Method for enzymatic synthesis of retinoic acid ceramide ester and use thereof

Abstract

The application discloses a method for synthesizing ceramide ester of retinoic acid by catalysis of an enzyme and application thereof, and belongs to the technical field of cosmetic raw material synthesis. The method uses retinoic acid and ceramide NP as substrates, adopts specific immobilized lipase as a catalyst, and efficiently synthesizes a target product in a non-aqueous organic solvent through the steps of raw material preparation, an oxygen-free reaction system building, directional esterification, post-treatment and purification detection. The method solves the problems of low substrate solubility, many by-products and difficult catalyst recovery in traditional processes, has mild reaction conditions, can retain the activity of raw materials, has high product purity, has the effects of anti-aging and barrier repair, and can be widely used in the preparation of anti-wrinkle and repair cosmetic raw materials.

Description

Technical Field

[0001] This invention relates to the field of cosmetic raw material synthesis technology, and in particular to a method for enzyme-catalyzed synthesis of retinoic acid ceramide ester and its application. Background Technology

[0002] In the field of cosmetic raw material synthesis, the development of active ingredients with both anti-aging and skin barrier repair functions has always been a research hotspot. Retinoic acid (vitamin A acid), as a classic bioactive substance, has the effect of significantly regulating epidermal cell proliferation and differentiation and promoting collagen synthesis, making it one of the core ingredients for anti-wrinkle and anti-aging. Ceramides, on the other hand, are key lipids constituting the skin barrier, effectively maintaining skin moisture and repairing damaged barriers. The synergistic effect of these two ingredients can simultaneously meet the dual needs of skin anti-aging and repair, showing broad application prospects. However, retinoic acid itself has inherent defects such as poor stability and strong skin irritation, while ceramides face the problem of insufficient solubility. These shortcomings severely limit the direct application and efficacy of these two ingredients in cosmetic formulations.

[0003] To address the aforementioned issues, the industry has attempted to combine retinoic acid and ceramides through esterification to form retinoic acid-ceramide ester compounds. These products not only significantly improve the stability of retinoic acid and reduce its irritation, but also retain the synergistic skincare effects of both, demonstrating promising application potential. Currently, the main technical pathways for achieving this esterification reaction include traditional chemical esterification and existing bio-enzyme catalysis methods, but both have significant limitations: traditional chemical esterification requires high temperature, strong acid, or strong alkali conditions, which not only generates a large number of byproducts leading to low product purity but also poses environmental pollution problems. Furthermore, extreme reaction conditions can easily induce isomerization of retinoic acid, causing it to lose its biological activity. While existing bio-enzyme catalysis systems have improved the mildness of reaction conditions, they generally suffer from low substrate solubility and insufficient reaction conversion rates. Additionally, the free enzyme is difficult to recover and reuse, increasing production costs. These technical bottlenecks restrict the large-scale preparation and industrial application of retinoic acid-ceramide esters. Therefore, developing a method for preparing retinoic acid-ceramides with mild reaction conditions, high selectivity, high product purity, and controllable cost has become an urgent technical need in this field. Summary of the Invention

[0004] To achieve the above objectives, this invention provides an enzyme-catalyzed method for synthesizing retinoic acid ceramide esters. Using retinoic acid and ceramide NP as reaction substrates and an immobilized lipase as a catalyst, the target product is synthesized via a directed esterification reaction in a non-aqueous organic solvent. The method includes steps such as raw material preparation, reaction system construction, esterification reaction, post-treatment, purification, and detection. The target product is a solid retinoic acid ceramide ester with a purity greater than 98%.

[0005] In the raw material preparation steps: the molar ratio of ceramide NP to retinoic acid is 1:1 to 1.5:1; the immobilized lipase is selected from one or two of Candida antarcticis lipase type B (Novozym 435) and Lipozyme TL IM, and the total amount of immobilized lipase added is 2% to 5% of the total weight of the reaction substrate;

[0006] The reaction system construction steps are as follows: add retinoic acid and ceramide NP to the reaction vessel, pour in a non-aqueous organic solvent and stir until the substrate is fully dissolved, then add immobilized lipase and molecular sieve, seal the reaction vessel and purge with nitrogen to create an anaerobic reaction environment;

[0007] In the esterification reaction step: the reaction is carried out in a constant temperature shaker at a temperature of 30~50℃, a shaking rate of 150-200rpm, and a reaction time of 8~24 hours. Lipase specifically catalyzes the esterification reaction between the carboxyl group of retinoic acid and the primary hydroxyl group of ceramide.

[0008] The post-processing steps are as follows: After the reaction is completed, the reaction system is filtered, the immobilized lipase and molecular sieve are recovered, the filtrate is collected and evaporated under reduced pressure at 40~45℃ to remove the non-aqueous organic solvent, and crude retinyl ceramide ester is obtained.

[0009] The purification and detection steps are as follows: Dichloromethane is added to the crude retinyl ceramide ester product for dissolution, and the product is purified by silica gel column chromatography with gradient elution using petroleum ether / ethyl acetate as eluent. The target eluent fraction is collected and dried. The purity is detected by high performance liquid chromatography (HPLC), and the product with a purity greater than 98% is collected as the target product.

[0010] Furthermore, when the two immobilized lipases were used in combination, the mass ratio of Candida antarcticis lipase type B (Novozym435) to Lipozyme TL IM was 1:1.

[0011] Furthermore, the non-aqueous organic solvent is selected from one or more mixtures of tert-butanol, n-hexane, and acetone.

[0012] Furthermore, the molecular sieve is a 3Å or 5Å molecular sieve.

[0013] Furthermore, the recovered immobilized lipase can be reused in subsequent batches of reactions.

[0014] This invention also claims the use of the retinoic acid ceramide ester synthesized according to the above method in the preparation of raw materials for anti-wrinkle and repair cosmetics.

[0015] The enzyme-catalyzed synthesis method of retinoic acid ceramide ester provided by this invention, through the scientific selection of immobilized lipase as catalyst, suitable non-aqueous organic solvent and dehydrating agent, combined with a rationally constructed reaction system and optimized reaction conditions, not only effectively solves the technical pain points of traditional synthesis processes such as poor substrate solubility, excessive by-product generation, insufficient product purity, and difficulty in catalyst recovery and reuse, but also protects the core active structure of the raw materials with a mild reaction environment, avoiding the destruction and loss of active ingredients. The prepared retinoic acid ceramide ester product has both the anti-aging effect of retinoic acid and the skin barrier repair function of ceramide, and the catalyst can be reused, significantly reducing production costs. It has good selectivity and practicality, providing reliable support for the large-scale preparation and industrial application of retinoic acid ceramide ester, and can be widely adapted to the production scenarios of anti-wrinkle and repair cosmetic raw materials.

[0016] The following will further explain the concept, specific structure, and technical effects of the present invention in conjunction with the accompanying drawings, so as to fully understand the purpose, features, and effects of the present invention. Attached Figure Description

[0017] Figure 1 This is a process flow diagram of the present invention. Detailed Implementation

[0018] The following description, with reference to the accompanying drawings, illustrates several preferred embodiments of the present invention to make its technical content clearer and easier to understand. The present invention can be embodied in many different forms, and the scope of protection of the present invention is not limited to the embodiments mentioned herein.

[0019] See Figure 1 This invention discloses an enzyme-catalyzed method for synthesizing retinoic acid ceramide esters. The method uses retinoic acid and ceramide as reaction substrates and immobilized lipase as a catalyst to efficiently synthesize the target product through a directed esterification reaction in a non-aqueous organic solvent. The specific steps are as follows:

[0020] Raw material preparation: Accurately weigh the corresponding amounts of retinoic acid and ceramide NP according to the molar ratio of ceramide to retinoic acid of 1:1 to 1.5:1; at the same time, prepare the catalyst, non-aqueous organic solvent and dehydrating agent. The catalyst is an immobilized lipase, selected from one or a mixture of two of Candida antarctica lipase type B (Novozym 435) and Thermophilic lipase of Lipozyme TL IM, and the amount of enzyme added is 2% to 5% of the total weight of the substrate; the non-aqueous organic solvent is selected from one or a mixture of tert-butanol, n-hexane and acetone, and the dehydrating agent is a 3Å or 5Å molecular sieve.

[0021] Reaction system setup: Weigh out retinoic acid and ceramide NP and add them to a dry reaction vessel. Pour in a pre-set amount of non-aqueous organic solvent and stir to fully dissolve the substrate. Then add the prepared immobilized lipase and molecular sieve. After sealing the reaction vessel, purge with nitrogen to create an anaerobic reaction environment and avoid oxidation of raw materials that could affect reaction efficiency and product quality.

[0022] Esterification reaction: Place the sealed reaction vessel in a constant temperature shaker, set the reaction temperature to 30~50℃, the shaking rate to 200rpm, and continue the reaction for 8~24 hours. During this period, maintain stable reaction conditions to ensure that lipase specifically catalyzes the esterification reaction between the carboxyl group of retinoic acid and the primary hydroxyl group of ceramide.

[0023] Post-processing: After the reaction is completed, the reaction system is filtered while hot to recover the immobilized lipase and molecular sieve. The recovered catalyst can be reused in subsequent batches of reaction. The filtrate is collected and subjected to vacuum rotary evaporation at 40-45℃ to remove the non-aqueous organic solvent, yielding crude retinoic acid ceramide ester.

[0024] Purification and detection: A small amount of dichloromethane was added to the crude product to dissolve it. The product was purified by silica gel column chromatography with gradient elution using petroleum ether / ethyl acetate as eluent. The target eluent was collected and dried. The purity of the dried product was detected by high performance liquid chromatography (HPLC). The white to pale yellow solid with a purity greater than 98% was collected as the finished product of retinyl ceramide ester.

[0025] This method effectively solves the problems of low substrate solubility, numerous by-products, insufficient product purity, and difficulty in catalyst recovery in traditional synthesis processes by rationally selecting catalysts, solvents, and reaction conditions. The reaction conditions are mild and can protect the core active structure of the raw materials from damage. Moreover, the product has both the anti-aging effect of retinoic acid and the barrier repair function of ceramide, and can be widely used in the preparation of raw materials for anti-wrinkle and repair cosmetics.

[0026] Example 1

[0027] A method for the enzyme-catalyzed synthesis of retinoic acid ceramide esters, the specific steps of which are as follows:

[0028] Raw material preparation: Accurately weigh 300mg (1.0mmol) of retinoic acid and 1mg (1.0mmol) of ceramide NP581mg to ensure that the purity of the raw materials meets the standards for cosmetic raw material synthesis; prepare 44.05mg of immobilized Candida antarcticis lipase (Novozym435), 1.0g of 3Å molecular sieve and 30mL of anhydrous tert-butanol.

[0029] Reaction system setup: Weigh out retinoic acid and ceramide NP and add them to a 100mL dry reaction flask. Pour in 30mL of anhydrous tert-butanol and stir to fully dissolve the raw materials. Then add 44.05mg of immobilized Candida antarcticis lipase and 1.0g of 3Å molecular sieve. Seal the reaction flask and purge the air in the flask with nitrogen to eliminate oxygen interference with the reaction.

[0030] Esterification reaction: Place the sealed reaction flask in a constant temperature shaker at 30°C, set the shaking rate to 200 rpm, and continue the reaction for 8 hours under nitrogen protection. During this period, keep the shaker temperature stable to ensure consistent reaction conditions.

[0031] Post-processing and purification: After the reaction is completed, the reaction system is filtered while hot to recover the immobilized enzyme and molecular sieve. The recovered enzyme can be used for subsequent batch reactions. The filtrate is collected and subjected to vacuum rotary evaporation at 40-45℃ to remove the solvent tert-butanol, yielding the crude product. A small amount of dichloromethane is added to the crude product to dissolve it, and the product is purified by silica gel column chromatography with petroleum ether / ethyl acetate gradient elution. The target eluent is collected and dried.

[0032] Product detection: After separation and purification, a white to pale yellow solid product, retinyl ceramide ester, was obtained. The purity of the product was greater than 98% by high performance liquid chromatography (HPLC), and the calculated yield was approximately 87%.

[0033] Example 2

[0034] A method for the enzyme-catalyzed synthesis of retinoic acid ceramide esters, the specific steps of which are as follows:

[0035] Raw material preparation: Weigh out 300 mg (1.0 mmol) of retinoic acid and 1.5 mg (1.5 mmol) of ceramide NP87. Select 23.43 mg of immobilized Lipozyme TL IM, 1.0 g of 5 Å molecular sieve and 30 mL of anhydrous tert-butanol. All raw materials are used after purity verification.

[0036] Reaction system setup: Place retinoic acid and ceramide NP into a 100mL dry reaction flask, add 30mL of anhydrous tert-butanol, and stir until the raw materials are completely dissolved; then add 23.43mg of immobilized cottony thermophilic lipase and 1.0g of 5Å molecular sieve, seal the reaction flask and purge with nitrogen to completely replace the air in the flask to ensure an anaerobic environment for the reaction.

[0037] Esterification reaction: Place the reaction flask in a constant temperature shaker at 50℃, adjust the shaking speed to 200 rpm, and continue the reaction for 24 hours under nitrogen protection. During the reaction, observe the state of the reaction system regularly to ensure that there is no leakage or abnormality.

[0038] Post-processing and purification: After the reaction, the mixture was filtered immediately to recover the immobilized enzyme and molecular sieve; the filtrate was collected and tert-butanol was removed by rotary evaporation under reduced pressure at 40-45℃ to obtain the crude product; the crude product was dissolved in a small amount of dichloromethane and purified by silica gel column chromatography with petroleum ether / ethyl acetate as a gradient eluent; the target product fraction was collected and dried.

[0039] Product detection: The final product was a white to pale yellow solid retinoic acid ceramide ester. HPLC analysis showed that the product purity was greater than 98% and the yield was approximately 76%.

[0040] Example 3

[0041] A method for the enzyme-catalyzed synthesis of retinoic acid ceramide esters, the specific steps of which are as follows:

[0042] Raw material preparation: Accurately weigh 300 mg (1.0 mmol) of retinoic acid and 97.2 mg (1.2 mmol) of ceramide NP6. Prepare 30.0 mg of immobilized Lipozyme TL IM, 1.0 g of 5 Å molecular sieve and 30 mL of acetone. All raw materials meet the purity requirements of the synthesis reaction.

[0043] Reaction system setup: Add retinoic acid and ceramide NP to a 100mL dry reaction flask, add 30mL acetone, and stir to fully dissolve the raw materials; then add 30.0mg immobilized cottony thermophilic lipase and 1.0g 5Å molecular sieve, seal the reaction flask and purge with nitrogen to replace the air to prevent oxidation reaction.

[0044] Esterification reaction: Place the reaction flask in a 40°C constant temperature shaker, set the shaking speed to 200 rpm, and react for 12 hours under nitrogen protection. Maintain a uniform and stable temperature in the shaker to ensure efficient reaction.

[0045] Post-processing and purification: After the reaction, filter while hot to recover the immobilized enzyme and molecular sieve; collect the filtrate and remove acetone by rotary evaporation under reduced pressure at 40-45℃ to obtain the crude product; dissolve the crude product in a small amount of dichloromethane and purify it by silica gel column chromatography with petroleum ether / ethyl acetate gradient elution, collect the target eluent and dry it.

[0046] Product detection: The final product was a white to pale yellow solid retinoic acid ceramide ester. HPLC analysis showed that the purity of the product was greater than 98% and the yield was approximately 82%.

[0047] Example 4

[0048] A method for the enzyme-catalyzed synthesis of retinoic acid ceramide esters, the specific steps of which are as follows:

[0049] Raw material preparation: Weigh 300 mg (1.0 mmol) of retinoic acid and 9.1 mg (1.1 mmol) of ceramide NP63. Select 27.9 mg of immobilized Candida antarctica lipase (Novozym435), 1.0 g of 3 Å molecular sieve and 30 mL of n-hexane. The purity of the raw materials was tested and found to meet the reaction requirements.

[0050] Reaction system setup: Retinoic acid and ceramide NP were placed in a 100 mL dry reaction flask, 30 mL of n-hexane was added, and the mixture was stirred until the raw materials were completely dissolved. 27.9 mg of immobilized Candida antarcticis lipase and 1.0 g of 3 Å molecular sieve were added. After sealing the reaction flask, nitrogen gas was introduced to replace the air in the flask to create an anaerobic reaction environment.

[0051] Esterification reaction: Place the reaction flask in a constant temperature shaker at 35℃ with a shaking rate of 200 rpm and react for 16 hours under nitrogen protection. During this period, keep the reaction conditions stable and avoid temperature fluctuations from affecting the reaction efficiency.

[0052] Post-processing and purification: After the reaction, the immobilized enzyme and molecular sieve were recovered by filtration; the filtrate was collected and hexane was removed by rotary evaporation under reduced pressure at 40-45℃ to obtain the crude product; the crude product was dissolved in a small amount of dichloromethane, purified by silica gel column chromatography, eluted with petroleum ether / ethyl acetate gradient, the target product was collected and dried.

[0053] Product detection: A white to pale yellow solid retinoic acid ceramide ester was obtained, with a purity greater than 98% and a yield of approximately 80% as determined by HPLC.

[0054] Example 5

[0055] A method for the enzyme-catalyzed synthesis of retinoic acid ceramide esters, the specific steps of which are as follows:

[0056] Raw material preparation: Accurately weigh 300 mg (1.0 mmol) of retinoic acid and 55.3 mg (1.3 mmol) of ceramide NP7. Prepare 33.1 mg of a mixture of immobilized Candida antarcticis lipase (Novozym435) and immobilized Thermophilic lipozyme TL IM (mass ratio of the two enzymes 1:1), 1.0 g of 5 Å molecular sieve, and 30 mL of a mixed solvent of tert-butanol and acetone (volume ratio 1:1).

[0057] Reaction system setup: Add retinoic acid and ceramide NP to a 100mL dry reaction flask, pour in the mixed solvent, and stir to fully dissolve the raw materials; add 33.1mg of mixed enzyme and 1.0g of 5Å molecular sieve, seal the reaction flask and purge with nitrogen to replace the air, ensuring that the reaction is carried out under anaerobic conditions.

[0058] Esterification reaction: The reaction flask was placed in a constant temperature shaker at 45°C and the shaking rate was 200 rpm. The reaction was carried out for 24 hours under nitrogen protection, while maintaining a constant temperature in the reaction system.

[0059] Post-processing and purification: After the reaction, filter while hot to recover the mixed enzyme and molecular sieve; collect the filtrate and remove the mixed solvent by rotary evaporation under reduced pressure at 40-45℃ to obtain the crude product; dissolve the crude product in a small amount of dichloromethane, purify by silica gel column chromatography with petroleum ether / ethyl acetate gradient elution, collect the target component and dry it.

[0060] Product detection: The final product was a white to pale yellow solid retinoic acid ceramide ester, with a purity greater than 98% and a yield of approximately 84% as determined by HPLC.

[0061] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. A method for enzyme-catalyzed synthesis of retinoic acid ceramide ester, characterized in that, Using retinoic acid and ceramide NP as reaction substrates and immobilized lipase as a catalyst, the target product was synthesized via a directed esterification reaction in a non-aqueous organic solvent. The process included raw material preparation, reaction system construction, esterification reaction, post-processing, purification, and detection steps. The target product was a solid retinoic acid-ceramide ester with a purity greater than 98%. Raw material preparation steps: The molar ratio of ceramide NP to retinoic acid is 1:1 to 1.5:1; the immobilized lipase is selected from one or two of Novozym 435 and Lipozyme TL IM, and the total amount of immobilized lipase added is 2% to 5% of the total weight of the reaction substrate; Reaction system setup steps: Add retinoic acid and ceramide NP to the reaction vessel, pour in a non-aqueous organic solvent and stir until the substrate is fully dissolved. Then add immobilized lipase and molecular sieve. After sealing the reaction vessel, purge with nitrogen to create an anaerobic reaction environment. Esterification reaction steps: The reaction is carried out in a constant temperature shaker at a temperature of 30~50℃, a shaking rate of 150-200rpm, and a reaction time of 8~24 hours, so that lipase specifically catalyzes the esterification reaction between the carboxyl group of retinoic acid and the primary hydroxyl group of ceramide. Post-processing steps: After the reaction is completed, filter the reaction system, recover the immobilized lipase and molecular sieve, collect the filtrate and evaporate it under reduced pressure at 40~45℃ to remove the non-aqueous organic solvent, and obtain the crude product of retinoic acid ceramide ester. Purification and detection steps: Dichloromethane was added to the crude retinyl ceramide ester to dissolve it, and the product was purified by silica gel column chromatography with gradient elution using petroleum ether / ethyl acetate as eluent. The target eluent was collected and dried. The purity was detected by high performance liquid chromatography (HPLC), and the product with a purity greater than 98% was collected as the target product.

2. The method for enzyme-catalyzed synthesis of retinoic acid ceramide ester according to claim 1, characterized in that, When the two immobilized lipases are used in combination, the mass ratio of Novozym 435 to Lipozyme TL IM is 1:

1.

3. The method for enzyme-catalyzed synthesis of retinoic acid ceramide ester according to claim 1, characterized in that, The non-aqueous organic solvent is selected from one or more of tert-butanol, n-hexane, and acetone.

4. The method for enzyme-catalyzed synthesis of retinoic acid ceramide ester according to claim 1, characterized in that, The molecular sieve is a 3Å or 5Å molecular sieve.

5. The method for enzyme-catalyzed synthesis of retinoic acid ceramide ester according to claim 1, characterized in that, The recovered immobilized lipase was reused in subsequent batches of reactions.

6. The application of retinoic acid ceramide ester synthesized by the enzyme-catalyzed synthesis method according to any one of claims 1-5 in the preparation of raw materials for anti-wrinkle and repair cosmetics.

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