Preparation and application of a composite polymer material based on hyaluronic acid

Abstract

The present invention relates to the preparation and application of a composite polymer material based on hyaluronic acid, and specifically, provides a composite polymer material based on hyaluronic acid-the preparation and application of a hyaluronic acid derivative, wherein hyaluronic acid and tranexamic acid can be covalently bonded at room temperature, thereby avoiding high energy consumption loss. The hyaluronic acid derivative has the effects of both hyaluronic acid and tranexamic acid, improves the transdermal permeability of tranexamic acid, and also enhances the efficacy of tranexamic acid, and can be applied in the preparation process of cosmetics, food or medicine.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to the preparation and application of a composite polymer material based on hyaluronic acid. Background Art

[0002] Melasma, as a common chronic skin pigmentation disease, has an incidence rate of up to 30%, especially in Asian women of childbearing age. Genetic susceptibility, ultraviolet exposure and fluctuations in sex hormone levels are the three main factors for the disease. In addition, active synthesis of melanocytes, local vascular proliferation, inflammatory response and damage to the skin barrier are also involved. These factors are intertwined and jointly promote the formation and development of melasma. Due to its prominent position on the face and its persistent characteristics, melasma often becomes a major concern in patients' daily lives. It not only affects the patient's external beauty, but is also likely to penetrate into the psychological level, causing negative emotions such as inferiority, anxiety and even depression, which has a significant impact on the patient's quality of life and emotional health. Therefore, the prevention and treatment of melasma is not only a medical need, but also an important way to improve the quality of life of patients.

[0003] In recent years, numerous studies have revealed the great potential of tranexamic acid (TA) as an antifibrinolytic hemostatic drug in the treatment of melasma. TA, as a derivative of lysine, can competitively bind to the lysine site on plasminogen, effectively blocking its conversion to plasmin, inhibiting the subsequent increase in melanin expression caused by various inflammatory factors, and helping to alleviate the symptoms of melasma. In addition, the similarity between TA and tyrosine in chemical structure gives it the ability to competitively inhibit tyrosinase activity, further reducing the production of melanin. However, due to the hydrophilic nature of TA, its permeability in the skin is limited, and it is difficult to directly act on melanocytes in the basal layer of the epidermis. This limitation has become a key issue that needs to be urgently addressed in the treatment of melasma.

[0004] Hyaluronic acid (HA) is a linear polymeric polysaccharide with multifunctional pharmacological properties. It has shown excellent effects in anti-aging, moisturizing, skin repair, anti-inflammatory, tissue regeneration and wound healing. In particular, low molecular weight HA, due to its small molecular size and high affinity with HA receptors on epidermal keratinocytes, has excellent skin penetration ability.

[0005] Combining HA and TA through covalent bonds is an innovative strategy. This method takes advantage of the penetration of HA, allowing TA to penetrate the stratum corneum of the skin more deeply and reach the basal layer. Through this ingenious molecular design, the combination of HA and TA can act more effectively on target cells, thereby reducing melanin production while promoting skin health and repair. This method provides a safer and more effective treatment option for the treatment of pigmented skin diseases such as melasma. Summary of the invention

[0006] The purpose is to provide a composite polymer material based on hyaluronic acid and a preparation method and application thereof based on the existing prior art problems; more specifically, the present invention provides a low-molecular hyaluronic acid derivative, and the low-molecular hyaluronic acid and tranexamic acid are connected by a covalent bond. The low-molecular hyaluronic acid derivative provided by the present invention has good stability and sustained-release transdermal effect, solves the problems of poor stability of hyaluronic acid and poor transdermal absorption of tranexamic acid in existing applications, and the hyaluronic acid derivative has the effects of both hyaluronic acid and tranexamic acid, and has strong moisturizing, repairing, whitening and anti-inflammatory effects.

[0007] The technical solution adopted by the present invention to achieve the above-mentioned purpose is:

[0008] A composite polymer material based on hyaluronic acid has a structure shown in the following formula I:

[0009]

[0010] I

[0011] Wherein, m≥1, n≥0; R is selected from at least one of small molecule amino acid derivatives, polypeptides, oligopeptides, collagen and functional molecules.

[0012] Preferably, R1, R2, R3, and R4 are each selected from a hydrogen group or an acetyl group; or, n≥1.

[0013] Preferably, R is selected from small molecule amino acid derivatives, and small molecule amino acid derivatives are selected from methyl tranexamic acid hydrochloride and L-tryptophan methyl ester hydrochloride; or, R is selected from functional molecules, and functional molecules include 2-aminothiazole-5-carboxylate. Wherein, when methyl tranexamic acid hydrochloride is involved in the preparation of a composite polymer material based on hyaluronic acid, the prepared composite polymer material based on hyaluronic acid shows excellent effects in transdermal, repair, whitening and anti-inflammatory. The present invention allows the amino acid derivatives methyl tranexamic acid hydrochloride, L-tryptophan methyl ester hydrochloride and the functional molecule 2-aminothiazole-5-carboxylate to participate in the preparation of a composite polymer material based on hyaluronic acid, and the prepared composite polymer material based on hyaluronic acid has significantly improved transdermal, repair, whitening and anti-inflammatory effects.

[0014] Preferably, the molecular weight of the composite polymer material based on hyaluronic acid is 3 kDa to 300 kDa; or, the molecular weight of the composite polymer material based on hyaluronic acid is 5 kDa to 20 kDa.

[0015] Preferably, the degree of substitution of R in the composite polymer material based on hyaluronic acid is 0.05-1.0; or, the degree of substitution of R is 0.6-1.0.

[0016] Preferably, a method for preparing a composite polymer material based on hyaluronic acid comprises:

[0017] S1, mixing sodium hyaluronate and an activator to react to generate intermediate 1;

[0018] S2, mixing the intermediate 1 with the R reagent to generate the intermediate 2; the R reagent is selected from at least one of small molecule amino acid derivatives, polypeptides, oligopeptides, collagen and functional molecules;

[0019] S3, performing dialysis separation on the intermediate 2 to obtain a composite polymer material based on hyaluronic acid.

[0020] More preferably, the activator is prepared by reacting 2-chloro-methylpyridinium iodide with an imidazole derivative, and the imidazole derivative includes at least one of N-methylimidazole, N-phenylimidazole and N-(4-methoxyphenyl)imidazole.

[0021] More preferably, a neutralizing agent is used in S2 to adjust the pH of the reaction system, and the neutralizing agent includes at least one of triethylamine aqueous solution, DIEA aqueous solution, KHCO3 aqueous solution and K2CO3 aqueous solution.

[0022] More preferably, the dialysis separation is sequentially treated with an aqueous solution, a NaOH solution, a NaCl solution and an aqueous solution.

[0023] More preferably, the composite polymer material based on hyaluronic acid is used in the preparation of medical materials.

[0024] The present invention provides a hyaluronic acid tranexamic acid derivative, which is a structure formed by covalent bonding of small molecule hyaluronic acid and tranexamic acid. The hyaluronic acid tranexamic acid derivative has a structure shown in the following formula II:

[0025]

[0026] II

[0027] Among them, m≥1, n≥1.

[0028] Preferably, in the structure of the hyaluronic acid and tranexamic acid derivative shown in Formula II, R1, R2, R3, and R4 are selected from hydrogen and acetyl groups, respectively.

[0029] More preferably, R1, R2, R3, and R4 are all hydrogen groups.

[0030] Preferably, R in the structure of the hyaluronic acid and tranexamic acid derivative is at least one of a small molecule amino acid derivative, a polypeptide, an oligopeptide, a collagen and a functional molecule.

[0031] More preferably, the R reagent is at least one of tranexamic acid methyl ester hydrochloride, L-tryptophan methyl ester hydrochloride, 2-aminothiazole-5-carboxylate and L-alanine ethyl ester hydrochloride.

[0032] Preferably, a method for preparing a hyaluronic acid derivative comprises the preparation of AMP and the preparation of a hyaluronic acid derivative.

[0033] More preferably, a method for preparing AMP is specifically:

[0034] 2-Chloro-methylpyridinium iodide (CMPI) is dissolved in acetonitrile, N-methylimidazole is added, reacted at room temperature, filtered, separated to remove the solvent acetonitrile, and freeze-dried to obtain an improved Mukaiyama reagent named AMP.

[0035] Preferably, in a method for preparing AMP, the amount of 2-chloro-methylpyridinium iodide (CMPI) used is 240-280 parts by weight.

[0036] Preferably, in a method for preparing AMP, the amount of acetonitrile used is 3800-4200 parts by volume, and the amount of 2-chloro-methylpyridinium iodide (CMPI) and acetonitrile used is in the ratio of 240-280 g: 3800-4200 ml.

[0037] Preferably, in a method for preparing AMP, the amount of N-methylimidazole used is 150-180 parts by weight.

[0038] Preferably, in a method for preparing AMP, the reaction time is 1 to 3 h.

[0039] Preferably, in a method for preparing AMP, the freeze-drying temperature is -70 to -90°C.

[0040] More preferably, a method for preparing a hyaluronic acid derivative comprises:

[0041] Sodium hyaluronate is dissolved in water, cooled in an ice bath, and then an activator is added to react to generate intermediate 1; R reagent is continuously added to intermediate 1, and the pH is adjusted with a neutralizer, and the reaction system is heated and stirred to generate intermediate 2; after the reaction, intermediate 2 is dialyzed, and after the dialysis is completed, lyophilized to obtain a hyaluronic acid derivative, and the reaction is carried out under an argon atmosphere.

[0042] Preferably, in a method for preparing a hyaluronic acid derivative, the molecular weight of sodium hyaluronate is 3-200 kDa.

[0043] Preferably, in a method for preparing a hyaluronic acid derivative, the amount of sodium hyaluronate used is 180-220 parts by weight.

[0044] Preferably, in a method for preparing a hyaluronic acid derivative, the argon gas is replaced 1 to 5 times.

[0045] Preferably, in a method for preparing a hyaluronic acid derivative, the activator is AMP, and the amount of AMP used is 180-220 parts by weight.

[0046] Preferably, in the method for preparing a hyaluronic acid derivative, the reaction time is 1 to 3 h.

[0047] Preferably, in a method for preparing a hyaluronic acid derivative, the R reagent is at least one of tranexamic acid methyl ester hydrochloride, L-tryptophan methyl ester hydrochloride, 2-aminothiazole-5-carboxylate and L-alanine ethyl ester hydrochloride.

[0048] More preferably, in a method for preparing a hyaluronic acid derivative, the R agent is methyl tranexamic acid hydrochloride.

[0049] More preferably, in a method for preparing a hyaluronic acid derivative, the R reagent is tranexamic acid methyl ester hydrochloride, L-tryptophan methyl ester hydrochloride and 2-aminothiazole-5-carboxylate.

[0050] More preferably, in a method for preparing a hyaluronic acid derivative, the R agent is methyl tranexamic acid hydrochloride, L-tryptophan methyl ester hydrochloride, 2-aminothiazole-5-carboxylate and L-alanine ethyl ester hydrochloride. Among them, when L-alanine ethyl ester hydrochloride is involved in the preparation of hyaluronic acid derivatives, the effects of the prepared hyaluronic acid derivatives in terms of transdermal, repair, whitening and anti-inflammatory are significantly improved, mainly due to L-alanine ethyl ester hydrochloride as an R agent, which has good biocompatibility with hyaluronic acid. Therefore, the prepared hyaluronic acid derivatives are easily absorbed and utilized by the skin, thereby effectively improving the performance of the hyaluronic acid derivatives in terms of transdermal, repair, whitening and anti-inflammatory.

[0051] More preferably, in the method for preparing a hyaluronic acid derivative, the amount of methyl tranexamic acid hydrochloride used is 150-160 parts by weight.

[0052] More preferably, in the method for preparing a hyaluronic acid derivative, the amount of L-tryptophan methyl ester hydrochloride used is 1 to 5 parts by weight.

[0053] More preferably, in the method for preparing a hyaluronic acid derivative, the amount of 2-aminothiazole-5-carboxylate used is 1 to 5 parts by weight.

[0054] More preferably, in the method for preparing a hyaluronic acid derivative, the amount of L-alanine ethyl ester hydrochloride used is 1 to 5 parts by weight.

[0055] Preferably, in a method for preparing a hyaluronic acid derivative, the neutralizing agent is one or more of a triethylamine aqueous solution, a DIEA aqueous solution, a KHCO3 aqueous solution or a K2CO3 aqueous solution.

[0056] More preferably, in a method for preparing a hyaluronic acid derivative, the volume percentage of the K2CO3 aqueous solution is 5-20%.

[0057] More preferably, in a method for preparing a hyaluronic acid derivative, the amount of K2CO3 aqueous solution used is 2-8 parts by volume, and the amount of sodium hyaluronate and K2CO3 aqueous solution used is in the ratio of 180-220 g: 2-8 L.

[0058] Preferably, in the method for preparing a hyaluronic acid derivative, the heating temperature is 20-30°C.

[0059] Preferably, in the method for preparing a hyaluronic acid derivative, the stirring time is 10 to 20 h.

[0060] Preferably, in a method for preparing a hyaluronic acid derivative, the tool used for dialysis is selected from a dialysis bag, and the molecular weight cutoff of the dialysis bag is 900-1100 Da.

[0061] Preferably, in a method for preparing a hyaluronic acid derivative, an aqueous solution, a NaOH solution, a NaCl solution and an aqueous solution are sequentially used in dialysis separation.

[0062] More preferably, in the method for preparing a hyaluronic acid derivative, the dialysis time of the aqueous solution is 12 to 36 h.

[0063] More preferably, in a method for preparing a hyaluronic acid derivative, the molar concentration of the NaOH solution is 0.01-0.1M.

[0064] Preferably, in a method for preparing a hyaluronic acid derivative, the dialysis time of the NaOH solution is 1 to 3 h.

[0065] More preferably, in a method for preparing a hyaluronic acid derivative, the molar concentration of the NaCl solution is 0.05-0.2M.

[0066] More preferably, in the method for preparing a hyaluronic acid derivative, the dialysis time of the NaCl solution is 30 to 50 hours.

[0067] More preferably, in the method for preparing a hyaluronic acid derivative, the dialysis time of the aqueous solution is 60 to 80 h.

[0068] Preferably, in a method for preparing a hyaluronic acid derivative, the freeze-drying temperature is -70 to -90°C.

[0069] Preferably, in the method for preparing a hyaluronic acid derivative, the degree of substitution of R is 0.05-1.0.

[0070] The hyaluronic acid derivative provided by the present invention has the effects of low-molecular hyaluronic acid and tranexamic acid, has the effects of moisturizing, repairing, whitening, etc., delays the enzymatic hydrolysis rate of hyaluronic acid, and increases the transdermal permeability of tranexamic acid.

[0071] The present invention innovatively synthesizes hyaluronic acid derivatives by covalent bonding of hyaluronic acid and tranexamic acid. The hyaluronic acid derivatives have excellent stability and sustained-release transdermal effect, solving the problems of poor stability of hyaluronic acid and poor transdermal absorption of tranexamic acid in existing applications. In addition, the hyaluronic acid derivatives have the effects of both hyaluronic acid and tranexamic acid, and have strong moisturizing, repairing, whitening and anti-inflammatory effects.

[0072] The synthesis route of the invention is simple, and the reaction process does not involve high-boiling-point solvents such as ion exchange resin and DMF, etc., and can be carried out at room temperature. Moreover, the raw materials are economical and readily available, and are easy to industrially scale up for production. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figure 1 This is a graph showing the effect of the test samples on the PGE2 content.

[0074] Figure 2 This is a graph showing the effect of the test samples on tyrosinase activity.

[0075] Figure 3 This is a graph showing the effect of the test samples on melanin production.

[0076] ## indicates the significance analysis relative to the blank group, where ## indicates p < 0.01; *, ** indicate the significance analysis relative to the model group, where * indicates p < 0.05, ** indicates p < 0.01. DETAILED DESCRIPTION

[0077] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0078] The following first describes the concepts involved in the present application in conjunction with the accompanying drawings. It should be noted that the following description of each concept is only to make the content of the present application easier to understand, and does not limit the scope of protection of the present application; at the same time, the embodiments and features in the embodiments of the present application can be combined with each other in the absence of conflict. The present application will be described in detail with reference to the accompanying drawings and in conjunction with the embodiments.

[0079] Embodiment 1:

[0080] A method for preparing a hyaluronic acid tranexamic acid derivative.

[0081] 2-Chloro-methylpyridinium iodide (CMPI) is dissolved in acetonitrile, N-methylimidazole is added, reacted at room temperature, filtered, separated to remove the solvent acetonitrile, and freeze-dried to obtain an improved Mukaiyama reagent named AMP.

[0082] The amount of 2-chloro-methylpyridinium iodide (CMPI) used was 255.5 g, the amount of acetonitrile used was 4000 ml, the amount of N-methylimidazole used was 164.2 g, the reaction time was 2 h, and the freeze-drying temperature was -80°C.

[0083] Sodium hyaluronate was dissolved in water and added to a reactor. Argon was introduced for replacement until the air in the reactor was completely replaced. AMP was added after cooling in an ice bath for reaction. Then methyl tranexamic acid hydrochloride was added. The pH was adjusted with a K2CO3 aqueous solution. The reaction system was heated to room temperature and stirred. After the reaction was completed, dialysis was performed using a dialysis bag, in the order of the first water dialysis, NaOH solution dialysis, NaCl solution dialysis, and the second water dialysis. The solution was freeze-dried to obtain a hyaluronic acid tranexamic acid derivative. The molecular weight of sodium hyaluronate is 5-10kDa, the amount of sodium hyaluronate is 200.0 g, the argon replacement is performed twice, the amount of AMP is 200.0 g, the reaction time is 2h, the amount of methyl tranexamic acid hydrochloride is 155.0 g, the volume percentage of K2CO3 is 10%, the amount of K2CO3 aqueous solution is 5 L, the pH is adjusted to 8, the stirring time is 15 h, the molecular weight cutoff of the dialysis bag is 1000 Da, the first water dialysis time is 24h, the molar concentration of NaOH solution is 0.05M, the dialysis time of NaOH solution is 2 h, the molar concentration of NaCl solution is 0.1M, the dialysis time of NaCl solution is 40 h, the second water dialysis time is 72 h, the freeze-drying temperature is -80°C, and the degree of substitution of R is 0.94.

[0084] Embodiment 2:

[0085] A method for preparing a hyaluronic acid tranexamic acid derivative.

[0086] The difference between this embodiment and embodiment 1 is that the molecular weight of sodium hyaluronate is 20-40 kDa. Other than that, the other conditions are the same as those in embodiment 1.

[0087] The hyaluronic acid tranexamic acid derivative prepared in this example has a substitution degree of R of 0.91.

[0088] Embodiment 3:

[0089] A method for preparing a hyaluronic acid tranexamic acid derivative.

[0090] The difference between this embodiment and embodiment 1 is that the molecular weight of sodium hyaluronate is 100-150 kDa. Apart from this, the other conditions are the same as those of embodiment 1.

[0091] The hyaluronic acid tranexamic acid derivative prepared in this example has a substitution degree of R of 0.84.

[0092] Embodiment 4:

[0093] A method for preparing a hyaluronic acid derivative.

[0094] The difference between this embodiment and embodiment 1 is that L-tryptophan methyl ester hydrochloride and 2-aminothiazole-5-carboxylate are added during the preparation of the hyaluronic acid tranexamic acid derivative, specifically:

[0095] The preparation process of AMP is the same as that in Example 1.

[0096] Sodium hyaluronate is dissolved in water and added to a reactor. Argon is introduced for replacement until the air in the reactor is completely replaced. After cooling in an ice bath, AMP is added for reaction. Subsequently, tranexamic acid methyl hydrochloride, L-tryptophan methyl hydrochloride and 2-aminothiazole-5-carboxylate are added. The pH is adjusted with a K2CO3 aqueous solution. The reaction system is heated to room temperature and stirred. After the reaction is completed, dialysis is performed with a dialysis bag, in the order of the first water dialysis, NaOH solution dialysis, NaCl solution dialysis, and the second water dialysis. The solution is freeze-dried to obtain a hyaluronic acid derivative, i.e., a composite polymer material based on hyaluronic acid. The molecular weight of sodium hyaluronate is 5-10 kDa, the amount of sodium hyaluronate is 200.0 g, the number of argon replacement is 2 times, the amount of AMP is 200.0 g, the reaction time is 2 h, the amount of tranexamic acid methyl ester hydrochloride is 155.0 g, the amount of L-tryptophan methyl ester hydrochloride is 2 g, the amount of 2-aminothiazole-5-carboxylate is 2 g, the volume percentage of K2CO3 aqueous solution is 10%, the amount of K2CO3 aqueous solution is 5 L, the pH is adjusted to 8, the stirring time is 15 h, the molecular weight cutoff of the dialysis bag is 1000 Da, the first water dialysis time is 24 h, the molar concentration of NaOH solution is 0.05 M, the dialysis time of NaOH solution is 2 h, the molar concentration of NaCl solution is 0.1 M, the dialysis time of NaCl solution is 40 h, the second water dialysis time is 72 h, the freeze-drying temperature is -80°C, and the degree of substitution of R is 0.93.

[0097] Embodiment 5:

[0098] A method for preparing a hyaluronic acid derivative.

[0099] The difference between this embodiment and embodiment 4 is that in the preparation process of the hyaluronic acid tranexamic acid derivative, 4 g of L-tryptophan methyl ester hydrochloride and 4 g of 2-aminothiazole-5-carboxylate are added. Apart from this, the other conditions are the same as those in embodiment 4.

[0100] The hyaluronic acid derivative prepared in this example has a substitution degree of R of 0.94.

[0101] Embodiment 6:

[0102] A method for preparing a hyaluronic acid derivative.

[0103] The difference between this embodiment and embodiment 5 is that L-alanine ethyl ester hydrochloride is added during the preparation process of the hyaluronic acid tranexamic acid derivative, specifically:

[0104] The preparation process of AMP is the same as that in Example 1.

[0105] Sodium hyaluronate was dissolved in water and added to a reactor. Argon was introduced for replacement until the air in the reactor was completely replaced. After cooling in an ice bath, AMP was added for reaction. Then, methyl tranexamic acid hydrochloride, L-tryptophan methyl ester hydrochloride, 2-aminothiazole-5-carboxylate and L-alanine ethyl ester hydrochloride were added. The pH was adjusted with a K2CO3 aqueous solution. The reaction system was heated to room temperature and stirred. After the reaction was completed, dialysis was performed with a dialysis bag, in the order of the first water dialysis, NaOH solution dialysis, NaCl solution dialysis, and the second water dialysis. The solution was freeze-dried to obtain a hyaluronic acid derivative. The molecular weight of sodium hyaluronate is 5-10 kDa, the amount of sodium hyaluronate is 200.0 g, the number of argon replacement is 2 times, the amount of AMP is 200.0 g, the reaction time is 2 h, the amount of tranexamic acid methyl ester hydrochloride is 155.0 g, the amount of L-tryptophan methyl ester hydrochloride is 4 g, the amount of 2-aminothiazole-5-carboxylate is 4 g, the amount of L-alanine ethyl ester hydrochloride is 2 g, the volume percentage of K2CO3 aqueous solution is 10%, the amount of K2CO3 aqueous solution is 5 L, the pH is adjusted to 8, the stirring time is 15 h, the molecular weight cutoff of the dialysis bag is 1000 Da, the first water dialysis time is 24 h, the molar concentration of NaOH solution is 0.05 M, the dialysis time of NaOH solution is 2 h, the molar concentration of NaCl solution is 0.1 M, the dialysis time of NaCl solution is 40 h, and the second water dialysis time is 72 h, the freeze-drying temperature was -80 °C, and the substitution degree of R was 0.94.

[0106] Embodiment 7:

[0107] A method for preparing a hyaluronic acid derivative.

[0108] The difference between this example and Example 6 is that 4 g of L-alanine ethyl ester hydrochloride is added during the preparation of the hyaluronic acid tranexamic acid derivative. Other than that, the other conditions are the same as those of Example 6.

[0109] The hyaluronic acid derivative prepared in this example has a substitution degree of R of 0.95.

[0110] Comparative Example 1:

[0111] A method for preparing a hyaluronic acid and tranexamic acid mixture.

[0112] 200 g of 5-10 kDa sodium hyaluronate was dissolved in water, and then 118.0 g of tranexamic acid was added. The mixture was stirred until clear, and the solution was freeze-dried to obtain a hyaluronic acid-tranexamic acid mixture.

[0113] Comparative Example 2:

[0114] A method for preparing a hyaluronic acid and tranexamic acid mixture.

[0115] 200.0 g of 20-40 kDa sodium hyaluronate was dissolved in water, and then 118.0 g of tranexamic acid was added, stirred until clear, and the solution was freeze-dried to obtain a hyaluronic acid-tranexamic acid mixture.

[0116] Comparative Example 3:

[0117] A method for preparing a hyaluronic acid and tranexamic acid mixture.

[0118] 200.0 g of 100-150 kDa sodium hyaluronate was dissolved in water, and then 118.0 g of tranexamic acid was added. The mixture was stirred until clear, and the solution was freeze-dried to obtain a hyaluronic acid-tranexamic acid mixture.

[0119] Comparative Example 4:

[0120] A method for preparing a hyaluronic acid derivative.

[0121] The difference between this embodiment and embodiment 1 is that L-tryptophan methyl ester hydrochloride is added during the preparation process of the hyaluronic acid tranexamic acid derivative, specifically:

[0122] The preparation process of AMP is the same as that in Example 1.

[0123] Sodium hyaluronate was dissolved in water and added to a reactor. Argon was introduced for replacement until the air in the reactor was completely replaced. After cooling in an ice bath, AMP was added for reaction. Then, methyl tranexamic acid hydrochloride and L-tryptophan methyl ester hydrochloride were added. The pH was adjusted with a K2CO3 aqueous solution. The reaction system was heated to room temperature and stirred. After the reaction was completed, dialysis was performed with a dialysis bag, in the order of the first water dialysis, NaOH solution dialysis, NaCl solution dialysis, and the second water dialysis. The solution was freeze-dried to obtain a hyaluronic acid derivative. The molecular weight of sodium hyaluronate is 5-10 kDa, the amount of sodium hyaluronate is 200.0 g, the number of argon replacements is 2 times, the amount of AMP is 200.0 g, the reaction time is 2 h, the amount of tranexamic acid methyl ester hydrochloride is 155.0 g, the amount of L-tryptophan methyl ester hydrochloride is 2 g, the volume percentage of K2CO3 aqueous solution is 10%, the amount of K2CO3 aqueous solution is 5 L, the pH is adjusted to 8, the stirring time is 15 h, the molecular weight cutoff of the dialysis bag is 1000 Da, the first water dialysis time is 24 h, the molar concentration of NaOH solution is 0.05 M, the dialysis time of NaOH solution is 2 h, the molar concentration of NaCl solution is 0.1 M, the dialysis time of NaCl solution is 40 h, the second water dialysis time is 72 h, the freeze-drying temperature is -80°C, and the degree of substitution of R is 0.91.

[0124] Comparative Example 5:

[0125] A method for preparing a hyaluronic acid derivative.

[0126] The difference from Example 1 is that 2-aminothiazole-5-carboxylate is added during the preparation of the hyaluronic acid tranexamic acid derivative, specifically:

[0127] The preparation process of AMP is the same as that in Example 1.

[0128] Sodium hyaluronate was dissolved in water and added to the reactor. Argon was introduced for replacement until the air in the reactor was completely replaced. After cooling in an ice bath, AMP was added for reaction. Then, methyl tranexamic acid hydrochloride and 2-aminothiazole-5-carboxylate were added. The pH was adjusted with K2CO3 aqueous solution. The reaction system was heated to room temperature and stirred. After the reaction was completed, dialysis was performed with a dialysis bag, in the order of the first water dialysis, NaOH solution dialysis, NaCl solution dialysis, and the second water dialysis. The solution was freeze-dried to obtain a hyaluronic acid derivative. The molecular weight of sodium hyaluronate is 5-10 kDa, the amount of sodium hyaluronate is 200.0 g, the number of argon replacements is 2 times, the amount of AMP is 200.0 g, the reaction time is 2 h, the amount of methyl tranexamic acid hydrochloride is 155.0 g, the amount of 2-aminothiazole-5-carboxylate is 2 g, the volume percentage of K2CO3 aqueous solution is 10%, the amount of K2CO3 aqueous solution is 5 L, the pH is adjusted to 8, the stirring time is 15 h, the molecular weight cutoff of the dialysis bag is 1000 Da, the first water dialysis time is 24 h, the molar concentration of NaOH solution is 0.05 M, the dialysis time of NaOH solution is 2 h, the molar concentration of NaCl solution is 0.1 M, the dialysis time of NaCl solution is 40 h, the second water dialysis time is 72 h, the freeze-drying temperature is -80°C, and the degree of substitution of R is 0.92.

[0129] The hyaluronic acid derivative prepared by the present invention was subjected to a mouse experiment, and a single large dose of the hyaluronic acid derivative reagent was administered to observe the toxicity of the hyaluronic acid derivative reagent on the mice for safety assessment. The evaluation results showed that the hyaluronic acid derivative prepared by the present invention was practically non-toxic. The dosage of the hyaluronic acid derivative reagent was 1-6 g / kg·BW.

[0130] Test Example 1: Transdermal performance test

[0131] 1.1 Reagents and Materials

[0132] Bama pork rind, PBS

[0133] 1.2 Experimental instruments

[0134] TP-6 Intelligent Transdermal Tester

[0135] 1.3 Experimental samples

[0136] Drug administration group: Examples 1-7, sample concentration was 1%, dissolved in PBS.

[0137] Control group: Comparative Examples 1-5, the sample concentration was 1%, and the samples were dissolved in PBS.

[0138] Blank group: PBS solution.

[0139] 1.4 Experimental methods

[0140] One end of the diffusion cell was covered with Bama Xiang pig skin, with the skin stratum corneum facing the supply cell. 1 mL of sample was added to the supply cell, and 7 mL of pre-degassed PBS was injected into the receiving cell as the receiving solution. The stirring speed was 350 r / min and the temperature was (32±1)℃ for transdermal experiment. After 4 hours, the residual sample on the skin surface was rinsed with 20 ml of PBS solution, the skin was cut into pieces, 5 mL of PBS solution was added, homogenized, and centrifuged at 10000 r / min for 30 min to collect the supernatant, which was filtered through a 0.22 μm filter membrane to obtain a clarified skin homogenate; the subcutaneous permeate was filtered through a 0.22 μm filter membrane, and the sample concentrations of the skin homogenate and subcutaneous permeate were determined by HPLC.

[0141] 1.5 Experimental Results

[0142] Table 1 Transdermal performance results of test samples

[0143]

[0144] The diffusion percentage is the ratio of the peptide concentration in the skin homogenate or subcutaneous permeate to the initial concentration.

[0145] The experimental results show that small-molecule hyaluronic acid derivatives have the highest transdermal efficiency and can reach the subcutaneous tissue, followed by medium-molecule hyaluronic acid derivatives, and large-molecule hyaluronic acid derivatives cannot penetrate the skin due to their larger molecular weight; in the comparative example, the transdermal absorption varies due to the different degrees of hydration of the stratum corneum skin by different hyaluronic acids. Small-molecule hyaluronic acid has the highest degree of hydration and can slightly damage the skin barrier, making the diffusion ratio of tranexamic acid the largest. The transdermal permeability of covalently bonded hyaluronic acid derivatives is much better than that of a simple mixture of the two.

[0146] As can be seen from Table 1, the comparison of the experimental results of Example 1, Example 4 and Example 5 shows that the transdermal efficiency of the two groups of experiments in Example 4 and Example 5 is higher. The results show that the addition of L-tryptophan methyl ester hydrochloride and 2-aminothiazole-5-carboxylate in the preparation process of hyaluronic acid derivatives can effectively improve the transdermal efficiency of hyaluronic acid derivatives, and the higher the amount of L-tryptophan methyl ester hydrochloride and 2-aminothiazole-5-carboxylate, the more conducive to the improvement of transdermal efficiency; the comparison of the experimental results of Example 1, Example 6 and Example 7 shows that the transdermal efficiency of the two groups of experiments in Example 6 and Example 7 is higher, indicating that the addition of L-alanine ethyl ester hydrochloride in the preparation process of hyaluronic acid derivatives can effectively improve the transdermal efficiency of hyaluronic acid derivatives, and the more the amount of L-alanine ethyl ester hydrochloride is used, the more conducive to the improvement of transdermal efficiency. Comparison of the experimental results of Comparative Example 4, Comparative Example 5 and Example 4 shows that the addition of L-tryptophan methyl ester hydrochloride or 2-aminothiazole-5-carboxylate alone during the preparation of hyaluronic acid derivatives is not conducive to improving the transdermal efficiency of hyaluronic acid derivatives.

[0147] Test Example 2 PGE2 Inhibition Performance Test

[0148] 2.1 Experimental cells

[0149] Human keratinocytes HaCaT

[0150] 2.2 Experimental reagents

[0151] Fetal bovine serum, DMEM medium

[0152] 2.3 Experimental samples

[0153] Examples 1-7, Comparative Examples 1-5

[0154] 2.4 Experimental Methods

[0155] Human keratinocytes HaCaT were cultured to the logarithmic growth phase and the concentration of the cell suspension was adjusted to 1×10 6 500 μL of cell suspension was inoculated into a 24-well plate and placed in a 37°C, 5% CO2 incubator for 24 h. After 24 h of keratinocyte culture, except for the blank group, the remaining groups were placed in 30 J / cm 2 Irradiation was performed under UVB light. After irradiation, the waste liquid was discarded, 500 μL of culture medium was added to each well of the model group, and 500 μL of culture medium containing 1% of the sample to be tested was added to the sample group. After continued incubation in the incubator for 24 h, the supernatant was centrifuged and the concentration of PGE2 in the cell supernatant was detected according to the instructions of the Elisa kit.

[0156] 2.5 Experimental Results

[0157] Ultraviolet radiation induces the synthesis of plasminogen and the activity of plasmin in keratinocytes. Plasmin activates the precursor of phospholipase A2 (PLA2), which in turn produces arachidonic acid (AA). The released AA is further converted into prostaglandin E2 (PEG2) under the catalysis of cyclooxygenase (COX) and prostaglandin E synthase (PGES), which subsequently leads to the production of melanin. Therefore, testing the content of PGE2 can indirectly evaluate the effect of active ingredients in improving skin pigmentation. The experimental results are as follows Figure 1 As shown, compared with the blank control, the PGE2 content in the keratinocytes of the model group increased significantly, indicating that the model was successfully established. Compared with the model, Example 1 can significantly reduce the PGE2 induced by ultraviolet rays, indicating that hyaluronic acid derivatives can effectively repair pigmentation caused by ultraviolet rays. The repair effect of the covalently bonded hyaluronic acid derivative is far better than that of a simple mixture of the two.

[0158] Table 2 PGE2 inhibition performance results of test samples

[0159]

[0160] As can be seen from Table 2, the PEG2 content of the blank group is significantly lower, mainly because the blank group is not irradiated with UVB light source. Compared with the model group, Example 1 can significantly reduce the PGE2 induced by ultraviolet rays, indicating that hyaluronic acid derivatives can effectively repair pigmentation caused by ultraviolet rays. The four groups of experimental results of Example 1 and Comparative Example 1, Comparative Example 2 and Comparative Example 3 are compared. The PGE2 content in the keratinocytes in Example 1 is significantly lower, which shows that Example 1 can significantly reduce the PGE2 induced by ultraviolet rays, indicating that hyaluronic acid derivatives can more effectively repair pigmentation caused by ultraviolet rays than simple mixtures. The three groups of experimental results of Example 1, Example 2 and Example 3 are compared. The results show that the PGE2 content in the keratinocytes of the experimental group of the small molecule hyaluronic acid derivative prepared in Example 1 is significantly lower, indicating that the small molecule hyaluronic acid derivative is more conducive to repairing pigmentation caused by ultraviolet rays. Comparison of the experimental results of Example 1, Example 4, Example 5, Example 6 and Example 7 shows that the PGE2 content in the keratinocytes in Example 1 is higher, which indicates that the addition of L-tryptophan methyl hydrochloride, 2-aminothiazole-5-carboxylate and L-alanine ethyl hydrochloride in the preparation process of hyaluronic acid derivatives can effectively repair the pigmentation caused by ultraviolet rays, and the higher the usage of L-tryptophan methyl hydrochloride, 2-aminothiazole-5-carboxylate and L-alanine ethyl hydrochloride, the more conducive the prepared hyaluronic acid derivatives are to repair the pigmentation caused by ultraviolet rays. In addition, by comparing the experimental results of Example 4, Comparative Example 4 and Comparative Example 5, the results show that the addition of L-tryptophan methyl hydrochloride or 2-aminothiazole-5-carboxylate alone in the preparation of hyaluronic acid derivatives is not conducive to repairing the pigmentation caused by ultraviolet rays.

[0161] Test Example 3 Tyrosinase Activity Inhibition Performance Test

[0162] 3.1 Experimental cells

[0163] Mouse melanoma cell line B16

[0164] 3.2 Experimental reagents

[0165] Fetal bovine serum, DMEM medium, PBS

[0166] 3.3 Experimental samples

[0167] Examples 1-7, Comparative Examples 1-5

[0168] 3.4 Experimental Methods

[0169] Mouse melanoma B16 cells were cultured to the logarithmic growth phase and the concentration of the cell suspension was adjusted to 1×10 5 100 μL of cell suspension was inoculated into 96-well plates and placed in a 37°C, 5% CO2 incubator for 24 h. After 24 h of cell culture, except for the blank group, the remaining groups were placed in 30 J / cm 2 Irradiation was performed under UVB light source. After irradiation, the waste liquid was discarded, 100 μL of culture medium was added to each well of the model group, and 100 μL of culture medium containing 1% of the sample to be tested was added to the sample group. After continued incubation in the incubator for 24 h, the supernatant was discarded, washed 3 times with PBS solution, 100 μL of 1% Triton X-100 solution was added to each well, frozen at -80°C for 40 min, thawed and lysed at room temperature, the cell lysate was centrifuged, 100 μL of supernatant was taken, 100 μL of 0.1% L-DOPA solution was added, and placed at 37°C for 2 h. The absorbance of dopaquinone was measured at a wavelength of 475 nm on an ELISA reader to calculate the tyrosinase activity.

[0170] 3.5 Experimental Results

[0171] The experimental results are as follows Figure 2 As shown in the figure, the tyrosinase activity of the model group reached 139.72% under UVB induction, proving that the model was successfully established. The tyrosinase activity of the cells treated with the sample of Example 1 was significantly reduced and significantly different from that of the blank group (P < 0.01), indicating that Example 1 has the effect of inhibiting tyrosinase activity. The tyrosinase inhibition effect of Example 3 is the worst because the hyaluronic acid with a larger molecular weight cannot carry tranexamic acid into the cells to produce tyrosinase inhibition.

[0172] Table 3 Tyrosinase activity inhibition test results of test samples

[0173]

[0174] As can be seen from Table 3, the relative activity of tyrosinase in the blank group is relatively low, mainly because the blank group is not irradiated with UVB light source. Compared with the model group, the relative activity of tyrosinase in Example 1 is lower, indicating that hyaluronic acid derivatives can effectively inhibit tyrosinase activity. Comparison of the experimental results of Example 1 with Comparative Example 1, Comparative Example 2 and Comparative Example 3 shows that the relative activity of tyrosinase in Example 1 is significantly lower, which shows that Example 1 has a better effect of inhibiting tyrosinase activity, indicating that hyaluronic acid derivatives can better inhibit tyrosinase activity than simple mixtures. Comparison of the experimental results of Example 1, Example 2 and Example 3 shows that the relative activity of tyrosinase in the experimental group of small molecule hyaluronic acid derivatives prepared in Example 1 is significantly lower, indicating that small molecule hyaluronic acid derivatives are more conducive to inhibiting tyrosinase activity. Comparison of the experimental results of Example 1, Example 4, Example 5, Example 6 and Example 7 shows that the relative activity of tyrosinase in Example 1 is significantly higher, which indicates that the addition of L-tryptophan methyl hydrochloride, 2-aminothiazole-5-carboxylate and L-alanine ethyl hydrochloride in the preparation process of hyaluronic acid derivatives can effectively inhibit tyrosinase activity, and the higher the amount of L-tryptophan methyl hydrochloride, 2-aminothiazole-5-carboxylate and L-alanine ethyl hydrochloride, the more beneficial the hyaluronic acid derivatives prepared are to inhibit tyrosinase activity. In addition, by comparing the experimental results of Example 4, Comparative Example 4 and Comparative Example 5, the results show that the addition of L-tryptophan methyl hydrochloride or 2-aminothiazole-5-carboxylate alone in the preparation of hyaluronic acid derivatives is not conducive to inhibiting tyrosinase activity.

[0175] Test Example 4 Melanin production inhibition performance test

[0176] 4.1 Experimental cells

[0177] Mouse melanoma cell line B16

[0178] 4.2 Experimental reagents

[0179] Fetal bovine serum, DMEM medium, PBS

[0180] 4.3 Experimental samples

[0181] Examples 1-7, Comparative Examples 1-5

[0182] 4.4 Experimental methods

[0183] Mouse melanoma B16 cells were cultured to the logarithmic growth phase and the concentration of the cell suspension was adjusted to 1×10 5100 μL of cell suspension was inoculated into 96-well plates and placed in a 37°C, 5% CO2 incubator for 24 h. After 24 h of cell culture, except for the blank group, the remaining groups were placed in 30 J / cm 2 Irradiation was performed under UVB light source. After irradiation, the waste liquid was discarded, 100 μL of culture medium was added to each well of the model group, and 100 μL of culture medium containing 1% of the sample to be tested was added to the sample group. After continued incubation in the incubator for 24 h, the supernatant was discarded, and the cells were washed three times with PBS solution. 100 μL of 1.0 mol / L NaOH solution (containing 10% DMSO) was added to each well, and the cells were fully lysed by heating in a water bath at 80°C, and the supernatant was obtained by centrifugation. The absorbance of melanin was measured at a wavelength of 405 nm using an ELISA reader.

[0184] 4.5 Experimental Results

[0185] The experimental results are as follows Figure 3 As shown, the melanin content of the model group reached 136.18% under UVB induction, proving that the model was successfully established. The amount of melanin produced by cells treated with the sample of Example 1 was significantly reduced and significantly different from that of the blank group (P < 0.01), indicating that Example 1 has the effect of inhibiting melanin production. The melanin production inhibition effect of Example 3 is the worst because the hyaluronic acid with a larger molecular weight cannot carry tranexamic acid into the cells to produce a melanin production inhibition effect.

[0186] Table 4 Melanin production inhibition performance test results of test samples

[0187]

[0188] As can be seen from Table 4, the relative content of melanin in the blank group is relatively low, mainly because the blank group has not been irradiated with a UVB light source. Compared with the model group, Example 1 has a lower relative content of melanin in Example 1, indicating that hyaluronic acid derivatives can effectively inhibit melanin production. Comparison of the experimental results of Example 1 with Comparative Example 1, Comparative Example 2 and Comparative Example 3 shows that the relative content of melanin in Example 1 is significantly lower, which shows that Example 1 has a better effect of inhibiting melanin production, indicating that hyaluronic acid derivatives can more effectively inhibit melanin production than simple mixtures. Comparison of the experimental results of Example 1, Example 2 and Example 3 shows that the relative content of melanin in the experimental group of the small molecule hyaluronic acid derivative prepared in Example 1 is significantly lower, indicating that the small molecule hyaluronic acid derivative is more conducive to inhibiting melanin production. Comparison of the experimental results of Example 1, Example 4, Example 5, Example 6 and Example 7 shows that the relative content of melanin in Example 1 is significantly higher, which indicates that the addition of L-tryptophan methyl hydrochloride, 2-aminothiazole-5-carboxylate and L-alanine ethyl hydrochloride in the preparation process of hyaluronic acid derivatives can effectively inhibit melanin production, and the higher the amount of L-tryptophan methyl hydrochloride, 2-aminothiazole-5-carboxylate and L-alanine ethyl hydrochloride, the more beneficial the prepared hyaluronic acid derivative is to inhibit melanin production. In addition, by comparing the experimental results of Example 4, Comparative Example 4 and Comparative Example 5, the results show that the addition of L-tryptophan methyl hydrochloride or 2-aminothiazole-5-carboxylate alone in the preparation of hyaluronic acid derivatives is not conducive to inhibiting melanin production.

[0189] The embodiments and / or implementation methods described above are only used to illustrate the preferred embodiments and / or implementation methods for realizing the technology of the present invention, and are not intended to limit the implementation methods of the technology of the present invention in any form. Any technical personnel in this field may make slight changes or modifications to other equivalent embodiments without departing from the scope of the technical means disclosed in the content of the present invention, but they should still be regarded as technologies or embodiments that are essentially the same as the present invention.

[0190] This article uses specific examples to illustrate the principles and implementation methods of this application. The description of the above embodiments is only used to help understand the method and its core ideas of this application. The above is only the preferred implementation method of this application. It should be pointed out that due to the limitations of textual expression and the objective existence of infinite specific structures, ordinary technicians in this technical field can make several improvements, modifications or changes without departing from the principles of this application, and can also combine the above technical features in an appropriate manner; these improvements, modifications, changes or combinations, or the direct application of the concept and technical solution of the invention to other occasions without improvement, should be regarded as the scope of protection of this application.

Claims

1. A method for preparing a composite polymer material based on hyaluronic acid, comprising: S1, mixing sodium hyaluronate and an activator to react to generate an intermediate 1; the molecular weight of sodium hyaluronate is 3-10 kDa, the amount of sodium hyaluronate is 180-220 parts by weight, and the amount of the activator is 180-220 parts by weight; S2, intermediate 1 is mixed with R reagent to react to generate intermediate 2; R reagent is methyl tranexamic acid ester hydrochloride, L-tryptophan methyl ester hydrochloride and 2-aminothiazole-5-carboxylate; or, R reagent is methyl tranexamic acid ester hydrochloride, L-tryptophan methyl ester hydrochloride, 2-aminothiazole-5-carboxylate and L-alanine ethyl ester hydrochloride; The amount of tranexamic acid methyl ester hydrochloride is 150-160 parts by weight, the amount of L-tryptophan methyl ester hydrochloride is 1-5 parts by weight, the amount of 2-aminothiazole-5-carboxylate is 1-5 parts by weight, and the amount of L-alanine ethyl ester hydrochloride is 1-5 parts by weight; S3, performing dialysis separation on the intermediate 2 to obtain a composite polymer material based on hyaluronic acid; The activator is prepared by reacting 2-chloro-methylpyridine iodide with an imidazole derivative, and the imidazole derivative includes at least one of N-methylimidazole, N-phenylimidazole and N-(4-methoxyphenyl)imidazole.

2. A method for preparing a composite polymer material based on hyaluronic acid according to claim 1, characterized in that: In the S2, a neutralizing agent is used to adjust the pH of the reaction system, and the neutralizing agent includes at least one of a triethylamine aqueous solution, a DIEA aqueous solution, a KHCO3 aqueous solution and a K2CO3 aqueous solution.

3. The method for preparing a composite polymer material based on hyaluronic acid according to claim 1, characterized in that: In the dialysis separation, aqueous solution, NaOH solution, NaCl solution and aqueous solution are used in sequence.

4. The method for preparing a composite polymer material based on hyaluronic acid according to claim 1, characterized in that: The molecular weight of the hyaluronic acid-based composite polymer material is 5 kDa to 20 kDa.

5. The method for preparing a composite polymer material based on hyaluronic acid according to claim 1, characterized in that: The substitution degree of R is 0.6~1.

0.

6. Use of the hyaluronic acid-based composite polymer material prepared by the method according to any one of claims 1 to 5 in the preparation of medical materials.

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