A glycosphingolipid derivative and its preparation method and application

Abstract

The present invention relates to a glycosphingolipid derivative and a preparation method and application thereof. Pullulan is activated, DMEDA is used to selectively remove part of the acetyl group, and trichloroacetonitrile (Cl3CCN) is introduced to generate an active intermediate, and the hydroxyl group of phytosphingosine is protected to form a stable ketal structure to avoid the occurrence of side reactions. The activated pullulan and the hydroxyl-protected phytosphingosine are subjected to a condensation reaction under the action of an acidic catalyst, and then a high-purity glycosphingolipid derivative is prepared by deprotection treatment and purification. The compound has excellent amphiphilicity, exhibits good moisturizing properties and anti-photoaging ability, and can be widely used in the fields of cosmetics, medicine and functional materials. The method of the present invention has mild reaction conditions and high efficiency, and avoids the problems of poor reaction selectivity and many impurities in traditional technologies.

Description

Technical Field

[0001] The present invention relates to the technical field of glycosphingolipid compounds, and in particular to a glycosphingolipid derivative and a preparation method and application thereof. Background Art

[0002] Polysaccharide sphingolipids are an important class of bioactive substances, which are widely present in the cell membranes of animals and plants and play a key role in biological processes such as cell recognition and immune regulation. Their unique structure gives them potential application value in anti-inflammatory, anti-tumor, and immune enhancement. Polysaccharide sphingolipids can be divided into neutral sphingolipids and acidic sphingolipids according to the polar groups. Among them, neutral sphingolipids are widely used in medicine, cosmetics, and health foods due to their good biocompatibility. For example, galactosylceramide can activate keratinocytes to release ceramide, thereby improving dry skin and allergic symptoms; glucosylceramide can generate ceramide in keratinocytes to help maintain the skin barrier function. Moisturizing, as the basic function of skin care products, depends on the combined action of stratum corneum moisture and lipid membrane. At present, popular moisturizing raw materials on the market include water-soluble raw materials of polysaccharides and fat-soluble raw materials of ceramides. However, the moisturizing effect of polysaccharides in low humidity environments is limited, and the solubility of ceramide compounds has long been a technical problem in formula development.

[0003] In the prior art, a new type of polysaccharide sphingolipid is designed by combining polysaccharides with ceramide precursor, sphingosine. This structure not only overcomes the single-channel limitation of the moisturizing effect of polysaccharides, but also significantly improves the solubility of sphingosine. As an important component of the cell membrane of eukaryotic organisms, the structure of glycosphingolipids has potential medicinal value. However, natural glycosphingolipids have low content in organisms, complex and heterogeneous structures, and extremely low extraction efficiency from animals and plants. Therefore, it is necessary to develop an efficient and simple method for synthesizing polysaccharide sphingolipids.

[0004] A Chinese patent (CN201680020040.X) discloses a phytosphingosine derivative and a composition containing the same. In the invention, phytosphingosine and aldehyde disaccharide are used in a condensation reaction at a molar ratio of 1:0.8 to 1:2, an acid or a base is used as a catalyst, and methanol or ethanol is used as a solvent. By the above method, a water-soluble disaccharide with improved stability in solution is prepared, and has a good antibacterial effect. However, the aldehyde disaccharide (such as maltose or lactose) in the application contains multiple hydroxyl groups and a reducing end group, and its reducing end carbonyl group is protonated under acid catalysis to form a highly active electrophilic center. This electrophilic center not only reacts with phytosphingosine to generate the target product, but may also react with its own hydroxyl group or solvent molecules, resulting in polymerization, isomerization or further degradation of sugar molecules. For example, protonated disaccharides may undergo CC bond cleavage through the enolization pathway to generate small molecular aldehyde compounds (such as formaldehyde) or carboxylic acid by-products, and under acidic conditions, the glycosidic bonds in the aldodecaned disaccharides may be hydrolyzed to generate monosaccharides, which further participate in complex degradation reactions to form impurities that are difficult to separate. In addition, in acidic or alkaline environments, the hydroxyl groups of methanol or ethanol may undergo acetalization reactions with the carbonyl groups of the aldodecaned disaccharides to generate solvent-modified sugar derivatives, which increases the complexity of the by-products. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a glycosphingolipid derivative, the chemical formula of the glycosphingolipid derivative is as follows:

[0006]

[0007] It should be noted that the present invention forms a glycosphingolipid derivative with unique advantages by introducing pullulan into the molecular structure of phytosphingosine. This structure itself has both the hydrophilicity of polysaccharides and the fat solubility of sphingosine, so that the molecule exhibits amphiphilic characteristics, which helps to show excellent performance in both hydrophilic and hydrophobic environments. In addition, the natural biocompatibility and stability of pullulan can give glycosphingolipids higher chemical stability and functional diversity, and has significant advantages in the fields of moisturizing and penetration. Especially in low humidity environments, this structure can continuously adsorb and retain moisture, improving the defects of traditional moisturizers that are not effective under dry conditions. By replacing the hydroxyl group at the top of phytosphingosine, pullulan effectively blocks the hydroxyl site with higher reaction activity, thereby reducing the occurrence of non-specific side reactions and ensuring the high purity and structural controllability of the target product. This substitution not only simplifies the synthesis process, reduces the generation of by-products, but also improves the stability of the product. In addition, the linear structure of pullulan itself makes the molecule more uniform, which is conducive to subsequent purification and large-scale production, thereby significantly improving the operability and application performance of the product. It is worth mentioning that the amino structure in phytosphingosine is retained and is easily protonated to form -NH3 under specific pH conditions. + This property gives the molecule a positively charged characteristic, enabling it to more effectively form electrostatic adsorption with water molecules, further enhancing the molecule's hydrophilicity and moisturizing properties. Especially under low humidity conditions, the protonated amino group exhibits excellent hygroscopicity and can continuously lock in moisture, overcoming the limitations of traditional moisturizers. At the same time, the protonated amino group also improves the solubility and skin permeability of the molecule, allowing the glycosphingolipid molecules to electrostatically interact with the negatively charged phospholipid bilayer on the skin surface, reducing the molecular penetration resistance, promoting the penetration of active ingredients into the deep layers of the skin, and exerting a more lasting moisturizing and functional effect.

[0008] At the same time, by replacing the hydroxyl group at the top of phytosphingosine with pullulan, the entire molecular structure is made more stable, reducing the active hydroxyl sites that are easily degraded when exposed to light or oxidative environments. This structural optimization effectively reduces the risk of molecular decomposition under ultraviolet (UV) or thermal conditions, thereby enhancing the anti-photoaging properties of glycosphingolipid compounds. In addition, phytosphingosine itself has certain antioxidant activity. By retaining its amino structure, it can effectively capture free radicals, block oxidative chain reactions, and reduce the damage of reactive oxygen species (ROS) to skin cells. Protonated amino groups (-NH3 + ) can also form electrostatic interactions with free radicals under specific pH conditions, further improving the antioxidant capacity of the molecule. This property enables glycosphingolipid molecules to protect the skin from aging damage caused by free radicals under light conditions and delay the photoaging process of the skin.

[0009] The invention provides a preparation method of glycosphingolipid derivatives, comprising the following technical steps: S1. adding hydroxyl-protected phytosphingosine and dodecylbenzenesulfonic acid into a three-necked flask with stirring, heating to 80 to 100° C., adding a pullulan polysaccharide molecular intermediate containing an active functional group, and stirring for 15 to 20 min; S2. cooling to 60° C. and stirring, adding 0.1 mol / L to 1 mol / L CH3ONa solution to adjust the pH of the entire solution to 7 to 8; S3. filtering impurities in the reaction system using a vacuum filtration device, rinsing the filtered product with purified water for multiple times, adding 30wt% ethanol solution to the filtered product, stirring evenly, heating the system to 55° C. in a rotary evaporator, and rotary evaporating for 20-30 minutes.

[0010] It should be noted that in step S1, the hydroxyl-protected phytosphingosine and the acid catalyst dodecylbenzenesulfonic acid (DBSA) are added to the reaction system and the temperature is raised to 80-100°C. This temperature range provides suitable energy for the condensation reaction, allowing the reaction to proceed smoothly. Under the action of the acid catalyst, DBSA provides protons (H +) activates the active functional groups on the pullulan molecular intermediate, enhancing its electrophilicity and making it easy to be nucleophilically attacked by the hydroxyl group at the top of phytosphingosine. At this time, the top hydroxyl group of phytosphingosine acts as a nucleophilic group, combines with the active site of the pullulan intermediate, undergoes a condensation reaction, and forms a stable glycosphingolipid structure. The control of the reaction temperature is crucial: under the condition of 80-100℃, the reaction rate can be increased, while avoiding the degradation of sugar molecules or side reactions caused by high temperature, thereby ensuring the high yield and structural stability of the target product. In step S2, the reaction system is cooled to 60℃, and 0.1mol / L to 1mol / L CH3ONa solution is gradually added to adjust the pH of the system. Sodium methoxide, as a weak base, has two important functions: one is to neutralize the acid catalyst DBSA remaining in the reaction system, thereby terminating the acid-catalyzed condensation reaction and avoiding further side reactions; the other is to promote the deprotection reaction of the protecting group in the phytosphingosine molecule, so that the originally protected hydroxyl group is released again. In this step, the gradual addition of CH3ONa can accurately control the pH within the neutral range of 7-8, which not only ensures the structural stability of the target product, but also avoids the degradation of sugar molecules or sphingosine caused by excessive alkalinity, thereby maintaining the chemical integrity of the product. In step S3, the reaction system is filtered using a vacuum filtration device to remove unreacted impurities and by-products to obtain a preliminarily purified product. In order to further improve the purity of the product, a 30wt% ethanol solution is added to the filtered product. As an organic solvent, ethanol has the following dual functions: first, it dissolves low molecular weight impurities remaining in the reaction system through the solvent effect, such as unreacted pullulan or acidic catalyst; second, it helps to grade and separate the target product, further exclude non-target compounds, and the purification effect is more significant. Then, the mixture is heated to 55°C in a rotary evaporator, and ethanol and other volatile solvents are effectively removed under reduced pressure by 20-30 minutes of rotary evaporation to obtain a pure glycosphingolipid derivative. This step further improves the purity of the product by evaporating the solvent, ensuring that it is suitable for subsequent applications or characterization.

[0011] As a preferred technical solution for the preparation of glycosphingolipid derivatives, in step S1, the preparation method of the pullulan polysaccharide molecular intermediate containing active functional groups comprises the following preparation steps:

[0012] The pullulan molecule and acetyl chloride (AcCl) are mixed in an appropriate amount of solvent, stirred and reacted at 0 to 5°C to form an acetylated pullulan derivative, and the organic layer is separated by liquid separation, and dried with anhydrous sodium sulfate and then concentrated to obtain a completely acetylated pullulan; wherein the solvent is pyridine, and the mass ratio of pullulan, acetyl chloride and solvent is 1:(2 to 2.2):(5 to 7);

[0013] The fully acetylated pullulan is reacted with N,N-dimethylethylenediamine (DMEDA). After the reaction, the product is purified and concentrated to obtain partially protected pullulan molecules. Trichloroacetonitrile (Cl3CCN) is used to react with the partially protected sugar molecules at 0 to 5°C to generate sugar molecule intermediates containing active functional groups; wherein the mass ratio between the fully acetylated pullulan and N,N-dimethylethylenediamine is 1:(1.5 to 2), and the mass ratio between the partially protected pullulan molecules and trichloroacetonitrile is 1:(1.2 to 1.5).

[0014] It should be noted that the pullulan is reacted with acetyl chloride (AcCl) in pyridine, and the reaction temperature is controlled at 0-5°C. The reaction is an acetylation reaction, and acetyl chloride reacts with multiple hydroxyl groups (-OH) in the pullulan molecule by providing acetyl groups (CH3CO-) to generate fully acetylated pullulan derivatives. In this process, pyridine not only acts as a solvent, but also as a trap for acidic byproducts (HCl), preventing the generated hydrogen chloride from further reacting with hydroxyl groups in the reaction system, resulting in the formation of byproducts. Low temperature (0-5°C) helps control the reaction rate and avoid degradation of the polysaccharide chain, while ensuring selective acetylation of the hydroxyl groups. After the reaction is completed, excess acetyl chloride and byproducts are removed by separation, and the organic layer is dried with anhydrous sodium sulfate to remove trace moisture and prevent product degradation. The fully acetylated pullulan is reacted with N,N-dimethylethylenediamine (DMEDA). DMEDA has strong nucleophilicity and can selectively attack acetyl groups at specific positions, causing deacetylation reactions, thereby exposing some hydroxyl groups and forming partially protected pullulan molecules. The mechanism of this process is that through the nucleophilic substitution of amine groups, DMEDA displaces certain acetyl groups to generate free hydroxyl groups, while acetyl groups at other positions remain unreacted, ensuring that the structural part of the molecule is protected. This selective deprotection provides specific reaction sites for the introduction of subsequent functional groups.

[0015] As a preferred technical solution for the preparation of a glycosphingolipid derivative, in step S1, the preparation method of the hydroxyl-protected phytosphingosine comprises the following steps:

[0016] The phytosphingosine is dissolved in toluene, acetone is added to the phytosphingosine solution under stirring, and then toluenesulfonic acid is slowly added, and the mixed solution is stirred and reacted at room temperature for 1 to 2 hours. After the reaction is completed, the excess solvent and acetone are removed by reduced pressure evaporation to obtain hydroxyl-protected phytosphingosine.

[0017] It should be noted that phytosphingosine was dissolved in toluene. Toluene, as a non-polar solvent, provided a good dissolving environment to ensure that the phytosphingosine molecules were evenly dispersed. Subsequently, acetone was added to the solution. As a carbonyl compound, the carbonyl group (C=O) of acetone is easily protonated under acidic conditions, thereby increasing the electrophilicity of the carbonyl carbon. When toluenesulfonic acid (PTSA) was slowly added as a catalyst, the toluenesulfonic acid provided a proton (H + ), further promoting the nucleophilic addition reaction of acetone with the hydroxyl group in phytosphingosine. During the reaction, the two adjacent hydroxyl groups on the phytosphingosine molecule act as nucleophilic groups and attack the activated carbonyl carbon in the acetone molecule through nucleophilic attack to form a hemiketal intermediate. Then, the intermediate loses a molecule of water and further forms a stable five-membered ring ketal ring structure. This structure can effectively block the adjacent hydroxyl groups of phytosphingosine, so that it no longer participates in other chemical reactions in subsequent reactions, thereby achieving selective protection of hydroxyl groups. Controlling the reaction temperature at room temperature (25-30°C) helps to improve the reaction selectivity of ketal formation and avoid degradation or other side reactions of phytosphingosine or acetone under high temperature conditions. At the same time, the reaction time is controlled at 1-2 hours to ensure that the reaction is fully carried out and the hydroxyl groups are completely protected. After the reaction is completed, the excess solvent (toluene) and acetone in the reaction system are removed by reduced pressure evaporation. Evaporation under reduced pressure can remove volatile substances at a lower temperature, avoid degradation of phytosphingosine due to long-term high temperature, and finally obtain phytosphingosine with protected hydroxyl groups.

[0018] As a preferred technical solution for the preparation of glycosphingolipid derivatives, in step S1, the mass ratio of the pullulan molecular intermediate containing active functional groups to the hydroxyl-protected phytosphingosine is 1:2 to 1:4.

[0019] It should be noted that the reaction is carried out efficiently and the yield and purity of the target product are improved.

[0020] As a preferred technical solution for the preparation of glycosphingolipid derivatives, in step S1, dodecylbenzenesulfonic acid accounts for 5 to 8 wt % of the total mass of the hydroxyl-protected phytosphingosine.

[0021] It should be noted that dodecylbenzenesulfonic acid (DBSA) accounts for 5 to 8 wt% of the total mass of the hydroxyl-protected phytosphingosine. Under this condition, DBSA provides a suitable acidic catalytic environment, which can effectively activate the active site of the pullulan molecule and promote the nucleophilic attack of the top hydroxyl group of phytosphingosine, thereby efficiently generating a stable glycosphingolipid structure.

[0022] As a preferred technical solution for the preparation of glycosphingolipid derivatives, the reaction conditions of fully acetylated pullulan and N,N-dimethylethylenediamine (DMEDA) are 30 to 50°C.

[0023] It should be noted that pullulan is a polysaccharide compound, and the sugar chain is prone to degradation or breakage at high temperatures, resulting in reduced molecular weight and structural damage, thus affecting the functionality of the target product. Secondly, mild reaction conditions contribute to the selectivity of the reaction, ensuring that DMEDA only deprotects the acetyl group at a specific position without damaging other chemical bonds (such as glycosidic bonds) or protecting groups. In addition, temperature control can also effectively inhibit the occurrence of side reactions, prevent molecular cross-linking or unnecessary structural changes caused by excessive reaction, and further improve the purity and uniformity of the product.

[0024] Another inventive point of this application is that the glycosphingolipid derivatives prepared by the present invention are used for skin moisturizing, repairing and anti-photoaging. In terms of structure, the glycosphingolipid derivatives combine the hydrophilicity of pullulan and the fat solubility of phytosphingosine to form a molecule with amphiphilic characteristics. This property enables glycosphingolipids to form a flexible protective film on the skin surface, effectively reducing water evaporation, while providing continuous moisturizing by adsorbing water molecules in the environment. In addition, the amino structure retained in the molecule can be protonated to form -NH3 under specific pH conditions. + , so that the molecule has a positive charge, thereby forming electrostatic adsorption with water molecules, further improving the molecule's hygroscopicity and water-locking ability, especially in low humidity environments. It can still show excellent moisturizing performance, overcoming the limitation that traditional moisturizers are not effective under dry conditions. As a key component of sphingolipid compounds, phytosphingosine can promote the synthesis of lipids in the stratum corneum of the skin and repair damaged skin barriers. Glycosphingolipid derivatives penetrate into the skin surface and gradually release phytosphingosine, which enhances the lipid content of the stratum corneum and improves the barrier function of the skin, thereby reducing damage to the skin caused by external stimuli, enhancing the skin's ability to lock in water, and maintaining the skin's health. This barrier repair effect works synergistically with the moisturizing function to effectively increase the skin's water content and softness, and alleviate skin dryness and barrier damage. In addition, the protonated amino group (-NH3 +) has significant antioxidant function. Protonated amino groups can capture free radicals generated in the skin due to ultraviolet irradiation or external pollutant stimulation, block oxidation chain reactions, reduce the damage of reactive oxygen species (ROS) to skin cells, delay the degradation of collagen and lipids, and prevent skin from wrinkling, pigmentation and other photoaging phenomena. At the same time, the molecular structure of glycosphingolipids has good photostability, which can reduce the damage of ultraviolet rays to the molecules themselves and improve the durability of anti-photoaging. It is worth mentioning that glycosphingolipid molecules carry positive charges and electrostatically interact with the negatively charged areas of the phospholipid bilayer on the skin surface, thereby reducing the molecular penetration resistance and promoting the penetration of molecules and their active ingredients into the deep layers of the skin. This penetration enhancement effect enables glycosphingolipid derivatives to repair the skin barrier more efficiently, provide lasting moisturizing and antioxidant protection, and achieve a more comprehensive skin care effect. In summary, the glycosphingolipid derivatives of the present invention effectively delay the skin photoaging process through the synergistic effects of moisturizing, barrier repair and anti-oxidation, meet the skin's repair and anti-aging needs under dry and external stimulation conditions, and show excellent skin care performance and broad application prospects.

[0025] The present invention successfully designs an efficient method for preparing glycosphingolipid derivatives by activating pullulan and protecting the hydroxyl groups of phytosphingosine. In the activation treatment step, the acetyl group of pullulan is selectively removed by N, N-dimethylethylenediamine (DMEDA), and trichloroacetonitrile (Cl3CCN) is introduced to generate an intermediate containing an active functional group, which greatly improves the reaction activity of the polysaccharide molecule and avoids the problem of insufficient reaction due to non-activation, thereby achieving a high conversion rate of the polysaccharide. In the hydroxyl protection step of phytosphingosine, a stable ketal structure is formed by reacting with acetone, which avoids the nonspecific reaction of multiple hydroxyls and reduces the possibility of side reactions, providing a guarantee for subsequent high-purity synthesis. In the condensation reaction step, by reacting the activated pullulan with the hydroxyl-protected phytosphingosine, combined with the precisely controlled reaction temperature (such as 30-50°C) and the amount of catalyst, the efficient combination of the sugar molecule and the sphingosine molecule is ensured, and finally a glycosphingolipid derivative with amphiphilicity and high purity is generated. Finally, impurities are further removed by deprotection treatment under alkaline conditions and multiple washing and solvent purification to ensure the chemical stability and biological activity of the target product. In summary, the present invention effectively solves the problems of low reaction efficiency, many side reactions, and difficulty in improving purity in traditional technologies. The obtained glycosphingolipid derivatives not only have excellent moisturizing properties and anti-photoaging capabilities, but also provide high-quality raw materials for the application of skin care products, drugs and functional materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 A process flow chart of the glycosphingolipid derivatives prepared in Examples 1 to 12;

[0027] Figure 2 The process flow chart of the glycosphingolipid derivative prepared in Comparative Example 1; DETAILED DESCRIPTION

[0028] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0029] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0030] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0031] Example 1

[0032] Example 1 provides a glycosphingolipid derivative and a preparation method thereof, wherein:

[0033] The structural formula of glycosphingolipid derivatives:

[0034]

[0035] Preparation method of glycosphingolipid derivatives:

[0036] Step 1: Protection of hydroxyl groups of phytosphingosine

[0037] Dissolve phytosphingosine in toluene, add acetone to the phytosphingosine solution under stirring, and then slowly add toluenesulfonic acid, wherein the toluenesulfonic acid accounts for 6wt% of the mass of the phytosphingosine, and stir the mixed solution for reaction at room temperature for 2 hours. After the reaction is completed, remove excess solvent and acetone by reduced pressure evaporation to obtain hydroxyl-protected phytosphingosine;

[0038] Step 2: Activation of Pullulan

[0039] Mixing pullulan molecules with acetyl chloride (AcCl) in pyridine, with the mass ratio of pullulan, acetyl chloride and solvent being 1:2:7, stirring and reacting at 0 to 5°C to form an acetylated pullulan derivative, separating the organic layer by liquid separation, drying with anhydrous sodium sulfate and concentrating to obtain fully acetylated pullulan;

[0040] The fully acetylated pullulan is reacted with N,N-dimethylethylenediamine (DMEDA) at 30°C, and the mass ratio between the fully acetylated pullulan and N,N-dimethylethylenediamine is 1:1.5. After the reaction is completed, the product is purified and concentrated to obtain a partially protected pullulan molecule, and trichloroacetonitrile (Cl3CCN) is used to react with the partially protected sugar molecule at 0 to 5°C, and the mass ratio between the partially protected pullulan molecule and trichloroacetonitrile is 1:1.5 to generate a sugar molecule intermediate containing an active functional group;

[0041] Step 3: Synthesis of glycosphingolipids

[0042] Add hydroxyl-protected phytosphingosine and dodecylbenzenesulfonic acid into a three-necked flask with stirring, the dodecylbenzenesulfonic acid accounts for 5wt% of the mass of the hydroxyl-protected phytosphingosine, heat to 90°C, add pullulan molecular intermediate containing active functional groups, stir for 15 min, the mass ratio of pullulan molecular intermediate containing active functional groups to hydroxyl-protected phytosphingosine is 1:2; cool to 60°C and stir, add 0.1mol / L CH3ONa solution to adjust the pH of the whole solution to 7 to 8; use vacuum filtration equipment to filter impurities in the reaction system, rinse the filtered product with purified water for multiple times, add 30wt% ethanol solution to the filtered product, stir evenly, heat the system to 55°C in a rotary evaporator, and rotary evaporate for 20-30 minutes.

[0043] The process flow chart for the preparation of glycosphingolipid derivatives is as follows Figure 1 shown.

[0044] Example 2

[0045] Example 2 provides a glycosphingolipid derivative and a preparation method thereof, wherein:

[0046] The structural formula of glycosphingolipid derivatives:

[0047]

[0048] Preparation method of glycosphingolipid derivatives:

[0049] Step 1: Protection of hydroxyl groups of phytosphingosine

[0050] Dissolve phytosphingosine in toluene, add acetone to the phytosphingosine solution under stirring, and then slowly add toluenesulfonic acid, wherein the toluenesulfonic acid accounts for 6wt% of the mass of the phytosphingosine, and stir the mixed solution for reaction at room temperature for 1 hour. After the reaction is completed, remove excess solvent and acetone by reduced pressure evaporation to obtain hydroxyl-protected phytosphingosine;

[0051] Step 2: Activation of Pullulan

[0052] Mixing pullulan molecules with acetyl chloride (AcCl) in pyridine, with the mass ratio of pullulan, acetyl chloride and solvent being 1:2.2:7, stirring and reacting at 0 to 5°C to form an acetylated pullulan derivative, separating the organic layer by liquid separation, drying with anhydrous sodium sulfate and concentrating to obtain fully acetylated pullulan;

[0053] The fully acetylated pullulan is reacted with N,N-dimethylethylenediamine (DMEDA) at 50°C, and the mass ratio between the fully acetylated pullulan and N,N-dimethylethylenediamine is 1:2. After the reaction is completed, the product is purified and concentrated to obtain a partially protected pullulan molecule. Trichloroacetonitrile (Cl3CCN) is used to react with the partially protected sugar molecule at 0 to 5°C, and the mass ratio between the partially protected pullulan molecule and trichloroacetonitrile is 1:1.2 to generate a sugar molecule intermediate containing an active functional group.

[0054] Step 3: Synthesis of glycosphingolipids

[0055] Add hydroxyl-protected phytosphingosine and dodecylbenzenesulfonic acid into a three-necked flask with stirring, the dodecylbenzenesulfonic acid accounts for 8wt% of the mass of the hydroxyl-protected phytosphingosine, heat to 80°C, add pullulan molecular intermediate containing active functional groups, stir for 20 min, the mass ratio of pullulan molecular intermediate containing active functional groups to hydroxyl-protected phytosphingosine is 1:4; cool to 60°C and stir, add 1 mol / L CH3ONa solution to adjust the pH of the whole solution to 7 to 8; use vacuum filtration equipment to filter impurities in the reaction system, rinse the filtered product with purified water for multiple times, add 30wt% ethanol solution to the filtered product, stir evenly, heat the system to 55°C in a rotary evaporator, and rotary evaporate for 20-30 minutes.

[0056] The process flow chart for the preparation of glycosphingolipid derivatives is as follows Figure 1 shown.

[0057] Example 3

[0058] Example 3 provides a glycosphingolipid derivative and a preparation method thereof, wherein:

[0059] The structural formula of glycosphingolipid derivatives:

[0060]

[0061] Preparation method of glycosphingolipid derivatives:

[0062] Step 1: Protection of hydroxyl groups of phytosphingosine

[0063] Dissolve phytosphingosine in toluene, add acetone to the phytosphingosine solution under stirring, and then slowly add toluenesulfonic acid, wherein the toluenesulfonic acid accounts for 6wt% of the mass of the phytosphingosine, and stir the mixed solution for reaction at room temperature for 1 hour. After the reaction is completed, remove excess solvent and acetone by reduced pressure evaporation to obtain hydroxyl-protected phytosphingosine;

[0064] Step 2: Activation of Pullulan

[0065] Mixing pullulan molecules with acetyl chloride (AcCl) in pyridine, with the mass ratio of pullulan, acetyl chloride and solvent being 1:2.2:5, stirring and reacting at 0 to 5°C to form an acetylated pullulan derivative, separating the organic layer by liquid separation, drying with anhydrous sodium sulfate and concentrating to obtain fully acetylated pullulan;

[0066] The fully acetylated pullulan is reacted with N,N-dimethylethylenediamine (DMEDA) at 40°C, and the mass ratio between the fully acetylated pullulan and N,N-dimethylethylenediamine is 1:1.8. After the reaction is completed, the product is purified and concentrated to obtain a partially protected pullulan molecule. Trichloroacetonitrile (Cl3CCN) is used to react with the partially protected sugar molecule at 0 to 5°C, and the mass ratio between the partially protected pullulan molecule and trichloroacetonitrile is 1:1.3, to generate a sugar molecule intermediate containing an active functional group;

[0067] Step 3: Synthesis of glycosphingolipids

[0068] Add hydroxyl-protected phytosphingosine and dodecylbenzenesulfonic acid into a three-necked flask with stirring, wherein dodecylbenzenesulfonic acid accounts for 8wt% of the mass of hydroxyl-protected phytosphingosine; after heating to 100°C, add a pullulan polysaccharide molecular intermediate containing an active functional group, and stir for 18 minutes; the mass ratio of the pullulan polysaccharide molecular intermediate containing an active functional group to the hydroxyl-protected phytosphingosine is 1:3; then cool to 60°C and stir, add 1 mol / L CH3ONa solution to adjust the pH of the entire solution to 7 to 8; use a vacuum filtration device to filter impurities in the reaction system, rinse the filtered product with purified water for multiple times, add 30wt% ethanol solution to the filtered product, stir evenly, heat the system to 55°C in a rotary evaporator, and rotary evaporate for 20-30 minutes.

[0069] The process flow chart for the preparation of glycosphingolipid derivatives is as follows Figure 1 shown.

[0070] Example 4

[0071] Example 4 provides a glycosphingolipid derivative and a preparation method thereof, wherein:

[0072] The structural formula of glycosphingolipid derivatives:

[0073]

[0074] Preparation method of glycosphingolipid derivatives:

[0075] Step 1: Protection of hydroxyl groups of phytosphingosine

[0076] Dissolve phytosphingosine in toluene, add acetone to the phytosphingosine solution under stirring, and then slowly add toluenesulfonic acid, wherein the toluenesulfonic acid accounts for 6wt% of the mass of the phytosphingosine, and stir the mixed solution for reaction at room temperature for 2 hours. After the reaction is completed, remove excess solvent and acetone by reduced pressure evaporation to obtain hydroxyl-protected phytosphingosine;

[0077] Step 2: Activation of Pullulan

[0078] Mixing pullulan molecules with acetyl chloride (AcCl) in pyridine, with the mass ratio of pullulan, acetyl chloride and solvent being 1:2.1:6, stirring and reacting at 0 to 5°C to form an acetylated pullulan derivative, separating the organic layer by liquid separation, drying with anhydrous sodium sulfate and concentrating to obtain fully acetylated pullulan;

[0079] The fully acetylated pullulan is reacted with N,N-dimethylethylenediamine (DMEDA) at 40°C, and the mass ratio between the fully acetylated pullulan and N,N-dimethylethylenediamine is 1:1.6. After the reaction is completed, the product is purified and concentrated to obtain a partially protected pullulan molecule. Trichloroacetonitrile (Cl3CCN) is used to react with the partially protected sugar molecule at 0 to 5°C, and the mass ratio between the partially protected pullulan molecule and trichloroacetonitrile is 1:1.4, to generate a sugar molecule intermediate containing an active functional group;

[0080] Step 3: Synthesis of glycosphingolipids

[0081] Add hydroxyl-protected phytosphingosine and dodecylbenzenesulfonic acid into a three-necked flask with stirring, wherein dodecylbenzenesulfonic acid accounts for 7wt% of the mass of hydroxyl-protected phytosphingosine; after heating to 100°C, add pullulan molecular intermediate containing active functional groups, stir for 18 minutes, and the mass ratio of pullulan molecular intermediate containing active functional groups to hydroxyl-protected phytosphingosine is 1:4; then cool to 60°C and stir, add 1 mol / L CH3ONa solution to adjust the pH of the entire solution to 7 to 8; use vacuum filtration equipment to filter impurities in the reaction system, rinse the filtered product with purified water for multiple times, add 30wt% ethanol solution to the filtered product, stir evenly, heat the system to 55°C in a rotary evaporator, and rotary evaporate for 20-30 minutes.

[0082] The process flow chart for the preparation of glycosphingolipid derivatives is as follows Figure 1 shown.

[0083] Example 5

[0084] Example 5 provides a glycosphingolipid derivative and a preparation method thereof, wherein:

[0085] The structural formula of glycosphingolipid derivatives:

[0086]

[0087] Preparation method of glycosphingolipid derivatives:

[0088] Step 1: Protection of hydroxyl groups of phytosphingosine

[0089] Dissolve phytosphingosine in toluene, add acetone to the phytosphingosine solution under stirring, and then slowly add toluenesulfonic acid, wherein the toluenesulfonic acid accounts for 6wt% of the mass of the phytosphingosine, and stir the mixed solution for reaction at room temperature for 1 hour. After the reaction is completed, remove excess solvent and acetone by reduced pressure evaporation to obtain hydroxyl-protected phytosphingosine;

[0090] Step 2: Activation of Pullulan

[0091] Mixing pullulan molecules with acetyl chloride (AcCl) in pyridine, with the mass ratio of pullulan, acetyl chloride and solvent being 1:2:6, stirring and reacting at 0 to 5°C to form an acetylated pullulan derivative, separating the organic layer by liquid separation, drying with anhydrous sodium sulfate and concentrating to obtain fully acetylated pullulan;

[0092] The fully acetylated pullulan is reacted with N,N-dimethylethylenediamine (DMEDA) at 10°C, and the mass ratio between the fully acetylated pullulan and N,N-dimethylethylenediamine is 1:2. After the reaction is completed, the product is purified and concentrated to obtain a partially protected pullulan molecule. Trichloroacetonitrile (Cl3CCN) is used to react with the partially protected sugar molecule at 0 to 5°C, and the mass ratio between the partially protected pullulan molecule and trichloroacetonitrile is 1:1.3, to generate a sugar molecule intermediate containing an active functional group;

[0093] Step 3: Synthesis of glycosphingolipids

[0094] Add hydroxyl-protected phytosphingosine and dodecylbenzenesulfonic acid into a three-necked flask with stirring, the dodecylbenzenesulfonic acid accounts for 7wt% of the mass of the hydroxyl-protected phytosphingosine, heat to 90°C, add pullulan molecular intermediate containing active functional groups, stir for 15 min, the mass ratio of pullulan molecular intermediate containing active functional groups to hydroxyl-protected phytosphingosine is 1:2; cool to 60°C and stir, add 1 mol / L CH3ONa solution to adjust the pH of the whole solution to 7 to 8; use vacuum filtration equipment to filter impurities in the reaction system, rinse the filtered product with purified water for multiple times, add 30wt% ethanol solution to the filtered product, stir evenly, heat the system to 55°C in a rotary evaporator, and rotary evaporate for 20-30 minutes.

[0095] The process flow of preparing glycosphingolipid derivatives is as follows Figure 1 shown.

[0096] Examples 6 to 12

[0097] The difference between Examples 6 to 12 and Example 5 is mainly reflected in the different reaction temperatures of fully acetylated pullulan and N,N-dimethylethylenediamine (DMEDA).

[0098] Table 1 is the parameters of the reaction conditions of Examples 1 to 12

[0099]

[0100] Note: A represents the mass ratio of pullulan, acetyl chloride and solvent; B represents the reaction temperature of fully acetylated pullulan and N,N-dimethylethylenediamine; C represents the mass ratio of fully acetylated pullulan and N,N-dimethylethylenediamine; D represents the mass ratio of partially protected pullulan molecules and trichloroacetonitrile; E represents the mass of dodecylbenzenesulfonic acid to hydroxyl-protected phytosphingosine; F represents the temperature required to heat up when hydroxyl-protected phytosphingosine and dodecylbenzenesulfonic acid are added to a three-necked flask with stirring; G represents the stirring time required when hydroxyl-protected phytosphingosine and dodecylbenzenesulfonic acid are added to a three-necked flask with stirring; H represents the mass ratio of pullulan molecular intermediates containing active functional groups to hydroxyl-protected phytosphingosine.

[0101] Comparative Example 1

[0102] The control example is a glycosphingolipid derivative and a preparation method thereof, wherein:

[0103] The structural formula of glycosphingolipid derivatives:

[0104]

[0105] Preparation method of glycosphingolipid derivatives:

[0106] Step 1: Activation of Pullulan

[0107] Mixing pullulan molecules with acetyl chloride (AcCl) in pyridine, with the mass ratio of pullulan, acetyl chloride and solvent being 1:2:6, stirring and reacting at 0 to 5°C to form an acetylated pullulan derivative, separating the organic layer by liquid separation, drying with anhydrous sodium sulfate and concentrating to obtain fully acetylated pullulan;

[0108] The fully acetylated pullulan is reacted with N,N-dimethylethylenediamine (DMEDA) at 40°C, and the mass ratio between the fully acetylated pullulan and N,N-dimethylethylenediamine is 1:2. After the reaction is completed, the product is purified and concentrated to obtain a partially protected pullulan molecule. Trichloroacetonitrile (Cl3CCN) is used to react with the partially protected sugar molecule at 0 to 5°C, and the mass ratio between the partially protected pullulan molecule and trichloroacetonitrile is 1:1.3, to generate a sugar molecule intermediate containing an active functional group;

[0109] Step 2: Synthesis of glycosphingolipids

[0110] Phytosphingosine and BF3·Et2O (boron trifluoride-ether complex) are added to a three-necked flask with stirring, the boron trifluoride-ether complex accounts for 7wt% of the mass of phytosphingosine, and the temperature is raised to 90°C, and then a pullulan polysaccharide molecular intermediate containing an active functional group is added, and stirred for 15 minutes, and the mass ratio of the pullulan polysaccharide molecular intermediate containing an active functional group to phytosphingosine is 1:2; then the temperature is lowered to 60°C and stirred, and 1 mol / L CH3ONa solution is added to adjust the pH of the entire solution to 7 to 8; impurities in the reaction system are filtered using a vacuum filtration device, and the filtered product is rinsed with purified water for multiple times, and a 30wt% ethanol solution is added to the filtered product, and stirred evenly, and the system is heated to 55°C in a rotary evaporator, and rotary evaporated for 20-30 minutes.

[0111] The process flow chart of the preparation of glycosphingolipid derivatives, such as Figure 2 shown.

[0112] Comparative Example 2

[0113] Preparation method of glycosphingolipid derivatives:

[0114] Step 1: Protection of hydroxyl groups of phytosphingosine

[0115] Dissolve phytosphingosine in toluene, add acetone to the phytosphingosine solution under stirring, and then slowly add toluenesulfonic acid, wherein the toluenesulfonic acid accounts for 6wt% of the mass of the phytosphingosine, and stir the mixed solution for reaction at room temperature for 1 hour. After the reaction is completed, remove excess solvent and acetone by reduced pressure evaporation to obtain hydroxyl-protected phytosphingosine;

[0116] Step 2: Synthesis of glycosphingolipids

[0117] Add hydroxyl-protected phytosphingosine and dodecylbenzenesulfonic acid into a three-necked flask with stirring, the dodecylbenzenesulfonic acid accounts for 7wt% of the mass of the hydroxyl-protected phytosphingosine, heat to 90°C, add pullulan, stir for 15 min, the mass ratio of pullulan molecules to hydroxyl-protected phytosphingosine is 1:2; cool to 60°C and stir, add 1 mol / L CH3ONa solution to adjust the pH of the whole solution to 7 to 8; use vacuum filtration equipment to filter impurities in the reaction system, rinse the filtered product with purified water for multiple times, add 30wt% ethanol solution to the filtered product, stir evenly, heat the system to 55°C in a rotary evaporator, and rotary evaporate for 20-30 minutes.

[0118] Comparative Example 3

[0119] Preparation method of glycosphingolipid derivatives:

[0120] Step 1: Activation of Pullulan

[0121] Mixing pullulan molecules with acetyl chloride (AcCl) in pyridine, with the mass ratio of pullulan, acetyl chloride and solvent being 1:2:6, stirring and reacting at 0 to 5°C to form an acetylated pullulan derivative, separating the organic layer by liquid separation, drying with anhydrous sodium sulfate and concentrating to obtain fully acetylated pullulan;

[0122] The fully acetylated pullulan is reacted with N,N-dimethylethylenediamine (DMEDA) at 40°C, and the mass ratio between the fully acetylated pullulan and N,N-dimethylethylenediamine is 1:2. After the reaction is completed, the product is purified and concentrated to obtain a partially protected pullulan molecule. Trichloroacetonitrile (Cl3CCN) is used to react with the partially protected sugar molecule at 0 to 5°C, and the mass ratio between the partially protected pullulan molecule and trichloroacetonitrile is 1:1.3, to generate a sugar molecule intermediate containing an active functional group;

[0123] Step 2: Synthesis of glycosphingolipids

[0124] Add phytosphingosine and dodecylbenzenesulfonic acid into a three-necked flask with stirring, the dodecylbenzenesulfonic acid accounts for 7wt% of the mass of phytosphingosine, heat to 90°C, add pullulan molecular intermediate containing active functional groups, stir for 15 min, the mass ratio of pullulan molecular intermediate containing active functional groups to phytosphingosine is 1:2; cool to 60°C and stir, add 1 mol / L CH3ONa solution to adjust the pH of the whole solution to 7 to 8; use vacuum filtration equipment to filter impurities in the reaction system, rinse the filtered product with purified water for multiple times, add 30wt% ethanol solution to the filtered product, stir evenly, heat the system to 55°C in a rotary evaporator, and rotary evaporate for 20-30 minutes.

[0125] Performance Characterization Methods

[0126] Testing method for polysaccharides: DNS colorimetry is mainly used to determine the amount of residual sugar in the reaction system and the conversion rate of polysaccharides. Under alkaline conditions, the DNS reagent reacts with the unreacted reducing sugar to develop a color, the absorbance is determined by colorimetry, and the residual sugar content and conversion rate are calculated in combination with the standard curve. The specific steps include preparing the DNS reagent and the standard curve, taking a sample and diluting it to react with the DNS reagent, and then calculating the total sugar content and conversion rate by measuring the absorbance at 540 nm. The calculation formula for the polysaccharide conversion rate is: Polysaccharide conversion rate = 1-(residual sugar amount / initial sugar dosage). This method is easy to operate and can quickly and accurately evaluate the conversion efficiency of polysaccharides.

[0127] Testing method for glycosphingolipid purity: Using high performance liquid chromatography-mass spectrometry (LC-MS), the purity is determined by detecting the mass-to-charge ratio (m / z) and peak area ratio of the target product in the sample. After the sample is acetylated, the detection sensitivity and separation effect are improved. The test steps include sample processing, LC-MS detection and data analysis, in which the LC-MS instrument is set to ESI positive ion mode and the mobile phase is a mixed solvent of acetonitrile and TFA buffer. The purity of the target product is expressed as the proportion of the target molecule peak in the total ion flow. This method has high precision and high sensitivity, and can accurately evaluate the purity of glycosphingolipid derivatives, providing a reliable basis for product quality control.

[0128] Test method for moisturizing properties of creams containing glycosphingolipid derivatives

[0129] The preparation process of the cream is as follows: First, water and 1,2-hexanediol are mixed and heated to 75°C under stirring to form an aqueous phase; at the same time, squalane, CG68 and 16 / 18 alcohol are heated to 75°C in a water bath and stirred evenly to form an oil phase. At 75°C, the aqueous phase is slowly added to the oil phase and emulsified for 5 minutes using a high-speed stirrer to form a uniform cream base. After emulsification, continue stirring and cool to below 40°C, add 0.5% of glycosphingolipid derivatives, stir evenly, and fully disperse them. After cooling to room temperature, the cream is packaged into clean containers and sealed for storage. The entire process must maintain sterile operation and strict temperature control to ensure the uniformity and stability of the product texture.

[0130] The subjects cleaned the test area with dry tissue and rested for 20 minutes in a constant temperature and humidity environment. The tester first tested the initial values ​​of each area, and then applied the sample in the marked measurement area at a dosage of (2.0±0.1) mg / cm2. The water content of the stratum corneum, transepidermal water loss, and water content of the stratum corneum were tested at four time points: 1h, 2h, 4h, and 8h.

[0131] Test methods for photosensitivity and anti-aging of glycosphingolipid derivatives

[0132] 1. Main equipment

[0133] CO2 incubator (Thermo), microplate reader (Tecan), inverted microscope (CEWEI), water bath (SENCO), vortex shaker (DLAB), electronic scale (Sunny Optical), and pipette (DLAB).

[0134] 2. Materials and Reagents

[0135] T-SkinTM 3D in vitro reconstructed full-thickness skin model (EPISKIN), maintenance medium (EPISKIN), detection medium (EPISKIN), COLI antibody (Thermo Fisher), secondary antibody (Thermo Fisher).

[0136] 3. Test samples

[0137] DropCare® Phytos phytosphingosine: Prepare sample solution of corresponding concentration with sterile PBS, and filter the sample through 0.22μm filter membrane before testing.

[0138] 4. T-SkinTM 3D in vitro reconstruction of full-thickness skin model culture

[0139] The T-SkinTM three-dimensional in vitro reconstructed full-thickness skin model was removed from the agar medium, the adhered agar was wiped off with a sterile filter paper strip, and the model was transferred to a 6-well plate with 4 ml of maintenance medium added to each well, and cultured for 24 hours.

[0140] 5. UVA-UVB induced T-SkinTM 3D in vitro reconstruction of full-thickness skin model

[0141] The skin model was transferred to a 6-well plate with 4 ml of sterile PBS buffer added to each well, and 25 μl of the test substance was applied to each group. Except for the blank group, the T-SkinTM three-dimensional in vitro reconstructed full-thickness skin model was irradiated with a dose of 90 mJ UVB + 0.8 J UVA. After the irradiation, it was transferred to a 6-well plate with 4 ml of detection culture medium added to each well and cultured for 24 hours; the samples were added according to the groups in Table 2 below.

[0142] Table 2 Effects of samples on UVA-UVB-induced T-SkinTM three-dimensional in vitro reconstruction of full-thickness skin model

[0143] Effect of COLI expression on the preparation table

[0144]

[0145] Note: BC is the blank group, and NC is the model group.

[0146] 6. Detection of T-SkinTM 3D in vitro reconstructed full-thickness skin model indicators

[0147] After culturing the epidermal model for 24 hours, the T-SkinTM three-dimensional in vitro full-thickness skin model was fixed with 4% paraformaldehyde for 48 hours, dehydrated with gradient ethanol, transparentized with xylene, embedded in paraffin and sliced ​​with a thickness of 3 μm. The slices were dewaxed with xylene, rehydrated with gradient ethanol, and sealed with neutral gum after immunofluorescence staining. Photos were taken with an inverted microscope, and analyzed and measured with Image-Pro Plus6.0 image analysis software.

[0148] 7. Statistical Analysis

[0149] GraphPad Prism8 software was used. Count data were expressed as number of cases and / or percentage. Normal distribution measurement data were expressed as mean ± standard deviation (x ± s). Non-normal distribution measurement data were expressed as median and interquartile range M (Q, Qu). Multi-sample analysis of variance was used for comparison between groups with equal variance, and corrected analysis of variance was used for comparison between groups with unequal variance. If the differences between groups were statistically significant, LSD method was used for further pairwise comparison.

[0150] Table 3 is the experimental data of Examples 1 to 12

[0151]

[0152] Table 4 is the moisturizing test data of Example 8 and Control Example 1

[0153]

[0154] Table 5 is the anti-aging test data of Example 8 and Comparative Example 1

[0155]

[0156] Note: ΔΔΔ: p<0.001, compared with BC; ***: p<0.001, compared with NC

[0157] Combining Examples 1 to 4 and Table 3, it can be seen that Examples 1 to 4 all show high polysaccharide conversion rates (94.8% to 97.8%) and glycosphingolipid purity (96.7% to 99.6%). Through the process method of the present application, pullulan is activated and the hydroxyl groups of phytosphingosine are protected, and a glycosphingolipid derivative with a specific structure is successfully synthesized. The optimized design of this process includes the selective activation reaction of pullulan, the introduction of active functional groups to specific sites of sugar molecules, and the protection of hydroxyl groups of phytosphingosine. By forming a stable ketal structure, the occurrence of non-specific side reactions is avoided, thereby ensuring the high efficiency of the reaction. The two undergo a condensation reaction under suitable reaction conditions, which significantly improves the conversion rate of polysaccharides and the purity of glycosphingolipid derivatives. The precise control of the activation and protection steps not only reduces the generation of by-products, but also improves the structural uniformity and chemical stability of the target product.

[0158] Combining Examples 5 to 12 and Table 3, it can be seen that as the reaction temperature gradually increases, the polysaccharide conversion rate and the purity of the glycosphingolipid derivatives gradually increase. When the reaction temperature reaches 40°C, the polysaccharide conversion rate and the purity of the glycosphingolipid derivatives reach the maximum value. As the reaction temperature continues to increase, the polysaccharide conversion rate and the purity of the glycosphingolipid derivatives gradually decrease; the main reason is that under low temperature conditions (such as below 30°C), the reaction rate is significantly reduced, resulting in incomplete deprotection reaction of pullulan with N, N-dimethylethylenediamine (DMEDA), and the acetyl group at a specific position cannot be effectively and selectively removed. In this case, insufficient exposure of the active site will limit the activation efficiency of the subsequent reaction, and the conversion rate of the polysaccharide will therefore decrease. In addition, due to the incomplete reaction, the undeprotected polysaccharide residues may increase the impurity content in the target product, resulting in the purity of the glycosphingolipid derivatives being affected. Although low temperature can reduce the occurrence of side reactions, insufficient kinetic activation energy makes it difficult for the target reaction to proceed efficiently. On the contrary, under high temperature conditions (such as above 60°C), although the reaction rate is accelerated, it is easy to induce significant side reactions. As a polysaccharide compound, the glycosidic bonds of pullulan are easily broken at high temperatures, resulting in a decrease in molecular weight and structural damage, which not only affects the functionality of the target product, but may also produce uncontrollable degradation products. In addition, high temperature conditions may induce cross-linking reactions between polysaccharide molecules, generate unwanted by-products, and increase the impurity content, further reducing the purity of the target product. Overall, nonspecific reactions and degradation problems at high temperatures have a serious adverse effect on reaction selectivity and product quality. Therefore, under suitable mild conditions (such as 40°C to 50°C), the reaction rate and selectivity can achieve an optimal balance, which not only avoids the problem of incomplete reaction at low temperature, but also effectively inhibits side reactions caused by high temperature.

[0159] Combining Example 8, Comparative Example 2 and Table 3, it can be seen that the polysaccharide conversion rate and purity of the glycosphingolipid derivatives prepared in Example 8 are significantly higher than those in Comparative Example 2. This difference is mainly attributed to the activation treatment of pullulan in Example 8, while pullulan was not activated in Comparative Example 2. In Example 8, pullulan was subjected to a selective deprotection reaction of N, N-dimethylethylenediamine (DMEDA) and an activation treatment of trichloroacetonitrile (Cl3CCN) to expose active functional groups at specific sites of the polysaccharide molecule. This treatment greatly improves the reaction efficiency between pullulan and phytosphingosine, ensures a high conversion rate of polysaccharides, and reduces the occurrence of side reactions. In addition, the activation treatment makes the condensation reaction of polysaccharides with phytosphingosine more selective, thereby generating glycosphingolipid derivatives with higher purity. On the contrary, in Comparative Example 2, since pullulan was not activated, the reactivity of its hydroxyl group was low, resulting in a reduced reaction efficiency with phytosphingosine and a failure to fully carry out the condensation reaction. This deficiency not only leads to a decrease in the conversion rate of polysaccharides, but also increases the residues of unreacted raw materials and by-products, thereby reducing the purity of the final product.

[0160] Combining Example 8, Reference Example 3 and Table 3, it can be seen that the polysaccharide conversion rate and purity of the glycosphingolipid derivatives prepared in Example 8 are significantly higher than those in Reference Example 3. The main reason for this difference is that the hydroxyl group of phytosphingosine is protected in Example 8, while the hydroxyl group of phytosphingosine is not protected in Reference Example 3. In Example 8, the hydroxyl group of phytosphingosine forms a stable diketal structure through the ketal reaction of acetone and toluenesulfonic acid. This hydroxyl protection can effectively avoid the non-specific reaction of the polyhydroxyl groups of phytosphingosine in the condensation reaction, thereby improving the selectivity of the reaction. This treatment not only reduces the occurrence of side reactions, but also makes the condensation reaction of pullulan and phytosphingosine more efficient, thereby significantly improving the conversion rate of polysaccharides. At the same time, hydroxyl protection also avoids the generation of impurities, which significantly improves the purity of glycosphingolipid derivatives. On the contrary, in Reference Example 3, since the hydroxyl group of phytosphingosine is not protected, its polyhydroxyl structure may cause non-specific side reactions during the reaction. For example, the polyhydroxyl groups of phytosphingosine may undergo non-specific cross-linking with other parts of pullulan, resulting in an increase in by-products. In addition, unprotected hydroxyl groups may also lead to heterogeneity of molecular structure, further reducing the purity of glycosphingolipid derivatives and the conversion rate of polysaccharides.

[0161] Combining Example 8, Control Example 1 and Table 4, it can be seen that within 0-8h, the △TEWL value of Example 1 is the largest compared with Control Example 1. The larger the △TEWL value (transepidermal water loss value), the better the moisturizing effect of the sample. Therefore, Formula 1 has the best moisturizing ability, mainly because the amino structure of phytosphingosine in the derivative prepared in Example 8 is retained and is easily protonated to form -NH3 under specific pH conditions. + This property gives the molecule a positive charge, allowing it to more effectively form electrostatic adsorption with water molecules, further enhancing the molecule's hydrophilicity and moisturizing properties. Especially under low humidity conditions, the protonated amino group exhibits excellent hygroscopicity and can continuously lock in moisture, overcoming the limitations of traditional moisturizers. At the same time, the protonated amino group also improves the solubility and skin permeability of the molecule, allowing the glycosphingolipid molecules to electrostatically interact with the negatively charged phospholipid bilayer on the skin surface, reducing the molecular penetration resistance, promoting the penetration of active ingredients into the deep layers of the skin, and exerting a more lasting moisturizing effect.

[0162] Combining Example 8, Control Example 1 and Table 5, it can be seen that compared with the BC group, the NC group can significantly reduce the expression of COLI after UVA-UVB induction (p<0.001), with a reduction rate of 52.30%; compared with the NC group, the derivatives prepared in Example 8 and Control Example 1 can significantly increase the expression of COLI, with an increase rate of 58.17% (p<0.001) in Control Example 1 and 75.39% (p<0.001) in Example 8. This is mainly because the hydroxyl group at the top of phytosphingosine is replaced by pullulan, making the entire molecular structure more stable, reducing the active hydroxyl sites that are easily degraded under exposure to light or oxidation. This structural optimization effectively reduces the risk of decomposition of the molecule under ultraviolet (UV) or thermal conditions, thereby enhancing the anti-photoaging properties of glycosphingolipid compounds. In addition, phytosphingosine itself has certain antioxidant activity. By retaining its amino structure, it can effectively capture free radicals, block oxidation chain reactions, and reduce the damage of reactive oxygen species (ROS) to skin cells. Protonated amino groups (-NH3 + ) can also form electrostatic interactions with free radicals under specific pH conditions, further improving the antioxidant capacity of the molecule. This property enables glycosphingolipid molecules to protect the skin from aging damage caused by free radicals under light conditions and delay the photoaging process of the skin.

[0163] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention is described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A glycosphingolipid derivative, characterized in that: The chemical formula of the glycosphingolipid derivative is as follows: 。 2. The method for preparing the glycosphingolipid derivative according to claim 1, characterized in that: The technical steps include: S1. Add hydroxyl-protected phytosphingosine and dodecylbenzenesulfonic acid into a three-necked flask with stirring, heat to 80 to 100°C, add pullulan molecular intermediate containing active functional groups, and stir for 15 to 20 min; S2. The mixture was cooled to 60 °C and stirred, and 0.1 mol / L to 1 mol / L CH3ONa solution was added to adjust the pH of the whole solution to 7 to 8; S3. Use a vacuum filtration device to filter impurities in the reaction system, rinse the filtered product several times with purified water, add 30wt% ethanol solution to the filtered product, stir evenly, heat the system to 55 ° C in a rotary evaporator, and evaporate for 20-30 minutes; The structural formula of the pullulan polysaccharide molecular intermediate containing active functional groups is: The structural formula of the phytosphingosine with protected hydroxyl group is: 。 3. The preparation method according to claim 2, characterized in that: In step S1, the method for preparing the pullulan molecular intermediate containing active functional groups comprises the following preparation steps: Mixing pullulan molecules and acetyl chloride in an appropriate amount of solvent, stirring and reacting at 0 to 5° C. to form an acetylated pullulan derivative, separating the organic layer by liquid separation, and drying with anhydrous sodium sulfate and concentrating to obtain fully acetylated pullulan; wherein the solvent is pyridine, and the mass ratio of pullulan, acetyl chloride and solvent is 1:(2 to 2.2):(5 to 7); The fully acetylated pullulan is reacted with N,N-dimethylethylenediamine. After the reaction is completed, the product is purified and concentrated to obtain partially protected pullulan molecules. Trichloroacetonitrile is used to react with the partially protected sugar molecules at 0 to 5°C to generate sugar molecule intermediates containing active functional groups; wherein the mass ratio between the fully acetylated pullulan and N,N-dimethylethylenediamine is 1:(1.5 to 2), and the mass ratio between the partially protected pullulan molecules and trichloroacetonitrile is 1:(1.2 to 1.5).

4. The preparation method according to claim 2, characterized in that: In step S1, the method for preparing the hydroxyl-protected phytosphingosine comprises the following steps: The phytosphingosine is dissolved in toluene, acetone is added to the phytosphingosine solution under stirring, and then toluenesulfonic acid is slowly added, and the mixed solution is stirred and reacted at room temperature for 1 to 2 hours. After the reaction is completed, the excess solvent and acetone are removed by reduced pressure evaporation to obtain hydroxyl-protected phytosphingosine.

5. The preparation method according to claim 2, characterized in that: In step S1, the mass ratio of the pullulan molecular intermediate containing active functional groups to the hydroxyl-protected phytosphingosine is 1:2 to 1:

4.

6. The preparation method according to claim 2, characterized in that: In step S1, dodecylbenzenesulfonic acid accounts for 5 to 8 wt % of the mass of the hydroxyl-protected phytosphingosine.

7. The preparation method according to claim 3, characterized in that: The reaction conditions of fully acetylated pullulan and N,N-dimethylethylenediamine are 30°C to 50°C.

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