups;

[0021] 4) As shown in formula II, wherein R is formula B, in which Y is 1 to 2, M is an oxygen atom, Z is 57, and R 1 , R 2 , R 3 and R 4 are all hydrogen atoms, R 5 , R 6 , R 7 and R 8 All are hydroxyl groups;

[0022] 5) As shown in formula III, wherein R is formula A, in which X is 2 to 3, M is a nitrogen atom, Z is 38, and R 1 , R 2 , R 3 and R 4 are all hydrogen atoms, R 5 , R 6 , R 7 and R 8 All are methyl, R 9 , R 10 , R 11 and R 12 is phenyl;

[0023] 6) As shown in formula III, wherein R is formula B, in which Y is 1 to 2, M is a sulfur atom, Z is 38, and R 1 , R 2 , R 3 and R 4 are all hydrogen atoms, R 5 , R 6 , R 7 and R 8 All are sulfonic acid groups, R 9 , R 10 , R 11 and R 12 is phenyl;

[0024] 7) As shown in Formula IV, wherein R is Formula A, wherein X is 3 to 5, M is a nitrogen atom, Z is 29, and R 1 , R 2 , R 3 , R 4 , R5 , R 6 , R 7 and R 8 All are sulfonic acid groups, R 9 , R 10 , R 11 , R 12 , R 13 , R 14 , R 15 and R 16 is a hydrogen atom;

[0025] 8) As shown in formula IV, wherein R is formula B, in which Y is 2 to 4, M is a nitrogen atom, Z is 29, and R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 All are sulfonic acid groups, R 9 , R 10 , R 11 , R 12 , R 13 , R 14 , R 15 and R 16 A hydrogen atom.

[0026] Preferably, the molecular weight of the polyethylene glycol is 200-800, for example, 200, 400, 600, or 800.

[0027] Preferably, the mass ratio of the polyethylene glycol carboxyl activated ester to lysozyme is (0.01-1):(0.01-8). The (0.01-1) may be, for example, 0.01, 0.1, 0.2, 0.4, 0.6, 0.8 or 1. The (0.01-8) may be, for example, 0.01, 0.1, 1, 2, 3, 4, 5, 6, 7 or 8.

[0028] In the present invention, the microstructure formed by cross-linking the polyethylene glycol carboxyl activated ester and lysozyme can form a cross-linked structure for all lysozymes within the above-mentioned ratio range, thereby maximizing the retention time of all lysozymes and further improving the effect.

[0029] Preferably, the mass ratio of chitosan to lysozyme is (0.1-20):(0.1-20), and the (0.1-20) can be, for example, 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, etc.

[0030] In the present invention, chitosan is a product obtained by removing some acetyl groups from natural polysaccharide chitin, has strong antibacterial and moisturizing functions, and does not cause any allergic irritation reactions. Within the above-mentioned proportion range, it can greatly reduce the influence of external factors on lysozyme activity, further improving the use effect.

[0031] In a second aspect, the present invention provides a stomatitis inhibitor based on micro-cross-linked polyethylene glycol lysozyme, wherein the stomatitis inhibitor comprises the stomatitis composition based on micro-cross-linked polyethylene glycol lysozyme described in the first aspect, a dissolving agent, chlorhexidine acetate, L-lysine, water-soluble vitamins and trace elements.

[0032] Preferably, the dissolving agent comprises sterile water and / or a buffer.

[0033] Preferably, the buffer comprises any one or a combination of at least two of phosphate buffer, phosphate-buffered saline, Tris-hydrochloric acid buffer, glycine-hydrochloric acid buffer, disodium hydrogen phosphate-sodium citrate buffer, citric acid-sodium hydroxide-hydrochloric acid buffer, citric acid-sodium citrate buffer, disodium hydrogen phosphate-potassium dihydrogen phosphate buffer, potassium dihydrogen phosphate-sodium hydroxide buffer, boric acid-borax buffer, borax-sodium hydroxide buffer or sodium carbonate-sodium bicarbonate buffer.

[0034] Preferably, the concentration of the buffer solution is 0.001-2M, for example, 0.001M, 0.01M, 0.1M, 0.5M, 1M, 1.5M or 2M.

[0035] Preferably, the pH of the dissolving agent is 7-8, for example, it can be 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8, etc.

[0036] In the present invention, the pH of the solvent will also affect the cross-linking reaction between the polyethylene glycol carboxyl activated ester and the lysozyme. If the pH is too high, the polyethylene glycol carboxyl activated ester is easily hydrolyzed and inactivated. If the pH is too low, the amino or thiol groups in the lysozyme are easily protonated, resulting in the obstruction of the cross-linking reaction.

[0037] Preferably, the concentration of the stomatitis composition in the stomatitis inhibitor is 0.1-75%, for example, it can be 0.1%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75%.

[0038] In the present invention, the mass volume of the stomatitis composition is the key factor affecting the effect. If the concentration of the stomatitis composition is too low, the effect will be poor, and if the concentration of the stomatitis composition is too high, the effect will not be improved much.

[0039] In the present invention, lysozyme is the main antibacterial component, but in order to ensure a more efficient antibacterial effect, a trace amount of auxiliary antibacterial agent can be added, including chlorhexidine acetate and L-lysine. Although chlorhexidine acetate belongs to a broad-spectrum antibacterial drug, L-lysine also has a good antibacterial effect on most bacteria, when treating stomatitis, the drug concentration is too low and the antibacterial effect is not ideal; and the drug concentration is too high, which is prone to increase in secretions and burning pain. Therefore, it is necessary to strictly control the concentration of these two substances. The two antibacterial substances are used in common with lysozyme, which can not only significantly reduce their concentrations and avoid undesirable side reactions, but also can be used as auxiliary antibacterial components to increase the final antibacterial effect of the preparation.

[0040] Preferably, the mass concentration of chlorhexidine acetate in the stomatitis inhibitor is 0.001%-0.005%, for example, it can be 0.001%, 0.002%, 0.003%, 0.004% or 0.005%.

[0041] Preferably, the mass concentration of L-lysine in the stomatitis inhibitor is 0.01%-2%, for example, it can be 0.01%, 0.1%, 0.2%, 0.4%, 0.6%, 0.8%, 1%, 1.2%, 1.4%, 1.6%, 1.8% or 2%, etc.

[0042] Preferably, the water-soluble vitamins include any one of vitamin B1, vitamin B2, vitamin B6, vitamin B12, and vitamin C, or a combination of at least two of them.

[0043] In the present invention, water-soluble vitamins and trace elements are mainly used as auxiliary additives, because they mainly act as coenzymes or prosthetic group components in the body and participate in material metabolism and energy metabolism. Therefore, adding these ingredients to the preparation can assist in regulating the oral inflammatory microenvironment.

[0044] Preferably, the mass concentration of the water-soluble vitamin in the stomatitis inhibitor is 0.01-1%, for example, it can be 0.01%, 0.1%, 0.2%, 0.4%, 0.6%, 0.8% or 1%, etc.

[0045] Preferably, the trace elements include any one of iron, copper and zinc or a combination of at least two of them.

[0046] Preferably, the mass concentration of the trace elements in the stomatitis inhibitor is 0.01-0.1%, for example, it can be 0.01%, 0.1%, 0.2%, 0.4%, 0.6%, 0.8% or 1%, etc.

[0047] In a third aspect, the present invention provides a method for preparing a stomatitis inhibitor based on micro-cross-linked polyethylene glycol lysozyme according to the second aspect, the preparation method comprising dissolving polyethylene glycol carboxyl activated ester, polyethylene glycol, chitosan, lysozyme, chlorhexidine acetate, L-lysine, water-soluble vitamins and trace elements in a solvent and then mixing.

[0048] In the present invention, the preparation method of the stomatitis inhibitor is quick and convenient, which reduces the preparation cost in industrial production and greatly improves the application scenarios of the preparation method.

[0049] Preferably, the preparation method comprises the steps of: adding polyethylene glycol carboxyl activated ester, polyethylene glycol, chitosan and lysozyme to a dissolving agent for a first mixing, and then adding chlorhexidine acetate, L-lysine, water-soluble vitamins and trace elements for a second mixing.

[0050] Preferably, the stirring speed of the first mixing is 200-1000 rpm, and the time is 1-10 min. The 200-1000 rpm may be, for example, 200 rpm, 400 rpm, 600 rpm, 800 rpm or 1000 rpm, etc. The 1-10 min may be, for example, 1 min, 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min or 10 min, etc.

[0051] Preferably, the stirring speed of the second mixing is 200-1000 rpm, and the time is 1-5 min. The 200-1000 rpm may be, for example, 200 rpm, 400 rpm, 600 rpm, 800 rpm or 1000 rpm, etc. The 1-5 min may be, for example, 1 min, 2 min, 3 min, 4 min or 5 min, etc.

[0052] In a fourth aspect, the present invention provides a method for preparing a stomatitis composition based on micro-cross-linked polyethylene glycol lysozyme according to the first aspect, a stomatitis inhibitor based on micro-cross-linked polyethylene glycol lysozyme according to the second aspect, or a stomatitis inhibitor based on micro-cross-linked polyethylene glycol lysozyme according to the third aspect, and use thereof in medicines or daily necessities for preventing and treating stomatitis.

[0053] Compared with the prior art, the present invention has at least the following beneficial effects:

[0054] The present invention prepares a composite spray through a micro-crosslinking reaction between polyethylene glycol carboxyl activated ester and lysozyme. On the one hand, after the polyethylene glycol carboxyl activated ester partial sites are cross-linked with lysozyme, the remaining uncross-linked activated ester sites continue to cross-link with proteins in the oral mucosa, thereby fixing the polyethylene glycol lysozyme complex on the tissue and prolonging the retention time of the lysozyme; on the other hand, by utilizing the encapsulation effect of the polymer microstructure, the risk of lysozyme being washed away due to direct contact with saliva can be reduced, the retention efficiency of lysozyme can be significantly enhanced, and the defect of easy loss of a single lysozyme can be overcome. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 This is an electron micrograph of a stomatitis inhibitor. DETAILED DESCRIPTION

[0056] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and through specific implementation methods. However, the following examples are only simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0057] Example 1

[0058] This embodiment provides a stomatitis inhibitor

[0059] The stomatitis composition is 3 parts of two-arm polyethylene glycol succinimidyl succinate 6000 (Formula 1), 8 parts of chitosan, 4 parts of polyethylene glycol 400 and 9 parts of lysozyme. Two-arm polyethylene glycol succinimidyl succinate 6000, chitosan, polyethylene glycol 400 and lysozyme are added to a phosphate buffer with a concentration of 0.1M, a pH of 7.4, and a stirring speed of 500rpm and a stirring time of 5min for the first mixing. Then, chlorhexidine acetate is added to a concentration of 0.002%, L-lysine is added to a concentration of 1%, water-soluble vitamin B1 is added to a concentration of 0.02%, vitamin B2 is 0.02%, vitamin B6 is 0.01%, vitamin B12 is 0.05%, vitamin C is 0.4%, trace elements iron are added to a concentration of 0.03%, copper is added to a concentration of 0.01%, and zinc is added to a concentration of 0.01%, and a second mixing is performed. The second mixing was performed at a stirring speed of 500 rpm and a stirring time of 5 min to finally obtain a stomatitis inhibitor. The mass concentration of the stomatitis composition in the stomatitis inhibitor was 10.5%.

[0060]

[0061] Example 2

[0062] This embodiment provides a stomatitis inhibitor

[0063] The stomatitis composition is 0.01 parts of two-arm polyethylene glycol succinimidyl succinate 6000 (Formula 1), 0.01 parts of chitosan, 0.1 parts of polyethylene glycol 200 and 0.01 parts of lysozyme. Four-arm polyethylene glycol succinimidyl succinate 8000, chitosan, polyethylene glycol 400 and lysozyme are added to a phosphate buffer with a concentration of 0.1M and a pH of 7.4, and the first mixing is performed at a stirring speed of 500 rpm and a stirring time of 10 minutes. Then, chlorhexidine acetate is added to a concentration of 0.005%, L-lysine is added to a concentration of 0.01%, water-soluble vitamin B1 is added to a concentration of 0.002%, vitamin B2 is 0.002%, vitamin B6 is 0.001%, vitamin B12 is 0.002%, vitamin C is 0.003%, trace elements iron is added to a concentration of 0.05%, copper is added to a concentration of 0.02%, and zinc is added to a concentration of 0.03%, and a second mixing is performed. The second mixing is performed at a stirring speed of 500 rpm and a stirring time of 2 minutes to finally obtain a stomatitis inhibitor. The mass concentration of the stomatitis composition in the stomatitis inhibitor is 0.13%.

[0064]

[0065] Example 3

[0066] This embodiment provides a stomatitis inhibitor

[0067] The stomatitis composition is 20 parts of six-arm polyethylene glycol succinimidyl succinate 10000 (Formula 2), 25 parts of chitosan, 10 parts of polyethylene glycol 600 and 20 parts of lysozyme. Two-arm polyethylene glycol succinimidyl succinate 6000, chitosan, polyethylene glycol 600 and lysozyme are added to a phosphate buffer with a concentration of 1.5M, a pH of 7.7, and a stirring speed of 500rpm for a stirring time of 10min for the first mixing. Then, chlorhexidine acetate is added to a concentration of 0.01%, L-lysine is added to a concentration of 2%, water-soluble vitamin B1 is added to a concentration of 0.1%, vitamin B2 is 0.2%, vitamin B6 is 0.2%, vitamin B12 is 0.2%, vitamin C is 0.3%, and trace element iron is added to a concentration of 0.01%, and a second mixing is performed. The second mixing is performed at a stirring speed of 500rpm for a stirring time of 1min to finally obtain a stomatitis inhibitor.

[0068] The mass concentration of the stomatitis composition in the stomatitis inhibitor is 75%.

[0069]

[0070] Example 4

[0071] This embodiment provides a stomatitis inhibitor

[0072] The stomatitis composition is 0.025 parts of six-arm polyethylene glycol succinimidyl succinate 10000 (Formula 2), 0.1 parts of chitosan, 5 parts of polyethylene glycol 400 and 20 parts of lysozyme. Four-arm polyethylene glycol succinimidyl succinate 10000, chitosan, polyethylene glycol 400 and lysozyme are added to a phosphate buffer with a concentration of 2M, a pH of 8, and a stirring speed of 500rpm and a stirring time of 1min for the first mixing. Then, chlorhexidine acetate is added to a concentration of 0.05%, L-lysine is added to a concentration of 0.1%, water-soluble vitamin B1 is added to a concentration of 0.02%, vitamin B2 is 0.02%, vitamin B6 is 0.02%, vitamin B12 is 0.04%, vitamin C is 0.4%, and trace element zinc is added to a concentration of 0.05%, and a second mixing is performed. The second mixing was performed at a stirring speed of 500 rpm and a stirring time of 5 min to finally obtain the stomatitis inhibitor.

[0073] The mass concentration of the stomatitis composition in the stomatitis inhibitor is 28.8%.

[0074]

[0075] Example 5

[0076] This example provides a stomatitis inhibitor, which is different from Example 1 only in that 1 part of two-arm polyethylene glycol succinimidyl succinate 6000 and 30 parts of lysozyme are used, and the rest is the same as Example 1.

[0077] Example 6

[0078] This example provides a stomatitis inhibitor, which is different from Example 1 only in that 1 part of two-arm polyethylene glycol succinimidyl succinate 6000 and 0.00001 part of lysozyme are used, and the rest is the same as Example 1.

[0079] Example 7

[0080] This embodiment provides a stomatitis inhibitor, which is different from the embodiment 1 only in that 0.005 parts of chitosan and 20 parts of lysozyme are used, and the rest is the same as the embodiment 1.

[0081] Example 8

[0082] This embodiment provides a stomatitis inhibitor, which is different from the embodiment 1 only in that 30 parts of chitosan and 10 parts of lysozyme are used, and the rest is the same as the embodiment 1.

[0083] Example 9

[0084] This embodiment provides a stomatitis inhibitor, which is different from Embodiment 1 only in that polyethylene glycol 400 is used to replace polyethylene glycol 100, and the rest is the same as Embodiment 1.

[0085] Example 10

[0086] This embodiment provides a stomatitis inhibitor, which is different from the embodiment 1 only in that the concentration of the stomatitis composition in the stomatitis inhibitor is 90%, and the rest is the same as the embodiment 1.

[0087] Comparative Example 1

[0088] This comparative example provides a stomatitis inhibitor, which is different from Example 1 only in that the stomatitis composition does not include two-arm polyethylene glycol succinimidyl succinate 6000, and the rest is consistent with Example 1.

[0089] Comparative Example 2

[0090] This comparative example provides a stomatitis inhibitor, which is different from Example 1 only in that the stomatitis composition does not include chitosan, and the rest is consistent with Example 1.

[0091] Comparative Example 3

[0092] This comparative example provides a stomatitis inhibitor, which is different from Example 1 only in that the stomatitis composition does not include polyethylene glycol 400, and the rest is consistent with Example 1.

[0093] Comparative Example 4

[0094] This comparative example provides a stomatitis inhibitor, which is different from Example 1 only in that the stomatitis composition does not include lysozyme, and the rest is consistent with Example 1.

[0095] Test Example 1

[0096] This test example tests the in vitro retention efficiency of lysozyme of the stomatitis inhibitors prepared in the above examples and comparative examples.

[0097] Ideally, human buccal mucosa tissue is the most suitable for in vitro studies related to oral mucosal administration, but it is rarely used due to ethics and difficulty in obtaining. In actual studies, in vitro animal tissue models with similar properties to human buccal mucosa are often used for related studies. The oral mucosa of experimental animals is separated from the oral cavity together with the submucosal connective tissue by surgical operation, and then the connective tissue is removed to obtain. In experimental animals, the buccal mucosa of rats and hamsters is keratinized tissue; only part of the buccal mucosa of rabbits is not keratinized, but the unkeratinized part is difficult to distinguish and separate from the keratinized part; the buccal mucosa tissue of monkeys, dogs and pigs is not keratinized, but the buccal mucosa of monkeys and dogs is thinner than that of humans, while the buccal mucosa of pigs is most similar to that of humans, and the cost is low, which is the most commonly used in vitro research animal model related to oral mucosal administration. Here, the buccal mucosa of pigs is selected for subsequent in vitro evaluation. The stomatitis inhibitors prepared in the above Examples 1-10 and Comparative Examples 1-4 were subjected to the in vitro retention efficiency of lysozyme, with 3 replicate samples in each group.

[0098] A layer of pig buccal mucosa tissue with a diameter of 1 cm was laid on the bottom of the 24-well plate to simulate the oral mucosal environment. Then 50 μL of the stomatitis inhibitors prepared in the above-mentioned Examples 1-10 and Comparative Examples 1-4 were added thereto and left to stand for 1 min. Then, sterile saline was used to rinse to simulate the flushing environment of oral fluid. The flushing time was used as a single research variable to evaluate the retention efficiency of lysozyme on the mucosa. The flushing time was set to 1 min, 2 min, 3 min, 4 min, and 5 min in sequence. After flushing, the 24-well plate was placed on an ELISA reader and its residual absorbance value was detected at 280 nm and 37 ° C. In addition, after adding 50 μL of the stomatitis inhibitors prepared in the above-mentioned Examples 1-10 and Comparative Examples 1-4, an absorbance test was performed before flushing with saline, and the test results were used as the original absorbance values ​​of the corresponding experimental groups. The retention efficiency of lysozyme was calculated according to the following formula: retention efficiency = (original absorbance - residual absorbance) / original absorbance × 100%.

[0099] The specific results are shown in Tables 1 to 5 below:

[0100] Table 1

[0101]

[0102]

[0103] Table 2

[0104]

[0105] Table 3

[0106]

[0107]

[0108] Table 4

[0109]

[0110]

[0111] Table 5

[0112]

[0113] The test results show that:

[0114] (1) By comparing Example 1 with Examples 2-4, it can be seen that the stomatitis preparations prepared within the scope of the present invention all have a high lysozyme retention efficiency, that is, after rinsing with physiological saline for 5 minutes, the in situ retention efficiency of lysozyme is still more than 85%.

[0115] (2) By comparing Example 1 with Examples 5-6, it can be seen that the polyethylene glycol carboxyl activated ester and lysozyme outside the scope of the invention will reduce the retention efficiency of lysozyme. This is because when the mass fraction of polyethylene glycol carboxyl activated ester or lysozyme is reduced, there are fewer cross-linking sites, and the micro-cross-linking structure formed is unstable, so the retention in the oral fluid environment is insufficient.

[0116] (3) By comparing Example 1 with Examples 7-8, it can be seen that chitosan and lysozyme outside the scope of the invention will reduce the retention efficiency of lysozyme. This is because an appropriate concentration of chitosan is conducive to increasing the viscosity of the microgel system formed by polyethylene glycol carboxyl activated ester and lysozyme, and improving the in-situ retention capacity of the gel. When the chitosan concentration is too high, it will hinder the cross-linking reaction between polyethylene glycol carboxyl activated ester and lysozyme; and when the chitosan concentration is too low, the viscosity contribution value is small.

[0117] (4) By comparing Example 1 with Example 9, it can be seen that the molecular weight of polyethylene glycol outside the scope of the invention will reduce the retention efficiency of lysozyme. This is because when the molecular weight of polyethylene glycol is low, the chain entanglement and intermolecular interaction of the macromolecular chain are weak, resulting in an unstable microstructure formed by the polyethylene glycol carboxyl activated ester and lysozyme; and when the molecular weight of polyethylene glycol is high, the steric hindrance effect of the macromolecule limits the cross-linking reaction between it and lysozyme.

[0118] (5) By comparing Example 1 with Example 10, it can be seen that the concentration of the stomatitis composition outside the scope of the invention will reduce the retention efficiency of lysozyme. This is because the covalent adhesion ability of polyethylene glycol lysozyme is closely related to its concentration. When the concentration is low, the liquid environment of the oral cavity will significantly weaken the covalent adhesion of polyethylene glycol lysozyme on the surface of the oral mucosa; when the concentration is high, the contact area between polyethylene glycol lysozyme and the mucosa is limited, resulting in a decrease in the actual adhesion efficiency. (6) By comparing Example 1 with Comparative Examples 1-4, it can be seen that polyethylene glycol carboxyl activated ester, chitosan, polyethylene glycol and lysozyme all play an important role in improving the retention efficiency of lysozyme in the prepared stomatitis preparation, and there is a synergistic effect between them.

[0119] Test Example 2

[0120] This test example tests the antibacterial effect of the stomatitis inhibitors prepared in the above examples and comparative examples.

[0121] The stomatitis inhibitors prepared in the above Examples 1-10 and Comparative Examples 1-4 and a blank control group (phosphate buffer with a concentration of 0.1 M) were tested for their in vitro antibacterial effects, with 3 replicate samples in each group.

[0122] A layer of pig buccal mucosa tissue with a diameter of 1 cm was spread on the bottom of a 24-well plate to simulate the oral mucosal environment. Then, 50 μL of blank control and the stomatitis inhibitors prepared in Examples 1-10 and Comparative Examples 1-4 were added thereto and allowed to stand for 1 minute. After rinsing with sterile saline for 5 minutes, bacterial solution was added. Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 25923) were used as Gram-negative and Gram-positive bacteria models. The bacterial solution in the logarithmic growth phase was diluted to 1×10 5 CFU / mL, 1 mL of bacterial solution was added to each well. Subsequently, the plates were transferred to a 37°C incubator for 4 h. After the incubation, the bacterial suspension was diluted 1000 times with phosphate buffer, and the diluted bacterial suspension was plated on agar medium. After 24 h of incubation at 37°C, the corresponding plates were photographed, and the colony counts were performed after visual inspection. The specific results are shown in Tables 6 and 7.

[0123] The inhibition rate was calculated according to the following formula: inhibition rate (%) = (blank control group - experimental group) / blank control group × 100%.

[0124] Table 6

[0125]

[0126]

[0127] Table 7

[0128]

[0129] The test results show that:

[0130] (1) By comparing Example 1 with Examples 2-4, it can be seen that the stomatitis preparation prepared within the limited scope of the present invention has a good antibacterial effect, and the antibacterial rates against Escherichia coli and Staphylococcus aureus are both over 85%.

[0131] (2) By comparing Example 1 with Examples 5-6, it can be seen that the polyethylene glycol carboxyl activated ester and lysozyme outside the scope of the invention will reduce the antibacterial effect of the stomatitis preparation. This is because outside the scope of the invention, the in situ retention efficiency of lysozyme decreases, resulting in a decrease in its effective antibacterial ability.

[0132] (3) By comparing Example 1 with Examples 7-8, it can be seen that chitosan and lysozyme outside the scope of the invention will reduce the antibacterial effect of the stomatitis preparation.

[0133] (4) By comparing Example 1 with Example 9, it can be seen that the molecular weight of polyethylene glycol outside the specified range of the invention will reduce the antibacterial effect of the stomatitis preparation.

[0134] (5) By comparing Example 1 with Example 10, it can be seen that the concentration of the stomatitis composition is outside the range specified by the invention, which will reduce the antibacterial effect of the stomatitis preparation.

[0135] (6) By comparing Example 1 with Comparative Examples 1-4, it can be seen that polyethylene glycol carboxyl activated ester, chitosan, polyethylene glycol and lysozyme all play an important role in improving the antibacterial effect of the prepared stomatitis preparation.

[0136] In summary, outside the scope of the invention, the in situ retention efficiency of lysozyme decreases, which leads to a decrease in its effective antibacterial ability, ultimately leading to the above conclusions (2)-(6).

[0137] Test Example 3

[0138] This test case detects the micro-cross-linked structure of stomatitis inhibitors

[0139] The morphology of the samples was analyzed using a Lorentz transmission electron microscope (TEM, instrument model Talos F200X, Thermo Fisher). Figure 1 As shown in the figure, pure lysozyme appears as dispersed tiny particles, and there are some irregular small aggregates, while polyethylene glycol lysozyme appears as evenly dispersed spheres, and its size is significantly larger than the original. This is because polyethylene glycol carboxyl activated ester is a polymer modifier. After modifying lysozyme, lysozyme presents a macromolecular aggregation morphology, so the size increases, and the microscopic morphology tends to the ordered morphology of macromolecular self-assembly.

[0140] In summary, the stomatitis composition prepared by the present invention through polyethylene glycol carboxyl activated ester, polyethylene glycol, chitosan and lysozyme increases the retention time of lysozyme, thereby significantly improving the antibacterial effect.

[0141] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A stomatitis composition based on micro-cross-linked polyethylene glycol lysozyme, characterized in that: The stomatitis composition comprises polyethylene glycol carboxyl activated ester, polyethylene glycol, chitosan and lysozyme.

2. The stomatitis composition according to claim 1, characterized in that The stomatitis composition comprises, by weight, 0.001-20 parts of polyethylene glycol carboxyl activated ester, 0.1-10 parts of polyethylene glycol, 0.01-25 parts of chitosan and 0.0001-20 parts of lysozyme; Preferably, the polyethylene glycol carboxyl activated ester may be any one of Formula I, Formula II, Formula III and Formula IV: Wherein, in each formula, Z is a natural number between 20 and 300, and may be 29 to 228, 29, 34, 38, 45, 57, 136, 181 or 228; the group R is formula A or formula B, in which M represents a nitrogen atom, an oxygen atom, a sulfur atom or a phosphorus atom, preferably an oxygen atom, a nitrogen atom or a sulfur atom, connected to the end of the polyethylene glycol, and X or Y is an integer between 1 and 5, such as 0, 2 or 3; the groups R1, R2, R3, R4, R5, R6, R7, R8, R9, R 10 , R 11 , R 12 , R 13 , R 14 , R 15 and R 16 are independently a hydrogen atom, a hydroxyl group, an alkyl group having 1 to 10 carbon atoms, a phenyl group or a sulfonic acid group; Preferably, the polyethylene glycol carboxyl activated ester may be any one of the following 1)-8): 1) As shown in formula I, wherein R is formula A, in formula A, X is 2 to 3, M is an oxygen atom, Z is 136, and R1, R2, R3 and R4 are all hydrogen atoms; 2) As shown in formula I, wherein R is formula B, in formula B, Y is 1 to 2, M is an oxygen atom, Z is 228, and R1, R2, R3 and R4 are all hydrogen atoms; 3) As shown in formula II, wherein R is formula A, in formula A, X is 2 to 3, M is an oxygen atom, Z is 57, R1, R2, R3 and R4 are all hydrogen atoms, and R5, R6, R7 and R8 are all hydroxyl groups; 4) As shown in formula II, wherein R is formula B, in formula B, Y is 1 to 2, M is an oxygen atom, Z is 57, R1, R2, R3 and R4 are all hydrogen atoms, and R5, R6, R7 and R8 are all hydroxyl groups; 5) As shown in formula III, wherein R is formula A, in which X is 2 to 3, M is a nitrogen atom, Z is 38, R1, R2, R3 and R4 are all hydrogen atoms, R5, R6, R7 and R8 are all methyl groups, R9, R 10 , R 11 and R 12 is phenyl; 6) As shown in formula III, wherein R is formula B, in which Y is 1 to 2, M is a sulfur atom, Z is 38, R1, R2, R3 and R4 are all hydrogen atoms, R5, R6, R7 and R8 are all sulfonic acid groups, R9, R 10 , R 11 and R 12 is phenyl; 7) As shown in Formula IV, wherein R is Formula A, in which X is 3 to 5, M is a nitrogen atom, Z is 29, R1, R2, R3, R4, R5, R6, R7 and R8 are all sulfonic acid groups, R9, R 10 , R 11 , R 12 , R 13 , R 14 , R 15 and R 16 is a hydrogen atom; 8) As shown in Formula IV, wherein R is Formula B, in Formula B, Y is 2 to 4, M is a nitrogen atom, Z is 29, R1, R2, R3, R4, R5, R6, R7 and R8 are all sulfonic acid groups, R9, R 10 , R 11 , R 12 , R 13 , R 14 , R 15 and R 16 is a hydrogen atom; Preferably, the molecular weight of the polyethylene glycol is 200-800.

3. The stomatitis composition according to claim 1, characterized in that The mass ratio of the polyethylene glycol carboxyl activated ester to lysozyme is (0.01-1):(0.01-8); Preferably, the mass ratio of chitosan to lysozyme is (0.1-20):(0.1-20).

4. A stomatitis inhibitor based on micro-cross-linked polyethylene glycol lysozyme, characterized in that: The stomatitis inhibitor comprises the stomatitis composition based on micro-cross-linked polyethylene glycol lysozyme according to any one of claims 1 to 3, a dissolving agent, chlorhexidine acetate, L-lysine, water-soluble vitamins and trace elements.

5. The stomatitis inhibitor according to claim 4, characterized in that The dissolving agent includes sterile water and / or buffer; Preferably, the buffer comprises any one or a combination of at least two of phosphate buffer, phosphate-buffered saline, Tris-hydrochloric acid buffer, glycine-hydrochloric acid buffer, disodium hydrogen phosphate-sodium citrate buffer, citric acid-sodium hydroxide-hydrochloric acid buffer, citric acid-sodium citrate buffer, disodium hydrogen phosphate-potassium dihydrogen phosphate buffer, potassium dihydrogen phosphate-sodium hydroxide buffer, boric acid-borax buffer, borax-sodium hydroxide buffer or sodium carbonate-sodium bicarbonate buffer; Preferably, the concentration of the buffer is 0.001-2M; Preferably, the pH of the dissolving agent is 7-8.

6. The stomatitis inhibitor according to claim 5, characterized in that The mass concentration of the stomatitis composition in the stomatitis inhibitor is 0.1-75%; Preferably, the mass concentration of chlorhexidine acetate in the stomatitis inhibitor is 0.001%-0.005%; Preferably, the mass concentration of L-lysine in the stomatitis inhibitor is 0.01%-2%; Preferably, the water-soluble vitamins include any one or a combination of at least two of vitamin B1, vitamin B2, vitamin B6, vitamin B12, and vitamin C; Preferably, the mass concentration of the water-soluble vitamins in the stomatitis inhibitor is 0.01-1%; Preferably, the trace elements include any one or a combination of at least two of iron, copper and zinc; Preferably, the mass concentration of the trace elements in the stomatitis inhibitor is 0.01-0.1%.

7. A method for preparing a stomatitis inhibitor based on micro-cross-linked polyethylene glycol lysozyme according to any one of claims 4 to 6, characterized in that: The preparation method comprises dissolving polyethylene glycol carboxyl activated ester, polyethylene glycol, chitosan, lysozyme, chlorhexidine acetate, L-lysine, water-soluble vitamins and trace elements in a solvent and then mixing them evenly.

8. The preparation method according to claim 7, characterized in that: The preparation method comprises the following steps: adding polyethylene glycol carboxyl activated ester, polyethylene glycol, chitosan and lysozyme into a dissolving agent for first mixing, and then adding chlorhexidine acetate, L-lysine, water-soluble vitamins and trace elements for second mixing.

9. The preparation method according to claim 8, characterized in that: The stirring speed of the first mixing is 200-1000 rpm, and the time is 1-10 min; Preferably, the stirring speed of the second mixing is 200-1000 rpm, and the time is 1-5 min.

10. Use of the stomatitis composition based on micro-cross-linked polyethylene glycol lysozyme according to any one of claims 1 to 3 or the stomatitis inhibitor based on micro-cross-linked polyethylene glycol lysozyme according to any one of claims 4 to 6 as a medicine or daily necessities for preventing and treating stomatitis.

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