Preparation method of naringenin functionalized chitosan
By grafting naringin onto chitosan, the problem of insufficient antioxidant capacity of chitosan was solved, expanding its application in the field of high antioxidant performance.
Patent Information
- Application Number
- CN202511511606.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-22
- Publication Date
- 2026-01-16
AI Technical Summary
Chitosan's antioxidant capacity is insufficient to meet requirements in some applications.
Naringenin is grafted onto chitosan via an affinity addition reaction of hydroxyl groups. The specific steps include dissolving chitosan and naringenin, adding EDC and NHS to activate the hydroxyl groups of naringenin, and then reacting them with the C2-NH2 groups of chitosan to form urea bonds.
The prepared naringenin-functionalized chitosan not only retains the inherent properties of chitosan, but also expands its application in fields requiring high antioxidant properties.
Abstract
Description
Technical Field
[0001] This invention relates to the field of chitosan derivative preparation technology, specifically to a method for preparing naringenin-functionalized chitosan. Background Technology
[0002] Chitosan, a polycationic polymer composed of N-acetylglucosamine and D-glucosamine linked by β-1,4-glycosidic bonds, has been widely used in pharmaceuticals, biotechnology, cosmetics, and many other fields due to its non-toxicity, good biocompatibility, biodegradability, and broad-spectrum antibacterial properties. Although chitosan itself possesses certain antioxidant capabilities, this property may be insufficient to meet the requirements in some applications. Summary of the Invention
[0003] To address the shortcomings of existing technologies, this invention provides a method for preparing naringenin-functionalized chitosan, specifically achieved through the following technical solution: A method for preparing naringin-functionalized chitosan includes the following steps: (1) Dissolve chitosan in an aqueous solution of glacial acetic acid; (2) Dissolve naringenin in dimethyl sulfoxide; (3) Weigh out the predetermined amounts of 1-ethyl-3-dimethylaminopropylcarbodiimide hydrochloride and hydroxysuccinimide and add them to the naringin solution in step (2) and stir until homogeneous; (4) Add the activated naringenin solution from step (3) to the chitosan glacial acetic acid aqueous solution, and then place the mixture in a magnetically stirred water bath at 75-80°C and stir in the dark for 20-24 hours. (5) After the reaction is complete, the solution is placed in a dialysis bag with a capacity of 8000-14000 Da and dialyzed with anhydrous ethanol for 1-2 days, then dialyzed with deionized water for 2-3 days. The dialysate is changed every 12-16 hours. Finally, the supernatant is collected by centrifugation and dried to obtain naringenin-functionalized chitosan.
[0004] In step (1), the glacial acetic acid aqueous solution is prepared according to the volume ratio of glacial acetic acid to water of 1-3:97-99; the mass-volume ratio of chitosan to glacial acetic acid aqueous solution is 2-3:100-150.
[0005] In step (2), the mass-volume ratio of naringenin to dimethyl sulfoxide is 2-3:100-150.
[0006] In step (3), the molar ratio of naringenin: EDC: NHS is 7-11:15-33:6-17.
[0007] In step (4), the volume ratio of chitosan glacial acetic acid aqueous solution to activated naringenin solution is 4-5:2-3.
[0008] In step (1), the degree of deacetylation of chitosan is ≥95%, and the viscosity is 100~200mPa.s.
[0009] The specific operation of step (1) is to mix chitosan and glacial acetic acid aqueous solution thoroughly, seal, and stir for 60 to 80 minutes to fully dissolve chitosan.
[0010] The specific operation of step (2) is to add naringenin to dimethyl sulfoxide, seal it, and stir for 8 to 10 minutes to fully dissolve the naringenin.
[0011] In step (4), the naringenin solution is added at a rate not exceeding 5% of the volume of the reaction system per minute.
[0012] The technical solution of this invention has the following advantages: This invention employs an affinity addition reaction of hydroxyl groups. First, the hydroxyl groups of naringenin are activated using EDC and NHS, and then reacted with the C2-NH2 groups of chitosan, thereby grafting naringenin onto chitosan. The naringenin-functionalized chitosan prepared in this way not only retains the inherent properties of chitosan but also expands its applications in fields requiring high antioxidant properties. This invention demonstrates promising application and development prospects in multiple fields, including pharmaceuticals, food, and cosmetics. Detailed Implementation
[0013] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, 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.
[0014] Furthermore, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.
[0015] This invention provides a method for preparing naringin-functionalized chitosan, specifically comprising the following steps: (1) Using chitosan as raw material, mix it thoroughly with glacial acetic acid aqueous solution in a reaction vessel, seal it to prevent solvent evaporation. Stir for 60-80 minutes to ensure the chitosan is fully dissolved.
[0016] (2) Using naringenin as raw material, mix it thoroughly with dimethyl sulfoxide in a reaction vessel, seal it to prevent solvent evaporation, and stir for 8 to 10 minutes to fully dissolve the naringenin.
[0017] (3) Weigh a certain amount of 1-ethyl-3-dimethylaminopropylcarbodiimide hydrochloride (EDC) and hydroxysuccinimide (NHS) and add them to the naringenin solution in step (2). Place the mixture on a magnetic stirrer and stir at 200-300 rpm in the dark for 60-90 minutes.
[0018] 1-Ethyl-3-dimethylaminopropylcarbodiimide hydrochloride acts as a protectant and buffer, while also activating the hydroxyl groups of naringenin.
[0019] Hydroxysuccinimide can effectively inhibit racemization.
[0020] (4) Slowly add the activated naringenin solution from step (3) to the chitosan glacial acetic acid aqueous solution, and then place the mixture in an environment of 75-80°C and stir at 200-300 rpm in the dark for 20-24 hours.
[0021] (5) After the reaction is complete, place the solution in a dialysis bag with a capacity of 8000-14000 Da. Dialyze with anhydrous ethanol for 1-2 days, then dialyze with deionized water for 2-3 days, changing the dialysate every 12-16 hours. Centrifuge to collect the supernatant, and dry to obtain pale green naringenin-functionalized chitosan.
[0022] Anhydrous ethanol dialysis is used to remove pigments and precipitates; deionized water dialysis is mainly used to remove melanin and other substances.
[0023] In step (1), the purity of glacial acetic acid is >99%, and the volume ratio of glacial acetic acid to water in the aqueous solution is 1-3:97-99 (v / v); the degree of deacetylation of chitosan is ≥95%, and the viscosity is 100-200 mPa.s; the mass-volume ratio of chitosan to aqueous glacial acetic acid is 2-3:100-150 (g / mL).
[0024] In step (2), the mass-to-volume ratio of naringenin to dimethyl sulfoxide is 2-3:100-150 (g / mL).
[0025] In step (3), the molar ratio of naringenin: EDC: NHS is 7-11:15-33:6-17 (n:n:n).
[0026] In step (4), the volume ratio of chitosan glacial acetic acid aqueous solution to activated naringenin solution is 4-5:2-3 (v / v).
[0027] In step (4), the naringenin solution is added at a rate not exceeding 5% of the volume of the reaction system per minute to prevent excessive foaming.
[0028] EDC, as a carbodiimide crosslinking agent, plays a crucial role in reacting with functional groups containing active hydrogen to form reactive intermediates. In the reaction system of this application, the N=C=N structure of EDC undergoes nucleophilic addition with the phenolic hydroxyl group of naringenin to form an O-acylisourea intermediate; NHS reacts with the above-mentioned O-acylisourea intermediate to form a stable N-hydroxysuccinimide ester intermediate, avoiding the hydrolysis side reaction of the O-acylisourea intermediate and significantly improving the activation efficiency of the phenolic hydroxyl group; the amino group (-NH2) at the C2 position of chitosan acts as a nucleophile, attacking the carbonyl carbon of the N-hydroxysuccinimide ester intermediate, undergoing a nucleophilic substitution reaction, releasing N-hydroxysuccinimide (NHS), and finally forming a urea bond (-NH-CO-NH-) between the phenolic hydroxyl group of naringenin and the amino group of chitosan, realizing the grafting of naringenin to chitosan.
[0029] The present invention will now be described in detail through specific embodiments.
[0030] Example 1 Mix 0.5 g of chitosan thoroughly with 25 mL of 2% (v / v) glacial acetic acid aqueous solution, seal, and stir for 80 minutes to ensure complete dissolution of the chitosan. Mix 0.3 g of naringenin thoroughly with 15 mL of dimethyl sulfoxide, seal, and stir for 8 minutes to ensure complete dissolution of the naringenin. Weigh 0.5083 g of EDC and 0.1787 g of NHS and add them to the naringenin solution. Place the solution on a magnetic stirrer and stir at 260 rpm in the dark for 90 minutes. Slowly add the activated naringenin solution to the chitosan-glacial acetic acid aqueous solution, then place the solution in an 80°C magnetically stirred water bath and stir at 250 rpm in the dark for 24 hours. After the reaction is complete, place the solution in a 14000 Da dialysis bag and dialyze with anhydrous ethanol for 1 day, followed by dialyze with deionized water for 2 days, changing the dialysate every 14 hours. Centrifuge and collect the supernatant, then dry at 50°C to obtain light green naringenin-functionalized chitosan.
[0031] In this embodiment, the naringenin solution was added at a rate of 2 mL / min.
[0032] Example 2 Mix 0.48 g of chitosan thoroughly with 25 mL of 3% glacial acetic acid aqueous solution (v / v), seal, and stir for 70 minutes to ensure complete dissolution of the chitosan. Mix 0.25 g of naringenin thoroughly with 12.5 mL of dimethyl sulfoxide, seal, and stir for 9 minutes to ensure complete dissolution of the naringenin. Weigh 0.4880 g of EDC and 0.1057 g of NHS and add them to the naringenin solution. Place the solution on a magnetic stirrer and stir at 230 rpm in the dark for 70 minutes. Slowly add the activated naringenin solution to the chitosan-glacial acetic acid aqueous solution, then place the solution in a 75°C magnetically stirred water bath and stir at 200 rpm in the dark for 22 hours. After the reaction is complete, place the solution in a 10000 Da dialysis bag. Dialyze with anhydrous ethanol for 2 days, then dialyze with deionized water for 3 days, changing the dialysate every 13 hours. Centrifuge and collect the supernatant, then dry at 45°C to obtain light green naringenin-functionalized chitosan.
[0033] In this embodiment, the naringenin solution was added at a rate of 1.9 mL / min.
[0034] Example 3 Mix 0.45 g of chitosan with 22.5 mL of 2% glacial acetic acid aqueous solution (v / v), seal, and stir for 60 minutes to ensure complete dissolution of the chitosan. Mix 0.2 g of naringenin with 10 mL of dimethyl sulfoxide, seal, and stir for 10 minutes to ensure complete dissolution of the naringenin. Weigh 0.4235 g of EDC and 0.0892 g of NHS and add them to the naringenin solution. Place the solution on a magnetic stirrer and stir at 260 rpm in the dark for 60 minutes. Slowly add the activated naringenin solution to the chitosan-glacial acetic acid aqueous solution, then place the solution in a 78°C magnetically stirred water bath and stir at 270 rpm in the dark for 20 hours. After the reaction is complete, place the solution in an 8000 Da dialysis bag. Dialyze with anhydrous ethanol for 1 day, then dialyze with deionized water for 3 days, changing the dialysate every 12 hours. Centrifuge and collect the supernatant, then dry at 60°C to obtain light green naringenin-functionalized chitosan.
[0035] In this embodiment, the naringenin solution was added at a rate of 1.7 mL / min.
[0036] Example 4 Mix 0.8 g of chitosan thoroughly with 40 mL of 1% (v / v) glacial acetic acid aqueous solution, seal, and stir for 75 minutes to ensure complete dissolution of the chitosan. Mix 0.6 g of naringenin thoroughly with 30 mL of dimethyl sulfoxide, seal, and stir for 9 minutes to ensure complete dissolution of the naringenin.
[0037] Weigh 0.952g of EDC and 0.228g of NHS and add them to the naringenin solution. Place the solution on a magnetic stirrer and stir at 200rpm in the dark for 80 minutes.
[0038] Slowly add 30 mL of activated naringenin solution to 40 mL of chitosan-acetic acid aqueous solution, then place it in a 77°C magnetically stirred water bath and stir at 280 rpm in the dark for 21 hours.
[0039] After the reaction was complete, the solution was placed in a 12000 Da dialysis bag and dialyzed with anhydrous ethanol for 1.5 days, followed by dialyzed with deionized water for 2.5 days, with the dialysate changed every 16 hours. The supernatant was collected by centrifugation and dried at 55°C to obtain pale green naringenin-functionalized chitosan.
[0040] In this embodiment, the naringenin solution was added at a rate of 3.5 mL / min.
[0041] Example 5 Mix 3.0 g of chitosan thoroughly with 100 mL of 2% (v / v) glacial acetic acid aqueous solution, seal, and stir for 75 minutes to ensure complete dissolution of the chitosan. Mix 1.8 g of naringenin thoroughly with 60 mL of dimethyl sulfoxide, seal, and stir for 9 minutes to ensure complete dissolution of the naringenin.
[0042] Weigh 2.453g of EDC and 0.587g of NHS and add them to the naringenin solution. Place the solution on a magnetic stirrer and stir at 300rpm in the dark for 80 minutes.
[0043] Slowly add 40 mL of activated naringenin solution to 100 mL of chitosan-acetic acid aqueous solution, and then place it in a magnetically stirred water bath at 76 °C and stir at 300 rpm in the dark for 23 hours.
[0044] After the reaction was complete, the solution was placed in a 13000 Da dialysis bag and dialyzed with anhydrous ethanol for 2 days, followed by dialyzed with deionized water for 2 days, with the dialysate changed every 15 hours. The supernatant was collected by centrifugation and dried at 52°C to obtain pale green naringenin-functionalized chitosan.
[0045] In this embodiment, the naringenin solution was added at a rate of 7 mL / min.
[0046] Example 6 Mix 2.0 g of chitosan thoroughly with 150 mL of 3% (v / v) glacial acetic acid aqueous solution, seal, and stir for 65 minutes to ensure complete dissolution of the chitosan. Mix 2.0 g of naringenin thoroughly with 150 mL of dimethyl sulfoxide, seal, and stir for 8 minutes to ensure complete dissolution of the naringenin.
[0047] Weigh 3.805g of EDC and 1.171g of NHS and add them to the naringenin solution. Place the solution on a magnetic stirrer and stir at 200rpm in the dark for 75 minutes.
[0048] Slowly add 90 mL of activated naringenin solution to 120 mL of chitosan-acetic acid aqueous solution, and then place it in a magnetically stirred water bath at 79 °C and stir at 220 rpm in the dark for 21.5 hours.
[0049] After the reaction was complete, the solution was placed in a 9000 Da dialysis bag and dialyzed with anhydrous ethanol for one day, followed by dialyzed with deionized water for three days, with the dialysate being changed every 14 hours. The supernatant was collected by centrifugation and dried at 58°C to obtain pale green naringenin-functionalized chitosan.
[0050] In this embodiment, the naringenin solution was added at a rate of 10.5 mL / min.
[0051] Example 7 Mix 0.6 g of chitosan thoroughly with 30 mL of 1% (v / v) glacial acetic acid aqueous solution, seal, and stir for 72 minutes to ensure complete dissolution of the chitosan. Mix 0.5 g of naringenin thoroughly with 25 mL of dimethyl sulfoxide, seal, and stir for 10 minutes to ensure complete dissolution of the naringenin.
[0052] Weigh 0.793g of EDC and 0.228g of NHS and add them to the naringenin solution. Place the solution on a magnetic stirrer and stir at 300rpm for 85 minutes in the dark.
[0053] Slowly add 12 mL of activated naringenin solution to 30 mL of chitosan-acetic acid aqueous solution, then place it in a 78°C magnetically stirred water bath and stir at 300 rpm in the dark for 22.5 hours.
[0054] After the reaction was complete, the solution was placed in a 11000 Da dialysis bag and dialyzed with anhydrous ethanol for 1.5 days, followed by dialyzed with deionized water for 2.5 days, with the dialysate changed every 16 hours. The supernatant was collected by centrifugation and dried at 48°C to obtain pale green naringenin-functionalized chitosan.
[0055] In this embodiment, the naringenin solution was added at a rate of 2.1 mL / min.
[0056] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.
Claims
1. A method for preparing naringenin functionalized chitosan, characterized by, The method comprises the following steps: (1) dissolving chitosan in an ice acetic acid aqueous solution; (2) dissolving naringin in dimethyl sulfoxide; (3) weighing a predetermined amount of 1-ethyl-3-dimethylaminopropyl carbodiimide hydrochloride and hydroxysuccinimide, adding them to the naringin solution in step (2), and stirring them uniformly; (4) adding the activated naringin solution in step (3) to the chitosan ice acetic acid aqueous solution, and then placing the mixture in a magnetic stirring water bath at 75-80℃, stirring in the dark for 20-24 hours; (5) after the reaction is completed, placing the solution in a dialysis bag with a molecular weight of 8000-14000 Da, dialyzing it in anhydrous ethanol for 1-2 days, and then dialyzing it in deionized water for 2-3 days, replacing the dialysis liquid every 12-16 hours, and finally centrifuging to obtain the supernatant, and drying to obtain naringin functionalized chitosan.
2. The method of preparing naringenin functionalized chitosan according to claim 1, characterized in that, In step (1), the ice acetic acid aqueous solution is prepared according to a volume ratio of ice acetic acid to water of 1-3:97-99; and the mass-volume ratio of chitosan to the ice acetic acid aqueous solution is 2-3:100-150.
3. The method of preparing naringenin functionalized chitosan according to claim 1, characterized in that, In step (2), the mass-volume ratio of naringin to dimethyl sulfoxide is 2-3:100-150.
4. The method of preparing naringenin functionalized chitosan according to claim 1, characterized in that, In step (3), the molar ratio of naringin to EDC to NHS is 7-11:15-33:6-17.
5. The method of preparing naringenin functionalized chitosan according to claim 1, characterized in that, In step (4), the volume ratio of the chitosan ice acetic acid aqueous solution to the activated naringin solution is 4-5:2-3.
6. The method of preparing naringenin functionalized chitosan according to claim 1, characterized in that, In step (1), the degree of deacetylation of chitosan is ≥95%, and the viscosity is 100-200 mPa.s.
7. The method of preparing naringenin functionalized chitosan according to claim 1, characterized in that, In step (1), the chitosan and the ice acetic acid aqueous solution are mixed uniformly, sealed, and stirred for 60-80 minutes to dissolve the chitosan.
8. The method of preparing naringenin functionalized chitosan according to claim 1, characterized in that, In step (2), the naringin is added to dimethyl sulfoxide, sealed, and stirred for 8-10 minutes to dissolve the naringin.
9. The method of preparing naringenin functionalized chitosan according to claim 1, characterized in that, In step (4), the naringin solution is added at a rate of no more than 5% of the volume of the reaction system per minute.