Hyaluronic acid tocopheryl conjugate, and preparation method and application thereof

Abstract

This invention belongs to the field of chemical synthesis technology, specifically relating to a hyaluronic acid-tocopherol conjugate, its preparation method, and its applications. The hyaluronic acid-tocopherol conjugate prepared by this invention combines the moisturizing properties of hyaluronic acid with the antioxidant effects of tocopherol, and can self-assemble into nanoparticles in water. It can be used to encapsulate hydrophobic cosmetic active ingredients to improve their water solubility and skin permeability, while avoiding skin problems such as clogged pores.

Description

Technical Field

[0001] This invention belongs to the field of chemical synthesis technology, specifically relating to a hyaluronic acid-tocopherol conjugate, its preparation method, and its application. Background Technology

[0002] Hyaluronic acid (HA) is widely used in the cosmetics industry due to its excellent moisturizing properties. As a natural polysaccharide, hyaluronic acid can absorb and lock in large amounts of moisture, maintaining skin hydration and thus improving problems such as dryness and fine lines. However, natural hyaluronic acid also has significant limitations in practical applications: it is easily degraded by hyaluronidase on the skin surface, resulting in insufficient durability; at the same time, its aqueous solution often has a strong sticky feel, affecting the user experience; in addition, its large molecular weight limits its skin penetration ability, making it difficult to effectively act on deeper layers of the skin. Summary of the Invention

[0003] In view of this, the present invention provides a hyaluronic acid-tocopherol conjugate, its preparation method and application. The hyaluronic acid-tocopherol conjugate provided by the present invention can improve the stickiness of hyaluronic acid and enhance its resistance to enzymatic degradation and skin penetration. It can also be used to encapsulate hydrophobic cosmetic active ingredients, thereby improving the water solubility and skin penetration of the active ingredients.

[0004] To achieve the above objectives, the present invention provides the following technical solution.

[0005] This invention provides a hyaluronic acid-tocopherol conjugate, with the structural formula shown in formula (I): Formula (I); In equation (I), n > 0, m ≥ 0; R1~R4 are independent , , , Or H, and R1~R4 are not all H at the same time.

[0006] Preferably, the total degree of substitution of tocopherol in the hyaluronic acid-tocopherol conjugate is 0.1-60%.

[0007] This invention also provides a method for preparing the hyaluronic acid-tocopherol conjugate described in the above technical solution, comprising the following steps: Tocopherol, an activator, and a first organic solvent are mixed and activated to obtain an activated intermediate. Hyaluronic acid, an activation intermediate, and a second organic solvent are mixed and coupled to obtain a hyaluronic acid-tocopherol conjugate.

[0008] Preferably, the tocopherol is one or more of α-tocopherol, β-tocopherol, γ-tocopherol, and δ-tocopherol; the activator is carbonyl diimidazole.

[0009] Preferably, the molar ratio of tocopherol to carbonyl diimidazole is 1:0.1~10; the activation temperature is 10~60℃ and the activation time is 0.2~12 h.

[0010] Preferably, the number-average molecular weight of the hyaluronic acid is 1~500 kDa; the molar ratio of tocopherol to hyaluronic acid is 20~0.1:1.

[0011] Preferably, the coupling reaction is carried out at a temperature of 20~100 ℃ for 6~72 h.

[0012] Preferably, the first or second organic solvent independently comprises at least one of methanol, ethanol, dimethyl sulfoxide, dichloromethane, chloroform, acetone, isopropanol, tetrahydrofuran, and N,N-dimethylformamide.

[0013] The present invention also provides the application of the hyaluronic acid tocopherol conjugate described in the above technical solution or the hyaluronic acid tocopherol conjugate prepared by the preparation method described in the above technical solution in cosmetics.

[0014] The present invention also provides a hyaluronic acid tocopherol conjugate material containing a water-insoluble active ingredient, comprising a hyaluronic acid tocopherol conjugate and a water-insoluble active ingredient loaded in the hyaluronic acid tocopherol conjugate; the mass ratio of the hyaluronic acid tocopherol conjugate to the water-insoluble active ingredient is 0.5~50:1; the hyaluronic acid tocopherol conjugate is the hyaluronic acid tocopherol conjugate described in the above technical solution or the hyaluronic acid tocopherol conjugate prepared by the preparation method described in the above technical solution.

[0015] The hyaluronic acid-tocopherol conjugate prepared by this invention combines the moisturizing properties of hyaluronic acid with the antioxidant effects of tocopherol, and can self-assemble into nanoparticles in water. It can be used to encapsulate hydrophobic cosmetic active ingredients to improve their water solubility and skin permeability, while avoiding skin problems such as clogged pores. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0017] Figure 1 The 1H NMR spectrum of the hyaluronic acid-tocopherol conjugate in Example 1; Figure 2 The 1H NMR spectrum of the hyaluronic acid-tocopherol conjugate in Example 2; Figure 3 The 1H NMR spectrum of the hyaluronic acid-tocopherol conjugate in Example 3; Figure 4 The infrared spectrum of the hyaluronic acid-tocopherol conjugate in Example 4; Figure 5 The UV-Vis spectrum of the hyaluronic acid-tocopherol conjugate in Example 5 is shown below. Figure 6 The particle size distribution diagrams are shown for the hyaluronic acid-tocopherol conjugate nanoparticles in Examples 6-10. Figure 7 The Zeta potential distribution diagrams are shown for the hyaluronic acid-tocopherol conjugate nanoparticles in Examples 11-12. Figure 8 The image shows the appearance (left) and Tyndall effect diagram (right) of the nanoparticles containing water-insoluble active ingredients encapsulated by the hyaluronic acid-tocopherol conjugate in Application Example 1. Detailed Implementation

[0018] This invention provides a hyaluronic acid-tocopherol conjugate, with the structural formula shown in formula (I): Formula (I); In equation (I), n > 0, m ≥ 0; R1~R4 are independent , , , Or H, and R1~R4 are not all H at the same time.

[0019] In this invention, the total degree of substitution of tocopherol in the hyaluronic acid-tocopherol conjugate can be 0.1-60%, specifically 1-40%.

[0020] This invention also provides a method for preparing the hyaluronic acid-tocopherol conjugate described in the above technical solution, comprising the following steps: Tocopherol, an activator, and a first organic solvent are mixed and activated to obtain an activated intermediate. Hyaluronic acid, an activation intermediate, and a second organic solvent are mixed and coupled to obtain a hyaluronic acid-tocopherol conjugate.

[0021] In this invention, the first or second organic solvent may independently include at least one of methanol, ethanol, dimethyl sulfoxide, dichloromethane, chloroform, acetone, isopropanol, tetrahydrofuran, and N,N-dimethylformamide, specifically dimethyl sulfoxide, dichloromethane, tetrahydrofuran, or N,N-dimethylformamide. In this invention, tocopherol may be one or more of α-tocopherol, β-tocopherol, γ-tocopherol, and δ-tocopherol; the activator may be carbonyl diimidazole; in this invention, the molar ratio of tocopherol to carbonyl diimidazole may be 1:0.1~10, specifically 1:0.5~5. In this invention, the activation temperature can be 10~60 ℃, specifically 20~40 ℃; the time can be 0.2~12 h, specifically 0.5~6 h; the activation is carried out under stirring conditions.

[0022] In this invention, the number-average molecular weight of the hyaluronic acid can be 1~500 kDa, specifically 5~200 kDa. In this invention, the molar ratio of tocopherol to hyaluronic acid can be 20~0.1:1, specifically 10~1:1; the temperature of the coupling reaction can be 20~100 ℃, specifically 30~80 ℃; the time can be 6~72 h, specifically 12~60 h; the coupling reaction can be carried out under stirring conditions.

[0023] In this invention, after the coupling reaction, the coupling reaction solution is further purified and dried; the purification includes sequential ethanol precipitation and washing; the drying is vacuum drying.

[0024] This invention couples hydrophobic tocopherol to hyaluronic acid. By optimizing the feeding ratio and reaction conditions between tocopherol and hyaluronic acid, a hyaluronic acid derivative with suitable hydrophilicity and hydrophobicity is obtained, which can improve the stickiness of hyaluronic acid and enhance its resistance to enzyme degradation and skin penetration.

[0025] The present invention also provides the application of the hyaluronic acid tocopherol conjugate described in the above technical solution or the hyaluronic acid tocopherol conjugate prepared by the preparation method described in the above technical solution in cosmetics.

[0026] The present invention also provides a hyaluronic acid tocopherol conjugate material containing a water-insoluble active ingredient, comprising a hyaluronic acid tocopherol conjugate and a water-insoluble active ingredient loaded in the hyaluronic acid tocopherol conjugate; the mass ratio of the hyaluronic acid tocopherol conjugate to the water-insoluble active ingredient is 0.5~50:1; the hyaluronic acid tocopherol conjugate is the hyaluronic acid tocopherol conjugate described in the above technical solution or the hyaluronic acid tocopherol conjugate prepared by the preparation method described in the above technical solution.

[0027] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0028] Example 1 210 mg of α-tocopherol was dissolved in dimethyl sulfoxide (DMSO) and mixed with 162 mg of carbonyl diimidazole dissolved in DMSO. The mixture was stirred at 25 °C for 15 min to obtain an activated intermediate solution. 380 mg of hyaluronic acid (number average molecular weight 1 kDa) was dissolved in DMSO and injected into the activated intermediate. The mixture was stirred at 40 °C for 12 h to obtain a crude product solution. The crude product solution was subjected to ethanol precipitation, washing, and vacuum drying to obtain the hyaluronic acid-tocopherol conjugate. The yield was 75.1% after weighing and calculation. The 1H NMR spectrum of the hyaluronic acid-tocopherol conjugate is shown below. Figure 1 As shown, the peaks at 1.1 ppm and 1.2 ppm are characteristic peaks of the methyl group in tocopherol, proving the successful synthesis of the coupling compound.

[0029] Example 2 420 mg of α-tocopherol was dissolved in dimethyl sulfoxide (DMSO) and mixed with 324 mg of carbonyl diimidazole dissolved in DMSO. The mixture was stirred at 30 °C for 30 min to obtain an activated intermediate solution. 380 mg of hyaluronic acid (number average molecular weight 10 kDa) was dissolved in DMSO and injected into the activated intermediate. The mixture was stirred at 60 °C for 24 h to obtain a crude product solution. The crude product solution was subjected to ethanol precipitation, washing, and vacuum drying to obtain the hyaluronic acid-tocopherol conjugate. The yield was 72.5% after weighing and calculation. The 1H NMR spectrum of the hyaluronic acid-tocopherol conjugate is shown below. Figure 2 As shown, the peaks at 1.1 ppm and 1.2 ppm are characteristic peaks of the methyl group in tocopherol, proving the successful synthesis of the coupling compound.

[0030] Example 3 840 mg of α-tocopherol was dissolved in dichloromethane and mixed with 500 mg of carbonyl diimidazole dissolved in dichloromethane. The mixture was stirred at 40 °C for 2 h to obtain an activated intermediate solution. 760 mg of hyaluronic acid (number average molecular weight 50 kDa) was dissolved in dichloromethane and injected into the above activated intermediate. The mixture was stirred at 80 °C for 36 h to obtain a crude product. The crude product solution was subjected to ethanol precipitation, washing, and vacuum drying to obtain the hyaluronic acid-tocopherol conjugate. The yield was 73.9% after weighing and calculation. The 1H NMR spectrum of the hyaluronic acid-tocopherol conjugate is shown below. Figure 3As shown, the peaks at 1.1 ppm and 1.2 ppm are characteristic peaks of the methyl group in tocopherol, proving the successful synthesis of the coupling compound.

[0031] Example 4 210 mg of β-tocopherol was dissolved in dimethyl sulfoxide (DMSO) and mixed with 324 mg of carbonyl diimidazole dissolved in DMSO. The mixture was stirred at 30 °C for 1 h to obtain an activated intermediate solution. 760 mg of hyaluronic acid (number average molecular weight 100 kDa) was dissolved in DMSO and injected into the above activated intermediate. The mixture was stirred at 30 °C for 12 h to obtain a crude product. The crude product solution was subjected to ethanol precipitation, washing, and vacuum drying to obtain the hyaluronic acid-tocopherol conjugate. The yield was 69.3% after weighing and calculation. The infrared spectrum of the hyaluronic acid-tocopherol conjugate is shown below. Figure 4 As shown, 1740-1800 cm -1 The peak at that position is a characteristic peak of carbonate bonds, further verifying the successful synthesis of the coupling compound.

[0032] Example 5 210 mg of γ-tocopherol was dissolved in dichloromethane and mixed with 162 mg of carbonyl diimidazole dissolved in dichloromethane. The mixture was stirred at 30 °C for 1 h to obtain an activation intermediate solution. 190 mg of hyaluronic acid (number average molecular weight 500 kDa) was dissolved in dichloromethane and injected into the above activation intermediate. The mixture was stirred at 40 °C for 12 h to obtain a crude product. The crude product solution was subjected to ethanol precipitation, washing, and vacuum drying to obtain the hyaluronic acid-tocopherol conjugate. The yield was 65.2% after weighing and calculation. The UV-Vis spectrum of the hyaluronic acid-tocopherol conjugate is shown below. Figure 5 As shown, the peak at 292 nm is the characteristic absorption peak of the benzene ring, which further verifies the successful synthesis of the conjugate. The degree of substitution of tocopherol can be calculated to be 4.8% by plotting the absorbance-concentration curve.

[0033] Example 6 420 mg of β-tocopherol was dissolved in dimethyl sulfoxide (DMSO) and mixed with 324 mg of carbonyl diimidazole dissolved in DMSO. The mixture was stirred at 40 °C for 2 h to obtain an activated intermediate solution. 190 mg of hyaluronic acid (number average molecular weight 1 kDa) was dissolved in DMSO and injected into the above activated intermediate. The mixture was stirred at 40 °C for 24 h to obtain a crude product. The crude product solution was subjected to ethanol precipitation, washing, and vacuum drying to obtain a hyaluronic acid-tocopherol conjugate. The yield was 67.8% after weighing and calculation. The conjugate was dissolved in pure water at a concentration of 10 mg / mL. The particle size distribution is shown in the figure. Figure 6As shown, the particle size is approximately 165 nm.

[0034] Example 7 840 mg of δ-tocopherol was dissolved in dimethyl sulfoxide (DMSO) and mixed with 648 mg of carbonyl diimidazole dissolved in DMSO. The mixture was stirred at 40 °C for 6 h to obtain an activated intermediate solution. 190 mg of hyaluronic acid (number average molecular weight 10 kDa) was dissolved in DMSO and injected into the above activated intermediate. The mixture was stirred at 60 °C for 36 h to obtain a crude product. The crude product solution was subjected to ethanol precipitation, washing, and vacuum drying to obtain a hyaluronic acid-tocopherol conjugate. The yield was 72.5% after weighing and calculation. The conjugate was dissolved in pure water at a concentration of 10 mg / mL. The particle size distribution is shown in the figure. Figure 6 As shown, the particle size is approximately 168 nm.

[0035] Example 8 210 mg of δ-tocopherol was dissolved in N,N-dimethylformamide and mixed with 648 mg of carbonyl diimidazole dissolved in N,N-dimethylformamide. The mixture was stirred at 25 °C for 2 h to obtain an activated intermediate solution. 380 mg of hyaluronic acid (number average molecular weight 100 kDa) was dissolved in N,N-dimethylformamide and injected into the above activated intermediate. The mixture was stirred at 40 °C for 12 h to obtain a crude product. The crude product solution was subjected to ethanol precipitation, washing, and vacuum drying to obtain a hyaluronic acid-tocopherol conjugate. The yield was 69.5% after weighing and calculation. The conjugate was dissolved in pure water at a concentration of 10 mg / mL. The particle size distribution is shown in the figure. Figure 6 As shown, the particle size is approximately 270 nm.

[0036] Example 9 210 mg of α-tocopherol and 210 mg of β-tocopherol were dissolved in dimethyl sulfoxide (DMSO) and mixed with 324 mg of carbonyl diimidazole dissolved in DMSO. The mixture was stirred at 30 °C for 2 h to obtain an activated intermediate solution. 380 mg of hyaluronic acid (number average molecular weight 300 kDa) was dissolved in DMSO and injected into the above activated intermediate. The mixture was stirred at 40 °C for 12 h to obtain a crude product. The crude product solution was subjected to ethanol precipitation, washing, and vacuum drying to obtain a hyaluronic acid-tocopherol conjugate. The yield was 67.6% after weighing and calculation. The conjugate was dissolved in pure water at a concentration of 10 mg / mL. The particle size distribution is shown in the figure. Figure 6 As shown, the particle size is approximately 280 nm.

[0037] Example 10 420 mg of α-tocopherol and 420 mg of β-tocopherol were dissolved in dimethyl sulfoxide (DMSO) and mixed with 648 mg of carbonyl diimidazole dissolved in DMSO. The mixture was stirred at 40 °C for 4 h to obtain an activated intermediate solution. 380 mg of hyaluronic acid (number average molecular weight 500 kDa) was dissolved in DMSO and injected into the above activated intermediate. The mixture was stirred at 40 °C for 24 h to obtain a crude product. The crude product solution was subjected to ethanol precipitation, washing, and vacuum drying to obtain a hyaluronic acid-tocopherol conjugate, which, after weighing and calculation, had a content of 71.6%. The conjugate was dissolved in pure water at a concentration of 10 mg / mL. Its particle size distribution is shown in the figure. Figure 6 As shown, the particle size is approximately 120 nm.

[0038] Example 11 105 mg of α-tocopherol and 105 mg of γ-tocopherol were dissolved in dimethyl sulfoxide (DMSO) and mixed with 162 mg of carbonyl diimidazole dissolved in DMSO. The mixture was stirred at 30 °C for 30 min to obtain an activation intermediate solution. 380 mg of hyaluronic acid (number average molecular weight 10 kDa) was dissolved in DMSO and injected into the above activation intermediate. The mixture was stirred at 40 °C for 12 h to obtain a crude product. The crude product solution was subjected to ethanol precipitation, washing, and vacuum drying to obtain a hyaluronic acid-tocopherol conjugate. The yield was calculated to be 64.3%. The conjugate was dissolved in pure water at a concentration of 10 mg / mL. Its Zeta potential diagram is shown below. Figure 7 As shown, the Zeta potential is approximately -12 mV.

[0039] Example 12 210 mg of α-tocopherol and 210 mg of γ-tocopherol were dissolved in dichloromethane and mixed with 648 mg of carbonyl diimidazole dissolved in dichloromethane. The mixture was stirred at 40 °C for 1 h to obtain an activated intermediate solution. 760 mg of hyaluronic acid (number average molecular weight 50 kDa) was dissolved in dichloromethane and injected into the above activated intermediate. The mixture was stirred at 40 °C for 12 h to obtain a crude product. The crude product solution was subjected to ethanol precipitation, washing, and vacuum drying to obtain a hyaluronic acid-tocopherol conjugate. The yield was 68.8% after weighing and calculation. The conjugate was dissolved in pure water at a concentration of 10 mg / mL, and its Zeta potential diagram is shown below. Figure 7 As shown, the Zeta potential is approximately -15 mV.

[0040] The total degree of substitution of tocopherol in the hyaluronic acid-tocopherol conjugates prepared in Examples 1-12 is shown in Table 1.

[0041] Table 1. Total degree of substitution of tocopherol in hyaluronic acid-tocopherol conjugates

[0042] Application Example 1 Weigh the hyaluronic acid-tocopherol conjugate obtained in Example 1 and dissolve it in pure water to prepare a 2 mg / mL solution. Weigh glycyrrhizin and dissolve it in anhydrous ethanol to prepare a 2 mg / mL ethanol solution. Measure 2 mL of the 2 mg / mL conjugate aqueous solution, and while stirring, add 200 μL of the water-insoluble active ingredient ethanol solution dropwise to the conjugate aqueous solution. After dialysis to remove the ethanol, self-assembled nanoparticles of hyaluronic acid-tocopherol conjugate encapsulating the water-insoluble active ingredient are obtained, with the appearance as shown. Figure 8 As shown on the left, the Tyndall effect under red laser illumination is as follows: Figure 8 As shown on the right.

[0043] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A hyaluronic acid-tocopherol conjugate, characterized in that, The structural formula is shown in equation (I): Equation (I); In equation (I), n > 0, m ≥ 0; R1~R4 are independent , , , Or H, and R1~R4 are not all H at the same time.

2. The hyaluronic acid-tocopherol conjugate as described in claim 1, characterized in that, The total degree of substitution of tocopherol in the hyaluronic acid-tocopherol conjugate is 0.1-60%.

3. The method for preparing the hyaluronic acid-tocopherol conjugate according to claim 1 or 2, characterized in that, Includes the following steps: Tocopherol, an activator, and a first organic solvent are mixed and activated to obtain an activated intermediate. Hyaluronic acid, an activation intermediate, and a second organic solvent are mixed and coupled to obtain a hyaluronic acid-tocopherol conjugate.

4. The preparation method according to claim 3, characterized in that, The tocopherol is one or more of α-tocopherol, β-tocopherol, γ-tocopherol, and δ-tocopherol; the activator is carbonyl diimidazole.

5. The preparation method according to claim 4, characterized in that, The molar ratio of tocopherol to carbonyl diimidazole is 1:0.1~10; the activation temperature is 10~60 °C, and the activation time is 0.2~12 h.

6. The preparation method according to claim 3, characterized in that, The number-average molecular weight of the hyaluronic acid is 1~500kDa; the molar ratio of tocopherol to hyaluronic acid is 20~0.1:

1.

7. The preparation method according to claim 6, characterized in that, The coupling reaction is carried out at a temperature of 20~100 ℃ for a time of 6~72 h.

8. The preparation method according to claim 3, characterized in that, The first or second organic solvent independently comprises at least one of methanol, ethanol, dimethyl sulfoxide, dichloromethane, chloroform, acetone, isopropanol, tetrahydrofuran, and N,N-dimethylformamide.

9. The application of a hyaluronic acid tocopherol conjugate according to claim 1 or 2, or a hyaluronic acid tocopherol conjugate prepared by any one of claims 3 to 8, in cosmetics.

10. A hyaluronic acid-tocopherol conjugate material encapsulating water-insoluble active ingredients, characterized in that, The invention comprises a hyaluronic acid-tocopherol conjugate and a water-insoluble active ingredient supported on the hyaluronic acid-tocopherol conjugate; the mass ratio of the hyaluronic acid-tocopherol conjugate to the water-insoluble active ingredient is 0.5-50:1; the hyaluronic acid-tocopherol conjugate is the hyaluronic acid-tocopherol conjugate according to claim 1 or 2 or the hyaluronic acid-tocopherol conjugate prepared by the preparation method according to any one of claims 3-8.

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