Resveratrol grafted chitosan as well as preparation method and application thereof

Abstract

The invention discloses resveratrol grafted chitosan as well as a preparation method and application thereof, and belongs to the technical field of medicines. The resveratrol grafted chitosan disclosed by the invention is prepared from resveratrol and chitosan, and the resveratrol and the chitosan are combined through a covalent bond. The chitosan grafted product is prepared from resveratrol and chitosan through a free radical method, and the preparation process is safe, green, efficient, simple and convenient; the prepared resveratrol grafted chitosan can inhibit sodium urate nucleation and crystal growth, and even eliminate sodium urate crystals.

Description

Technical Field

[0001] The invention belongs to the technical field of medicine, and particularly relates to resveratrol-grafted chitosan, a preparation method and an application thereof. Background Art

[0002] Gout, the most common arthritis, results from persistently elevated uric acid levels in the body, leading to the deposition of sodium urate crystals in joints and other tissues. This leads to inflammation, pain, joint deformities, bone damage, and other symptoms. Gout patients often require long-term treatment with uric acid-lowering and anti-inflammatory drugs, but these medications can cause liver and kidney damage. Severe gout patients may even face the risk of surgical removal of tophi or amputation.

[0003] As a crystalline disease, inhibiting or eliminating sodium urate crystals is another way to treat gout. It is reported that uricase can oxidize insoluble uric acid into soluble allantoin, which is then excreted from the body to achieve the purpose of treatment. However, uricase is extremely unstable and has strong immunogenicity, resulting in a high incidence of allergies and failure rate. Therefore, there is an urgent need to develop safer and more effective sodium urate crystal inhibitors. Based on the inventor's previous research, it is believed that polyphenol-grafted chitosan has great potential in inhibiting sodium urate crystallization. Resveratrol, as an alkaline polyphenol, may interact with uric acid and further inhibit sodium urate crystallization by reducing uric acid supersaturation. Therefore, the present invention prepared resveratrol-grafted chitosan and examined its effect on sodium urate nucleation and crystal growth. Summary of the Invention

[0004] The purpose of the present invention is to prepare resveratrol-grafted chitosan by an efficient, green, safe and environmentally friendly method. The prepared resveratrol-grafted chitosan can inhibit the nucleation and crystal growth of sodium urate and even eliminate sodium urate crystals.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0006] The invention discloses a resveratrol grafted chitosan, which is prepared from resveratrol and chitosan, wherein the resveratrol and chitosan are combined via a covalent bond.

[0007] The preparation method of the resveratrol-grafted chitosan comprises the following steps:

[0008] (1) dissolving chitosan in an acetic acid aqueous solution to obtain a chitosan solution;

[0009] (2) dissolving ascorbic acid in hydrogen peroxide to obtain a free radical solution;

[0010] (3) adding a free radical solution to the chitosan solution, stirring and reacting, and obtaining a free radicalized chitosan solution;

[0011] (4) adding resveratrol to the radicalized chitosan solution and stirring to react;

[0012] (5) The reaction solution is centrifuged, the supernatant is placed in a dialysis bag for dialysis, and the dialyzate is dried to obtain resveratrol-grafted chitosan.

[0013] Furthermore, in step (1), the deacetylation degree of chitosan is about 90%, and the concentration of the chitosan solution is 1% to 1.5% (m / v).

[0014] Furthermore, in step (1), the concentration of the acetic acid aqueous solution is 1% to 2% (v / v).

[0015] Furthermore, in step (2), the concentration of hydrogen peroxide is 1M to 1.2M, and the concentration of ascorbic acid is 50mg / mL to 60mg / mL.

[0016] Furthermore, in step (3), the volume ratio of the chitosan solution to the free radical solution is 25:1 to 50:1 (v / v).

[0017] Furthermore, in step (3), the reaction conditions are stirring for 0.5h to 1h (25°C to 40°C) under nitrogen protection.

[0018] Furthermore, in step (4), the molar ratio of resveratrol to chitosan is 1:1 to 1:2, and the resveratrol is added by dissolving it in ethanol or directly adding it as a powder; preferably, it is added after dissolving it in ethanol. The reason for adding it after dissolving it in ethanol is to increase the solubility of resveratrol in the system, make the reaction more complete, save reaction time, and improve reaction efficiency.

[0019] Furthermore, in step (4), the reaction conditions are stirring in the dark under nitrogen protection for 12 h to 36 h (25° C. to 40° C.).

[0020] Furthermore, in step (5), the centrifugation condition is 5000 rpm, 5 min; the molecular weight cutoff of the dialysis bag is 14 kDa, the dialysis is performed for 72 hours, and the water is changed more than 8 times; the drying method includes oven drying, vacuum drying, freeze drying and spray drying, preferably spray drying.

[0021] The application of the resveratrol grafted chitosan in the preparation of products for inhibiting sodium urate crystallization.

[0022] The above-mentioned resveratrol grafted chitosan is used as a sodium urate crystal inhibitor in the preparation of gout prevention and treatment products.

[0023] Beneficial effects:

[0024] (1) The present invention provides a new chitosan grafted product, which is prepared from resveratrol and chitosan by a free radical method. The preparation process is safe, green, efficient and simple.

[0025] (2) The chitosan grafted product provided by the present invention is a new sodium urate crystal inhibitor with a very strong nucleation and crystal growth inhibitory effect, and can even eliminate sodium urate crystals. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The UV spectra of resveratrol, chitosan and resveratrol-grafted chitosan are shown in Figure 2.

[0027] Figure 2 It is thin layer chromatography of resveratrol, chitosan and resveratrol grafted chitosan.

[0028] Figure 3 IR spectra of resveratrol, chitosan, physical mixture and resveratrol-grafted chitosan.

[0029] Figure 4 Differential scanning calorimetry and thermogravimetric spectra of resveratrol, chitosan, physical mixture and resveratrol-grafted chitosan.

[0030] Figure 5 This is the H NMR spectrum of chitosan and resveratrol-grafted chitosan.

[0031] Figure 6 The nucleation induction time of the sodium urate system was investigated in the blank and in the presence of several crystallization inhibitors.

[0032] Figure 7 The crystallization process of sodium urate system was observed in the blank and in the presence of several crystallization inhibitors.

[0033] Figure 8 Dynamic light scattering monitoring of the elimination of sodium urate crystals by resveratrol-grafted chitosan.

[0034] Figure 9 This is the observation of the decrystallization process of sodium urate by resveratrol grafted chitosan at different concentrations. DETAILED DESCRIPTION

[0035] The preferred embodiments of the present invention will be described in detail below with reference to the examples. It should be understood that the following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0036] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0037] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0038] Example 1

[0039] Preparation of resveratrol grafted chitosan

[0040] 0.5 g of chitosan was dissolved in 50 mL of acetic acid aqueous solution (1%, v / v) to obtain a chitosan solution. 0.1 g of ascorbic acid was dissolved in 2 mL of hydrogen peroxide solution (1 M) to obtain a free radical solution. The free radical solution was added to the chitosan solution (chitosan solution: free radical solution = 25:1, v / v) and stirred at 40°C under nitrogen protection for 1 hour. Subsequently, resveratrol powder (0.7075 g) was added thereto and stirred in the dark at 40°C under nitrogen protection for 12 hours. The reaction solution was centrifuged at 5000 rpm for 5 minutes, and the supernatant was placed in a 14 kDa dialysis bag for 72 hours, with the water changed more than 8 times. The dialysate was spray dried (the inlet temperature, air flow rate and spray flow rate were set to 120°C, 100% and 10 mL / min, respectively) to obtain a solid product.

[0041] Example 2

[0042] Preparation of resveratrol grafted chitosan

[0043] Dissolve 0.5g of chitosan in 50mL of acetic acid aqueous solution (1%, v / v) to obtain a chitosan solution. Take 0.1g of ascorbic acid and dissolve it in 2mL of hydrogen peroxide solution (1M) to obtain a free radical solution. Add the free radical solution to the chitosan solution (chitosan solution: free radical solution = 25:1, v / v) and stir at 40°C under nitrogen protection for 1 hour. Subsequently, add resveratrol solution (0.7075g) dissolved in 5mL of ethanol and stir in the dark at 40°C under nitrogen protection for 12 hours. The reaction solution is centrifuged at 5000rpm for 5min, and the supernatant is placed in a 14kDa dialysis bag for 72 hours, with the water changed more than 8 times. The dialysate is spray dried (inlet temperature, air flow rate and spray flow rate are set to 120°C, 100% and 10mL / min respectively) to obtain a solid product.

[0044] Example 3

[0045] Preparation of resveratrol grafted chitosan

[0046] 0.5 g of chitosan was dissolved in 50 mL of acetic acid aqueous solution (2%, v / v) to obtain a chitosan solution. 0.1 g of ascorbic acid was dissolved in 2 mL of hydrogen peroxide solution (1 M) to obtain a free radical solution. The free radical solution was added to the chitosan solution (chitosan solution: free radical solution = 50:1, v / v) and stirred at 25°C under nitrogen protection for 1 hour. Subsequently, resveratrol powder (0.3538 g) was added thereto and stirred in the dark at 25°C under nitrogen protection for 24 hours. The reaction solution was centrifuged at 5000 rpm for 5 minutes, and the supernatant was placed in a 14 kDa dialysis bag for 72 hours, with the water changed more than 8 times. The dialysate was spray dried (the inlet temperature, air flow rate and spray flow rate were set to 120°C, 100% and 10 mL / min, respectively) to obtain a solid product.

[0047] The resveratrol-grafted chitosan prepared in Example 1 was tested as follows:

[0048] 1. Ultraviolet full wavelength scanning spectrum

[0049] Instrument: TU-1901 UV spectrophotometer (Puxi General, Beijing)

[0050] Wavelength: 200-400nm.

[0051] UV spectra of resveratrol, chitosan and resveratrol-grafted chitosan Figure 1 Chitosan has no UV absorption in the range of 200-400 nm, while resveratrol-grafted chitosan exhibits UV absorption close to that of resveratrol, indicating that resveratrol was successfully grafted onto chitosan.

[0052] 2. Thin layer chromatography

[0053] Methods: 10 μL of 1 mg / mL sample solution (resveratrol, chitosan, and resveratrol-grafted chitosan) was spotted onto a silica gel plate and then eluted with a chloroform-ethyl acetate-acetic acid solution (50:50:1, v / v / v) at room temperature. When the eluate rose to the top of the plate, the plate was removed, dried, and then visualized using 365 nm UV light.

[0054] Results: The thin layer chromatography of resveratrol, chitosan and resveratrol grafted chitosan was as follows: Figure 2 As shown in the figure, resveratrol showed a bright spot at a distance from the baseline, while chitosan did not show any bright spot. The resveratrol-grafted chitosan showed a bright spot of resveratrol at the baseline and was not unfolded, proving that resveratrol was successfully grafted onto chitosan and that there was no free resveratrol in the product.

[0055] 3. Calculation of grafting rate

[0056] Methods: The grafting efficiency of resveratrol-grafted chitosan was determined using the classic Folin-phenol method. 0.5 mL of the resveratrol-grafted chitosan solution (20 mg / mL) to be tested was mixed with 0.5 mL of Folin-phenol reagent (1 M) and 2 mL of purified water and allowed to react in the dark for 5 minutes. Then, 2 mL of 7.5% sodium carbonate solution was added to the system and allowed to react in the dark at room temperature for 2 hours. The UV absorbance of the mixture was then measured at 760 nm. Resveratrol solutions of a series of concentrations (0.01, 0.025, 0.05, 0.075, 0.1, 0.15, 0.2, and 0.25 mg / mL) were subjected to the above procedure and the wavelength was measured at 760 nm. A standard curve was plotted and the grafting efficiency of resveratrol-grafted chitosan was calculated based on this. The grafting efficiency was expressed as milligrams of resveratrol equivalent per gram.

[0057] Results: The grafting rate of the prepared resveratrol-grafted chitosan was calculated to be about 7.55 mg / g.

[0058] 4. Infrared spectroscopy

[0059] Instrument: IRAffinity-1S Fourier transform infrared spectrometer (SHIMADU, Japan)

[0060] Results: The infrared spectra of resveratrol, chitosan, physical mixture (mixed by calculating the content ratio based on the grafting rate of resveratrol grafted chitosan) and resveratrol grafted chitosan are as follows: Figure 3 As shown. In the infrared spectrum of resveratrol, 3293 cm -1 and 1587cm -1 They are respectively attributed to the stretching vibration of -OH and C=C of benzene ring skeleton. In the infrared spectrum of chitosan, 3440cm -1 and 1643cm -1 The absorption peaks at 2960-2850 cm-1 belong to the stretching vibration and bending vibration of the -NH2 group; the absorption bands of the -CH- and -CO- skeleton stretching on the chitosan chain are located at 2960-2850 cm-1, respectively. -1 and 1170~1050cm -1 The infrared spectrum of the physical mixture of resveratrol and chitosan is the physical superposition of the infrared spectra of resveratrol and chitosan. In the infrared spectrum of resveratrol grafted chitosan, the vibration peaks of the -OH group in the resveratrol structure and the -NH2 group in the chitosan are combined at 3417 cm -1 The C=C stretching vibration of the benzene ring structure of resveratrol shows a peak at 1587 cm -1 Offset to 1593cm -1 ; In addition, the absorption intensity of -CH- and -CO- skeleton stretching in the infrared spectrum of resveratrol-grafted chitosan is weaker than that of chitosan, indicating that resveratrol is successfully grafted onto the chitosan skeleton.

[0061] 5.DSC and TGA

[0062] Instruments: DSC 3500 differential scanning calorimeter (NETZSCH, 135 Germany); TGA 4000 thermogravimetric analyzer (PerkinElmer, USA)

[0063] Heating rate: 20℃ / min

[0064] Temperature range: DSC set to 50 ~ 400 ° C; TGA set to 50 ~ 800 ° C

[0065] Results: The DSC and TGA of resveratrol, chitosan, physical mixture (with product grafting rate as reference) and resveratrol grafted chitosan were as follows: Figure 4 As shown. The DSC curve of resveratrol exhibits a sharp endothermic peak at 269.2°C, corresponding to its melting behavior. Combined with the TGA curve, it is clear that at this temperature, resveratrol also undergoes degradation while melting. Chitosan exhibits a broad endothermic peak at 103.1°C, indicating dehydration, followed by degradation at 300.8°C. The thermal behavior of resveratrol-grafted chitosan differs from that of chitosan, with a dehydration peak occurring at 106.8°C and degradation occurring at 245.4°C.

[0066] 6. H NMR

[0067] Instrument: Bruker NMR spectrometer (Bruker BioSpin, Germany)

[0068] Results: Resveratrol, chitosan, physical mixture (with product grafting rate as reference) and resveratrol grafted chitosan 1 H NMR spectra as Figure 5 As shown. Located at 3.0ppm, 3.6~3.8ppm and 4.7ppm 1 The H absorption peaks correspond to the hydrogen protons at C-2, C-3~6 and C-1 in the chitosan structure, and the absorption peak of the protons in the N-acetyl unit on chitosan is about 1.9ppm. 1 In the H NMR spectrum, in addition to the characteristic peaks of chitosan, a new peak at 6.0-10.0 ppm was 1The H absorption peaks originate from resveratrol. The single peak at 9.5 ppm and the double peak at 9.1 ppm are attributed to the protons at C-4' and C-3&5 positions on the RES structure, respectively. The double peak at 7.4 ppm corresponds to the protons at C-2'&6' on benzene ring B in the RES structure; the multiple absorption peaks at 6.7-6.9 ppm are related to the protons at Ca&b and C-3'&5' on the olefin in the RES structure; and the absorption peaks for the protons at C-2&6 and C-4 on benzene ring A in the RES are located at 6.4 ppm and 6.1 ppm, respectively.

[0069] 7. Determination of nucleation induction time

[0070] Method: Take an appropriate amount of uric acid powder and dissolve it in a 15mM NaOH aqueous solution using a 50°C water bath and ultrasound. At the same time, take an appropriate amount of NaCl powder and dissolve it in pure water. Mix the uric acid and NaCl solution to make the total concentration of uric acid in the mixed solution 8.8mM and NaCl 8.8mM. + The total concentration was 160 mM. The mixture was adjusted to pH 7.4 using 1 M HCl or NaOH solution. Resveratrol, chitosan, a physical mixture (using the grafting efficiency of the product as a reference), and resveratrol-grafted chitosan were then added to the solution, achieving a concentration of 20 μg / mL. The solution was then filtered through a 0.22 μm filter membrane. The filtrate was placed in a turbidity meter, and the turbidity was monitored in real time to determine the nucleation induction time.

[0071] Results: The effects of resveratrol, chitosan, physical mixture and resveratrol-grafted chitosan on the nucleation of sodium urate were shown in Figure 2. Figure 6 The nucleation induction time of blank sodium urate was 1.9 hours. When 20 μg / mL resveratrol was added to the system, the nucleation induction time was extended to 7.8 hours. At the same concentration, chitosan further extended the nucleation induction time to 22.5 hours. When the physical mixture was added, the nucleation induction time of sodium urate was 22.9 hours, showing a similar effect to chitosan. Under the influence of resveratrol-grafted chitosan, the turbidity of the sodium urate nucleation system did not increase after 250 hours, indicating that resveratrol-grafted chitosan can extend the nucleation behavior of sodium urate by more than 10 days, making it a very effective nucleation inhibitor.

[0072] 8. Effect of crystal inhibitors on sodium urate crystal growth

[0073] Method: Take an appropriate amount of uric acid powder and dissolve it in a 15mM NaOH aqueous solution using a 50°C water bath and ultrasound. At the same time, take an appropriate amount of NaCl powder and dissolve it in pure water. Mix the uric acid and NaCl solution until the uric acid concentration in the mixed solution is 8.8mM and the NaCl solution is 1. +The mixture was adjusted to a pH of 7.4 with a 1M HCl or NaOH solution at a concentration of 160 mM. An appropriate amount of micron-sized sodium urate crystals was then added as seeds. Resveratrol, chitosan, a physical mixture (using the grafting rate of the product as a reference), and resveratrol-grafted chitosan were then added to the solution, achieving a concentration of 20 μg / mL. The crystallization solution was allowed to stand, and the crystallization process was observed in real time.

[0074] Results: The effects of resveratrol, chitosan, physical mixture and resveratrol-grafted chitosan on the crystallization of sodium urate were shown in Figure 2. Figure 7 and Figure 8 As shown in the figure. At 0 hours, all crystallization systems resembled true solutions macroscopically, but a clear beam of light could be observed after laser irradiation, and only micron-sized particles could be observed under a microscope. These particles were the added sodium urate seeds. After 2 hours, distinct needle-shaped crystals were observed in the blank sodium urate, indicating that crystallization had occurred. Subsequently, the crystallization rate increased. After 20 hours, the number of blank sodium urate crystals no longer increased, and crystallization was essentially complete. Under the influence of resveratrol, the crystallization process of sodium urate was affected, and crystallization was delayed, with crystallization essentially complete after 30 hours. Chitosan, as a polymer, exhibited a superior inhibitory effect on nucleation and crystal growth than resveratrol, achieving complete crystallization after nearly 50 hours. The crystal size was shorter than that of blank sodium urate and sodium urate with resveratrol. Physical mixtures exhibited similar inhibitory effects to chitosan. Unlike all previous crystal inhibitors, after adding resveratrol-grafted chitosan to the system, no crystallization behavior was observed in the field of view over time, and the initial micron-sized particles gradually disappeared. After 10 days, no crystals were observed in the field of view, and even the added sodium urate seeds disappeared. Correspondingly, in the macroscopic image of the crystallization system, the beam under the irradiation of the laser pen gradually weakened, and no beam was observed after 10 days, indicating that the system is a true solution. By dynamic light scattering detection ( Figure 8 ) It can be seen that particles with a size of approximately 2 μm are present in the system at 0 h, and the particle size gradually decreases, reaching zero by the 10th day, which is consistent with the phenomena observed by microscopy and macroscopic images. In other words, resveratrol, chitosan, and physical mixtures can inhibit the crystallization of sodium urate, and resveratrol-grafted chitosan not only inhibits sodium urate crystallization but also eliminates it.

[0075] 9. Elimination of sodium urate crystals

[0076] Methods: To facilitate observation, resveratrol-grafted chitosan was added to a 0.35 mM sodium urate supersaturated solution (close to the uric acid concentration under normal human physiological conditions) to make its concentration in the system reach 0.7, 0.8, 0.9, and 1.0 mg / mL, respectively. Then, an appropriate amount of sodium urate crystals were directly added and the changes in the sodium urate crystals were observed under a microscope.

[0077] Results: The decrystallization process of resveratrol grafted chitosan on sodium urate was as follows: Figure 9 As shown in the figure. Since the solution is a supersaturated solution of sodium urate, the addition of sodium urate crystals does not cause any changes in the short term. When resveratrol-grafted chitosan is added to the system, the sodium urate crystals gradually disappear, demonstrating that resveratrol-grafted chitosan eliminates sodium urate crystals. Furthermore, the decrystallization of sodium urate by resveratrol-grafted chitosan is concentration-dependent; higher concentrations result in faster decrystallization rates.

Claims

1. A resveratrol-grafted chitosan, characterized in that: It is made of resveratrol and chitosan, wherein the resveratrol and chitosan are combined by covalent bonds.

2. The method for preparing resveratrol grafted chitosan according to claim 1, characterized in that: The following steps are involved: (1) dissolving chitosan in an acetic acid aqueous solution to obtain a chitosan solution; (2) dissolving ascorbic acid in hydrogen peroxide to obtain a free radical solution; (3) adding a free radical solution to the chitosan solution and stirring the solution to obtain a free radicalized chitosan solution; (4) Add resveratrol to the radicalized chitosan solution and stir to react; (5) The reaction solution is centrifuged, the supernatant is placed in a dialysis bag for dialyzation, and the dialyzate is dried to obtain resveratrol-grafted chitosan.

3. The preparation method according to claim 2, wherein In step (1), the concentration of the chitosan solution is 1% to 1.5% m / v, and the concentration of the acetic acid aqueous solution is 1% to 2% v / v.

4. The preparation method according to claim 2, wherein In step (2), the concentration of hydrogen peroxide is 1 M to 1.2 M, and the concentration of ascorbic acid is 50 mg / mL to 60 mg / mL.

5. The preparation method according to claim 2, wherein In step (3), the volume ratio of chitosan solution to free radical solution is 25:1~50:1, and the reaction conditions are nitrogen atmosphere, 0.5 h~1 h, 25 o C~40 o C.

6. The preparation method according to claim 2, wherein In step (4), the molar ratio of resveratrol to chitosan is 1:1~1:2, and the reaction conditions are nitrogen atmosphere, light-proof, 12 h~36 h, 25 o C~40 o C.

7. Use of the resveratrol-grafted chitosan according to claim 1 in preparing a product for inhibiting sodium urate crystallization.

8. Use of the resveratrol-grafted chitosan according to claim 1 as a sodium urate crystal inhibitor in the preparation of a gout prevention and treatment product.

9. A gout prevention and treatment product, characterized in that: The resveratrol-grafted chitosan according to claim 1 is used as the effective active ingredient.

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