Liposoluble selenoquinine and its preparation method and application

Abstract

The present application relates to natural antioxidant modification and food / cosmetic additive technical field, more particularly to a kind of liposoluble seleno catechin and its preparation method and application.The present application uses epicatechin as starting material, through the directional molecular modification strategy of group protection-etherification reaction-selenization modification-deprotection, first, epicatechin hydroxyl is protected using propionic anhydride, then 9-bromo-1-nonanol hydrophobic chain is introduced to improve liposolubility, selenization modification is realized using sodium selenite, finally deprotection, finally obtain seleno catechin derivative with high liposolubility and antioxidant activity.

Description

Technical Field

[0001] This invention relates to the field of natural antioxidant modification and food / cosmetic additives, and more specifically to a fat-soluble selenized catechin, its preparation method, and its application. Background Technology

[0002] Lipids, especially those rich in unsaturated fatty acids, are key raw materials in food processing and cosmetic formulations. Their carbon-carbon double bonds are easily oxidized under the influence of light, oxygen, high temperature, and metal ions, leading to rancidity, functional loss, and even the production of harmful substances, severely impacting product quality and safety. Synthetic antioxidants (such as BHA and BHT) are subject to strict limitations due to potential toxicity and safety concerns, making highly effective and safe natural antioxidants a hot topic.

[0003] Epicatechin (EC) is a natural polyphenolic compound extracted from tea leaves. It is a representative of natural antioxidants, with phenolic hydroxyl groups in its molecular structure exhibiting excellent free radical scavenging capabilities. It also demonstrates good physiological activity in preventing metabolic syndrome and cardiovascular diseases. However, the presence of multiple phenolic hydroxyl groups makes catechin highly water-soluble, difficult to dissolve and disperse uniformly in oil systems, and unable to fully exert its antioxidant effects, greatly limiting its application in food oil preservation and cosmetic oil matrix stabilization. To improve the lipid solubility of catechin, existing technologies mostly employ physical encapsulation or simple hydrophobic chain modification methods. Physical encapsulation refers to using carrier materials (such as liposomes, cyclodextrins, or nanoparticles) to physically encapsulate catechin within its interior or cavities without chemical bonding, thereby improving its lipid solubility. However, physical encapsulation suffers from problems such as large batch-to-batch variability and unstable drug-carrier interaction. While simple hydrophobic chain modification can improve lipid solubility, it does not enhance antioxidant activity. Furthermore, because catechin molecules have multiple connectable hydroxyl groups (-OH), it is impossible to precisely control the specific position of the hydrophobic chain during the chemical reaction, resulting in unclear modification sites and uncertain product structures. In addition, in the same batch of reactions, multiple mixed products with different sites or different numbers of hydrophobic chains may be generated, making it difficult to obtain a standard with a single composition and consistent properties, and thus failing to meet the needs of oil systems for highly effective antioxidants. Summary of the Invention

[0004] To address the aforementioned issues, this invention provides a fat-soluble selenized catechin, its preparation method, and its application. It develops a catechin derivative that combines high fat solubility, strong antioxidant synergy, and a stable preparation process, becoming a key breakthrough in overcoming the limitations of natural antioxidant applications. Furthermore, it provides a method for preparing fat-soluble selenized catechin that combines fat solubility and selenium fortification, and uses it as an antioxidant in oils and fats.

[0005] A method for preparing fat-soluble selenized catechins includes the following steps: Group protection: Using methyl tert-butyl ether as a solvent, epicatechin, propionic anhydride, and triethylamine undergo a group protection reaction in the solvent to give product 1, denoted as EC-OMe; wherein the molar ratio of epicatechin, propionic anhydride, and triethylamine is 1:4.5~4.8:8~8.3; the reaction process is shown below: ; Etherification reaction: Using N,N-dimethylformamide as solvent and potassium carbonate as acid-binding agent, product 1 and bromoethanol were etherified under the conditions of solvent and acid-binding agent to introduce a hydrophobic alkyl chain, yielding product 2, denoted as EC-OMe-C9; wherein the molar ratio of product 1 to bromoethanol was 1:2~2.5, and the molar ratio of epicatechin to potassium carbonate was 1:2~2.4; taking 9-bromo-1-nonanol as an example, the reaction process is as follows: ; Etherification reactions occur at different hydroxyl sites of epicatechin, resulting in the preparation of lipid-soluble selenized catechins modified at different sites; Selenization modification: Using N,N-dimethylformamide as a solvent, product 2 undergoes a selenization modification reaction with sodium selenite. After the reaction is complete, post-treatment yields product 3 (denoted as EC-OMe-C9-Se). The molar ratio of product 2 to sodium selenite is 1:3~3.3. The reaction process is shown below: ; Deprotection: EC-OMe-C9-Se reacts with hydrated hydrazine in anhydrous methanol to give product 4, denoted as EC-C9-Se; the molar ratio of product 3 to hydrated hydrazine is 1:6~7; the reaction process is shown below: .

[0006] The described protection-etherification-selenization-deprotection reaction pathway is suitable for preparing lipid-soluble selenized polyphenolic compounds, including epicatechin, catechin, or quercetin. When different substrates are selected, the number, position, and activity of their phenolic hydroxyl groups vary. Therefore, the amount of propionic anhydride used as the group protectant is adjusted proportionally according to the number of molar phenolic hydroxyl groups of the target substrate. The reagent types, reaction temperatures, and times of the remaining steps can remain unchanged. The specific amounts need to be recalculated based on the molecular weight of the substrate to ensure the selectivity of the modification sites.

[0007] The protection-etherification-selenization-deprotection method of this invention first selectively protects the highly active phenolic hydroxyl groups on the catechin nucleus with propionic anhydride, so that the subsequent etherification reaction can only occur at specific unprotected hydroxyl sites, thereby achieving site-directed modification and avoiding product heterogeneity caused by random substitution. Selenium (Se) is an essential trace element for the human body with extremely strong antioxidant activity. It can participate in the synthesis of antioxidant enzymes such as glutathione peroxidase and synergistically scavenge reactive oxygen free radicals in the body. After combining with catechin, it can achieve the dual function of "polyphenol antioxidant + selenium synergistic effect". Therefore, this invention introduces selenium-containing groups through selenization modification and finally deprotects to restore the phenolic hydroxyl structure, resulting in a fat-soluble selenized catechin that is both fat-soluble and selenium-fortified epicatechin. The preparation route in this invention ensures that the product structure is clear and the consistency between batches is strong by fixing the feed ratio and reaction conditions. This fundamentally overcomes the defects of physical embedding method, such as large batch differences, unclear sites of simple hydrophobic modification and no enhancement of antioxidant activity. It enables the product to be uniformly dispersed in the oil system, and the selenium-containing groups introduced by selenization modification work together with catechins to exert a highly efficient and synergistic antioxidant effect.

[0008] In another preferred embodiment, the group protection reaction is carried out at 25°C to 30°C for 15 to 20 hours.

[0009] In another preferred embodiment, the etherification reaction is carried out at a temperature of 55°C to 60°C for a reaction time of 24 to 36 hours.

[0010] In another preferred embodiment, the reaction temperature is 60°C to 70°C, and the reaction time is 4 to 5 hours.

[0011] In another preferred embodiment, the deprotection reaction takes 6 to 8 hours and is carried out at a temperature of 30°C to 40°C.

[0012] In another preferred embodiment, the post-processing includes gradient dialysis using a 300Da dialysis bag with 70% to 95% ethanol, followed by freeze-drying to obtain the product. Specifically, a 300Da dialysis bag is used, and impurities are removed by gradient dialysis with 95% ethanol, 80% ethanol, and 70% ethanol. The product in the dialysis bag is collected, and after freeze-drying, the target product, fat-soluble selenized catechin, is obtained.

[0013] In another preferred embodiment, by employing different protection and deprotection methods, the etherification reaction can occur at different phenolic hydroxyl sites of epicatechin, thereby obtaining lipid-soluble selenized catechins substituted at different sites such as C-5, C-7, C-3′, and C-4′. These site isomers all have improved lipid solubility and antioxidant activity.

[0014] The second aspect of the present invention provides a fat-soluble selenized catechin prepared by the preparation method described above, wherein the etherification reaction is carried out using bromools of different chain lengths to prepare different forms of fat-soluble selenized catechins; wherein the bromools of different chain lengths are any one of 6-bromo-1-hexanol, 9-bromo-1-nonanol, and 10-bromo-1-decanol.

[0015] The third aspect of this invention provides the application of the aforementioned fat-soluble selenized catechins in the preparation of oil antioxidants.

[0016] Compared with the prior art, the present invention has the following innovative advantages: (1) This invention utilizes the antioxidant properties of catechin phenolic hydroxyl groups and the synergistic effect of selenium to achieve molecular modification through a four-step directional modification process involving group protection, etherification reaction, selenization modification, and deprotection. This process achieves molecular modification with a fixed molar ratio, resulting in a simple assembly mode and high product uniformity. It avoids the batch-to-batch variability of physical embedding methods and the problem of ambiguous sites in simple hydrophobic modification. In particular, the group protection agent propionic anhydride selectively protects the highly active phenolic hydroxyl groups on the catechin nucleus, ensuring that the subsequent etherification reaction can only occur at specific unprotected hydroxyl sites, thereby achieving site-directed modification and effectively solving the problem of insufficient product uniformity. Furthermore, by introducing selenium-containing groups, the phenolic hydroxyl structure is finally restored through deprotection, resulting in a fat-soluble selenized catechin that combines fat solubility with selenium-enhanced epicatechin.

[0017] (2) The product of this invention is of natural origin. Existing synthetic fat-soluble antioxidants such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) are mainly derived from chemical raw materials and have certain toxicity. In contrast, the product of this invention is of natural origin and can be widely used in the fields of food oil preservation and cosmetic oil matrix stabilization, providing a safe solution for the functional application of natural antioxidants.

[0018] (3) Compared with the existing synthetic fat-soluble antioxidants butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT), the product of this invention introduces selenium, which enhances the antioxidant properties of catechins in the oil phase. Attached Figure Description

[0019] Figure 1 The NMR spectrum of lipid-soluble selenized catechins is shown in 1H NMR.

[0020] Figure 2 The NMR chromatogram of fat-soluble selenized catechins is shown.

[0021] Figure 3 Mass spectrometry of fat-soluble selenized catechins.

[0022] Figure 4 The image shows the results of hydrophilic angle determination for fat-soluble selenized catechins.

[0023] Figure 5 The graph shows the DPPH free radical scavenging results of different antioxidants.

[0024] Figure 6 The graph shows the oxidation inhibition rate of oils with different antioxidants.

[0025] Figure 7 The graph shows the acid value of linoleic acid affected by different antioxidants.

[0026] Figure 8 The graph shows the peroxide value of linoleic acid produced by different antioxidants.

[0027] Figure 9 The graph shows the carbonyl values ​​of linoleic acid affected by different antioxidants. Detailed Implementation

[0028] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] This invention prepares a lipid-soluble selenized catechin with well-defined site modifications. Using epicatechin as the starting material, this invention employs a targeted molecular modification strategy of "group protection-etherification reaction-selenization modification-deprotection". First, propionic anhydride is used to protect the hydroxyl groups of catechin. Then, a hydrophobic chain of 9-bromo-1-nonanol is introduced to enhance lipid solubility. Sodium selenite is used to achieve selenization modification. Finally, deprotection is performed to obtain a selenized catechin derivative (EC-C9-Se) with both high lipid solubility and antioxidant activity. This product can be directly applied to the antioxidant preservation of oil systems.

[0030] The epicatechin used in the following examples was purchased from Maclean Biotechnology Co., Ltd.

[0031] Example 1: A method for preparing fat-soluble selenized catechins, comprising the following steps: S1. Group Protection: Using methyl tert-butyl ether as a solvent, epicatechin reacts with propionic anhydride and triethylamine at room temperature for 15 hours to obtain product 1 (EC-OMe). The molar ratio of epicatechin, propionic anhydride, and triethylamine is 1:4.5:8; that is, 1.0 g of epicatechin, 2.02 g of propionic anhydride, 2.79 g of triethylamine, and 50 mL of methyl tert-butyl ether. The reaction principle is as follows:

[0032] .

[0033] S2. Etherification reaction: Using N,N-dimethylformamide as solvent, in the presence of potassium carbonate, EC-OMe and 9-bromo-1-nonanol undergo a hydrophobic chain introduction reaction at 60°C for 24 hours to obtain product 2 (EC-OMe-C9). The molar ratio of EC-OMe to 9-bromo-1-nonanol is 1:2, and the molar ratio of epicatechin to potassium carbonate is 1:2, i.e., EC-OMe is 1 g, potassium carbonate is 0.95 g, 9-bromo-1-nonanol is 1.54 g, and N,N-dimethylformamide is 40 mL. The reaction principle is as follows: .

[0034] S3. Selenization Modification: Using N,N-dimethylformamide as a solvent, acetic acid was added to a concentration of 1%. EC-OMe-C9 and sodium selenite underwent a selenization modification reaction at 70℃ for 4 hours. After the reaction was completed, post-treatment yielded product 3 (EC-OMe-C9-Se). The molar ratio of EC-OMe-C9 to sodium selenite was 1:3, i.e., 1.0 g of EC-OMe-C9, 1.79 g of sodium selenite, and 40 mL of N,N-dimethylformamide. The reaction principle is as follows: .

[0035] S4. Deprotection: Using anhydrous methanol as solvent, EC-OMe-C9-Se reacts with hydrazine hydrate in a methanol solution at 35°C for 6 hours to obtain product 4 (EC-C9-Se). The molar ratio of EC-OMe-C9-Se to hydrazine hydrate is 1:6, i.e., 1.0 g of EC-OMe-C9-Se, 1.03 g of hydrazine hydrate, and 50 mL of anhydrous methanol. The reaction principle is as follows: .

[0036] The specific process for characterizing the lipid-soluble selenized catechins prepared above is as follows: NMR detection of fat-soluble selenized catechins To verify whether selenization modification successfully introduced selenium-containing functional groups while preserving the catechin core structure, nuclear magnetic resonance (NMR) and mass spectrometry (MS) were used to systematically characterize the product. Results Figure 1 and Figure 2 As shown. 1 The H NMR spectrum showed proton signals from the epicatechin skeleton and proton signals from the introduced C9 alkyl chain, and the integral ratio of each proton peak was consistent with the theoretical structure. 13The CNMR spectrum also showed the corresponding carbon atom shifts, which preliminarily proves the successful synthesis of EC-C9-Se. The above characterization data jointly confirm that the present invention successfully synthesized the target selenized catechin derivative via the "protection-etherification-deprotection-selenization" route. The product retains the flavan-3-ol core structure and introduces hydrophobic carbon chains and selenium-containing functional groups.

[0037] EC-C9-Se was dissolved in methanol and diluted to approximately 1 μg / mL. Detection was performed using electrospray ionization-time-of-flight mass spectrometry in positive ion mode. Instrument settings: spray voltage 3.5 kV, scan range m / z 100–1000. Results are as follows: Figure 3 As shown, the mass spectrometry results indicate that the measured molecular ion peak of EC-C9-Se is 561.31 m / z, which is consistent with the theoretical value of 560.12 (C). 24 H 32 O 10 Se + H + The results are largely consistent, further confirming the successful synthesis of the target product.

[0038] Example 3: Hydrophilicity and hydrophobicity detection of fat-soluble selenized catechins EC and EC-C9-Se were each prepared into 5 mg / mL solutions with anhydrous ethanol. 20 μL of each solution was dropped onto a clean glass slide and allowed to air dry at room temperature to form films. The static contact angle of ultrapure water on the membrane surface was measured using an optical contact angle meter at 25 °C via the seated drop method. The results are as follows: Figure 4 As shown. From Figure 4 It can be seen that the water contact angle of the unmodified EC is 70.24±0.76°, exhibiting strong hydrophilicity. After introducing the C9 alkyl chain and selenization, the contact angle of EC-C9-Se significantly increased to 98.35±0.07°, indicating that its surface changed from hydrophilic to hydrophobic. This demonstrates that long-chain alkyl modification effectively improves the lipophilicity of catechins.

[0039] Example 4: In vitro DPPH free radical scavenging by fat-soluble selenized catechins Prepare a 0.2 mmol / L DPPH ethanol solution and store it protected from light. Take 1 mL of DPPH solution and add 1 mL of the test sample solution at different concentrations (0.2~1.0 mmol / L), vortex to mix, and react at room temperature in the dark for 30 min. Measure the absorbance at 517 nm. Use distilled water as a control instead of the sample solution. Calculate the DPPH scavenging rate (%) = (A0-A1) / A0×100%. The results are shown in the figure below. Figure 5As shown, in the highly polar ethanol / water system, the DPPH scavenging rate of each sample increased with increasing concentration. EC and commercially available PG had similar scavenging rates, while EC-C9 and EC-C9-Se had slightly lower scavenging rates than EC, but the differences were not significant. The results indicate that moderate hydrophobic modification and selenization modification have no significant negative impact on the free radical scavenging ability of EC.

[0040] Example 5: Evaluation of the antioxidant capacity of fat-soluble selenized catechin oils against β-carotene / linoleic acid oxidation Prepare a β-carotene-linoleic acid emulsion (containing 0.2% β-carotene, linoleic acid, and Tween-80). Take 5 mL of different concentrations (0.2 mmol / L, 0.4 mmol / L, 0.6 mmol / L, 0.8 mmol / L, 1.0 mmol / L) of epicatechin EC, epicatechin-9-nonyl ether derivative EC-C9, EC-C9-Se, and propyl gallate PG as test solutions, add 5 mL of the emulsion, mix well, and incubate in a 50°C water bath in the dark. Measure the absorbance at 470 nm at 0 h, 2 h, 4 h, and 6 h. Calculate the oxidation inhibition rate (%) = [A 样本(t) -A 空白(t) ] / [A 空白(0) -A 空白(t) ]×100%. The result is as follows: Figure 6 As shown, commercially available PG exhibits the strongest antioxidant capacity in oil systems; EC, due to its strong hydrophilicity, shows significantly weaker antioxidant capacity in oil systems; EC-C9 demonstrates higher antioxidant capacity than EC; and EC-C9-Se exhibits antioxidant capacity close to that of PG. These results indicate that hydrophobic modification can enhance the antioxidant performance of EC in oil systems, and selenization modification further strengthens its effect.

[0041] Example 6: Evaluation of the antioxidant capacity of fat-soluble selenized catechin oils against β-carotene / linoleic acid oxidation Different antioxidants at a concentration of 0.05 mg / mL, namely epicatechin EC, epicatechin-9-nonyl ether derivatives EC-C9 and EC-C9-Se, and propyl gallate PG, were added to linoleic acid and accelerated oxidation was performed according to the national standard method GB / T 21121-2024. The acid value (AV), peroxide value (POV), and carbonyl value (CV) of the samples after the reaction were measured. Linoleic acid without added antioxidants was used as a blank control. The results are as follows: Figures 7-9 As shown in the figure, compared with the blank control group, all antioxidant treatment groups effectively reduced the acid value, peroxide value, and carbonyl value of linoleic acid. Among them, EC had the worst effect, followed by EC-C9, while EC-C9-Se had the best effect, comparable to commercial PG. This indicates that EC-C9-Se can significantly delay the oxidative spoilage process of linoleic acid.

[0042] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0043] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A method for preparing fat-soluble selenized catechins, characterized in that, Includes the following steps: Using methyl tert-butyl ether as a solvent, epicatechin as a substrate undergoes a group protection reaction with propionic anhydride and triethylamine in the solvent to obtain product 1; wherein the molar ratio of epicatechin, propionic anhydride and triethylamine is 1:4.5~4.8:8~8.

3. Using N,N-dimethylformamide as solvent and potassium carbonate as acid-binding agent, product 1 and bromoethanol were subjected to an etherification reaction under the conditions of solvent and acid-binding agent to introduce a hydrophobic alkyl chain, thus obtaining product 2; wherein the molar ratio of product 1 to bromoethanol was 1:2~2.5, and the molar ratio of epicatechin to potassium carbonate was 1:2~2.

4. Using N,N-dimethylformamide as a solvent, product 2 undergoes a selenization modification reaction with sodium selenite in the solvent. After the reaction is completed, post-treatment is performed to obtain product 3; wherein the molar ratio of product 2 to sodium selenite is 1:3~3.

3. Using anhydrous methanol as a solvent, product 3 undergoes a deprotection reaction with hydrated hydrazine in the solvent to obtain product 4, which is the fat-soluble selenized catechin; wherein the molar ratio of product 3 to hydrated hydrazine is 1:6~7.

2. The method for preparing fat-soluble selenized catechins according to claim 1, characterized in that, The bromool is any one of 6-bromo-1-hexanol, 9-bromo-1-nonanol, and 10-bromo-1-decanol.

3. The method for preparing fat-soluble selenized catechins according to claim 1, characterized in that, The group protection reaction was carried out at 25℃~30℃ for 15h~20h.

4. The method for preparing fat-soluble selenized catechins according to claim 1, characterized in that, The etherification reaction is carried out at a temperature of 55℃~60℃ for 24h~36h.

5. The method for preparing fat-soluble selenized catechins according to claim 1, characterized in that, The selenization modification reaction is carried out at a temperature of 60℃~70℃ for 4h~5h.

6. The method for preparing fat-soluble selenized catechins according to claim 2, characterized in that, The deprotection reaction takes 6 to 8 hours and is carried out at a temperature of 30 to 40 degrees Celsius.

7. The method for preparing fat-soluble selenized catechins according to claim 1, characterized in that, The post-processing refers to dialysis using 300Da dialysis bags with a gradual gradient of 70% to 95% alcohol by mass, followed by freeze drying.

8. A fat-soluble selenized catechin prepared by the preparation method according to any one of claims 1 to 7.

9. The application of the fat-soluble selenized catechin according to claim 8 in the preparation of oil antioxidants.

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