EGCG-Cu complex, EGCG-Cu-Cs nano-particles and preparation method and application of EGCG-Cu complex and EGCG-Cu-Cs nano-particles

Abstract

The invention discloses an EGCG-Cu complex, an EGCG-Cu-Cs nanoparticle, a preparation method of the EGCG-Cu complex, and an application of the EGCG-Cu-Cs nanoparticle. The preparation method comprises the following steps: mixing an aqueous solution of EGCG and an aqueous solution containing copper ions, adjusting the pH value, stirring for reaction, dialyzing or centrifuging, and freeze-drying to obtain the EGCG-Cu complex; the EGCG-Cu complex is dissolved in water, a chitosan solution is added, stirring reaction, centrifugal washing and freeze drying are carried out, and the EGCG-Cu-Cs nanoparticles are obtained. The EGCG-Cu complex and the EGCG-Cu-Cs nanoparticles prepared by the invention are uniform in size and good in dispersity, and have excellent xanthine oxidase inhibition effect and free radical scavenging effect.

Description

Technical Field

[0001] The invention belongs to the technical field of complex chemistry, and specifically relates to an EGCG-Cu complex and EGCG-Cu-Cs nanoparticles, and a preparation method and application thereof. Background Art

[0002] Hyperuricemia is a metabolic disease caused by excessive uric acid concentration in the blood due to purine metabolism disorders or poor diet. Xanthine oxidase catalyzes xanthine and hypoxanthine to produce uric acid and superoxide anions. When the uric acid concentration in the body is too high, it will be deposited in the joints, which will cause gout symptoms. Therefore, how to reduce the uric acid concentration in the body has become an important issue in the study of hyperuricemia treatment. In addition, the accumulation of superoxide anions produced with uric acid will also cause partial oxidative stress damage to the body. Therefore, seeking a complex that has both uric acid-lowering and antioxidant effects is of great significance for alleviating hyperuricemia and its complications.

[0003] There are currently three main ways to reduce uric acid. The first is to inhibit the activity of xanthine oxidase, reduce the production of uric acid and thus achieve the goal of reducing concentration. Currently, the commonly used drugs on the market are allopurinol and febuxostat. The second method is to achieve the goal of reducing concentration by accelerating the excretion of uric acid. Currently, the commonly used drug on the market is benzbromarone. The last method is to alkalize uric acid, and the commonly used drug is colchicine. At present, the academic community generally believes that the first method has more potential. The drug benzbromarone used in the second method is banned by the US FDA because it was found that benzbromarone caused liver failure after taking it. Therefore, the first-line drug for the treatment of gout in the United States is still allopurinol. However, the uric acid-lowering effect of allopurinol and febuxostat is still not ideal, and they do not have antioxidant capacity, and cannot alleviate the oxidative stress damage caused by the increase of superoxide anions during the production of uric acid.

[0004] Some studies have found that transition metal ions have a good inhibitory effect on xanthine oxidase by combining transition metal ions with xanthine oxidase, among which the inhibitory effect of metal copper ions is the most ideal. Although copper ions are non-toxic at trace concentrations, they are still toxic to the human body when the concentration increases, which limits their further application in the field of biomedicine. Transition metal ions play an important role in the treatment of uric acid, but they cannot be used directly in clinical practice due to their high toxicity and strong irritation. However, if they are turned into complexes, their toxicity and irritation can be reduced, which is beneficial to human absorption, such as iron citrate complexes for the treatment of anemia; potassium antimony tartrate for the treatment of diabetes and schistosomiasis. However, at present, most of the prepared copper ion complexes have poor solubility, which limits their further application. Therefore, it is of great significance to prepare a copper ion complex with good effect and stable properties for the treatment of hyperuricemia. Considering that the actual application of the particles is oral delivery, chitosan encapsulation is adopted to reduce the impact of the gastrointestinal environment on the particles. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention aims to provide an EGCG-Cu complex and EGCG-Cu-Cs nanoparticles and their preparation method and application

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] A method for preparing an EGCG-Cu complex comprises the following steps:

[0008] The aqueous solution of EGCG (epigallocatechin gallate) and the aqueous solution containing copper ions are mixed, the pH is adjusted, the reaction is stirred, dialyzed or centrifuged, and freeze-dried to obtain an EGCG-Cu complex.

[0009] Preferably, the aqueous solution containing copper ions is an aqueous solution of copper chloride and / or copper sulfate;

[0010] Preferably, the concentration of copper ions in the aqueous solution containing copper ions is 1:10M.

[0011] Preferably, the concentration of EGCG in the aqueous solution of EGCG is 1:30M.

[0012] Preferably, the aqueous solution of EGCG is obtained by adding EGCG into water and stirring to dissolve it;

[0013] More preferably, the temperature for stirring and dissolving is 25-40°C.

[0014] Preferably, the molar ratio of copper ions to EGCG is 1 to 3:1.

[0015] Preferably, the pH is adjusted by adding an alkaline solution;

[0016] Further preferably, the alkaline solution is sodium hydroxide solution or sodium carbonate solution.

[0017] Preferably, the pH is adjusted to 3.5-7.5.

[0018] More preferably, the pH is adjusted to 3.2-4.2 or 6.8-7.2.

[0019] Preferably, the stirring reaction time is greater than or equal to 2 hours, and the temperature is 20-50°C.

[0020] More preferably, the stirring reaction time is 2 to 12 hours and the temperature is 25°C to 40°C.

[0021] More preferably, the stirring reaction time is 5 h and the temperature is 30°C.

[0022] Preferably, the dialysis is performed using deionized water, and the dialysis time is 24 to 48 hours.

[0023] More preferably, a dialysis bag of 8000 to 10000 D is used for dialysis.

[0024] Preferably, the centrifugal speed is 8000-10000r, and the time is 4-10min.

[0025] Preferably, the freeze-drying time is 24 to 48 hours and the temperature is -60°C to -80°C.

[0026] The EGCG-Cu complex prepared by the above preparation method.

[0027] A method for preparing EGCG-Cu-Cs nanoparticles comprises the following steps:

[0028] The above EGCG-Cu complex particles were dissolved in water, chitosan solution was added, stirred for reaction, centrifuged for washing, and freeze-dried to obtain EGCG-Cu-Cs nanoparticles (EGCG-Cu coated with chitosan).

[0029] Preferably, the dosage ratio of EGCG-Cu complex to water is 0.5-3 mg / mL.

[0030] Preferably, the chitosan is at least one of carboxymethyl chitosan and chitosan hydrochloride;

[0031] Preferably, the mass ratio of chitosan to EGCG-Cu complex is 1:1 to 1.5:1.

[0032] Preferably, the stirring reaction time is 2 to 5 hours and the temperature is 25 to 40°C.

[0033] Preferably, the speed of centrifugal washing is 8000-10000r, the temperature is 25-40°C, and the time is 4-10min;

[0034] Preferably, the freeze-drying time is 24 to 48 hours and the temperature is -60°C to -80°C.

[0035] More preferably, the freeze-drying time is 24 hours.

[0036] The EGCG-Cu-Cs nanoparticles prepared by the above preparation method.

[0037] Application of the above-mentioned EGCG-Cu complex or the above-mentioned EGCG-Cu-Cs nanoparticles in the preparation of uric acid-lowering, antioxidant drugs or foods.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] (1) The present invention improves the uric acid-lowering effect of copper ions by chemically combining copper ions with EGCG, achieves low toxicity through coordination, introduces the reducing property of EGCG, and imparts it with anti-aging effects.

[0040] (2) The EGCG-Cu-Cs powder prepared by the present invention has small size, excellent uric acid lowering effect, good antioxidant effect and stable properties, and has potential application value in the medical fields of lowering uric acid and delaying aging.

[0041] (3) The present invention has the advantages of simple preparation process and low price of raw materials used. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 It is the infrared spectrum of the EGCG-Cu complex in Example 1 of the present invention.

[0043] Figure 2 It is the ultraviolet absorption spectrum of the EGCG-Cu complex in Example 1 of the present invention.

[0044] Figure 3 It is the ultraviolet absorption spectrum of the EGCG-Cu complex in Example 3 of the present invention.

[0045] Figure 4a This is a diagram showing the uric acid lowering effect of the EGCG-Cu complex in Example 1 of the present invention.

[0046] Figure 4b It is a diagram showing the uric acid lowering effect of the EGCG-Cu complexes in Examples 1, 3, 4, and 5 of the present invention.

[0047] Figure 5a and Figure 5bThis is a test chart of the EGCG-Cu complex's ability to reduce DPPH free radicals in Example 1 of the present invention.

[0048] Figure 6 It is a release curve diagram of chitosan-coated EGCG-Cu-Cs nanoparticles and EGCG-Cu complex particles not coated with chitosan in Example 1 of the present invention in simulated digestive fluid. DETAILED DESCRIPTION

[0049] The specific implementation of the present invention is further described below in conjunction with examples, but the implementation and protection of the present invention are not limited thereto. It should be noted that if there are processes that are not particularly described in detail below, they can be implemented or understood by those skilled in the art with reference to the prior art. If the manufacturer of the reagents or instruments used is not indicated, they are deemed to be conventional products that can be purchased commercially.

[0050] Example 1

[0051] A method for preparing an EGCG-Cu complex and EGCG-Cu-Cs nanoparticles comprises the following steps:

[0052] Take 458 mg of EGCG and dissolve it in 30 mL of deionized water, take 170 mg of cupric chloride dihydrate and dissolve it in 10 mL of deionized water, mix the two solutions and adjust the pH to 7 with NaOH solution, react at 30°C for 5 h (no precipitation), dialyze with 8000D dialysis bag for 24 h, and then freeze-dry for 24 h to obtain a solid sample EGCG-Cu complex.

[0053] Weigh 50 mg of EGCG-Cu complex solid sample and dissolve it in 20 mL of deionized water. After mixing evenly, add 15 mL of chitosan hydrochloride solution (2 mg / mL). After stirring evenly, add 15 mL of carboxymethyl chitosan solution (2 mg / mL). React for 2 h, centrifuge and wash at 8000 r for 4 min, and then freeze-dry for 24 h to obtain solid sample EGCG-Cu-Cs nanoparticles.

[0054] In this example, the infrared spectrum of the prepared EGCG-Cu complex nanoparticles is as follows: Figure 1 As shown, 3381cm -1 1600cm is the stretching vibration absorption peak of hydroxyl group. Its intensity is lower than that of EGCG infrared spectrum and it moves to a higher wave number, indicating that phenolic hydroxyl group and copper ion have a complex reaction. -1 The left and right sides are the vibration absorption peaks of carbonyl, 1511cm -1 It is the stretching vibration absorption peak of the benzene ring. 1380cm -1 and 1063cm -1The deformation absorption peak of hydroxyl group is weakened, indicating that the copper ion is coordinated with two phenolic hydroxyl groups and EGCG is a bidentate ligand. Figure 2 As shown, the 280nm characteristic peak of EGCG red-shifted, which corresponds to the coordination reaction between the hydroxyl group of EGCG and copper ions.

[0055] Example 2

[0056] A method for preparing an EGCG-Cu complex comprises the following steps:

[0057] 458 mg of EGCG was dissolved in 50 mL of anhydrous ethanol. 170 mg of copper chloride dihydrate was added to the anhydrous ethanol solution of EGCG and stirred to obtain a mixed solution. The mixed solution was reacted at 30°C for 5 h and then rotary evaporated at 60°C to obtain a solid product. It was found that no EGCG-Cu complex was generated.

[0058] The EGCG-Cu complex cannot be prepared in this example.

[0059] Example 3

[0060] A method for preparing an EGCG-Cu complex comprises the following steps:

[0061] Take 458 mg of EGCG and dissolve it in 30 mL of deionized water, take 170 mg of cupric chloride dihydrate and dissolve it in 10 mL of deionized water, mix the two solutions and the pH value is about 3, adjust the pH to 4 with NaOH solution, react at 30 ° C for 5 h (with precipitation), centrifuge and wash at 8000 r, 4 min, and then freeze-dry for 24 h to obtain a solid sample EGCG-Cu complex.

[0062] In this embodiment, comparing the ultraviolet spectra ( Figure 3 ) found that although a new peak was generated, the 280nm characteristic peak of EGCG did not completely disappear, indicating that EGCG and copper formed a more complex complex.

[0063] Example 4

[0064] The preparation method is basically the same as that of Example 1, except that the pH is adjusted to 5.

[0065] Example 5

[0066] The preparation method is basically the same as that of Example 1, except that the pH is adjusted to 6.

[0067] Example 6

[0068] The preparation method is basically the same as that of Example 1, except that the molar ratio of EGCG to cupric chloride dihydrate is 1:2, that is, the amount of cupric chloride dihydrate is 340 mg.

[0069] Example 7

[0070] The preparation method is basically the same as that of Example 1, except that the molar ratio of EGCG to cupric chloride dihydrate is 1:3, that is, the amount of cupric chloride dihydrate is 510 mg.

[0071] Example 8

[0072] 458 mg of EGCG was dissolved in 30 mL of deionized water without adding any copper salt, the pH was adjusted to 7, and the reaction was carried out at 30°C for 5 h to obtain a brown solution.

[0073] The solution obtained in this example is produced by oxidation of EGCG, and no complex can be obtained.

[0074] Performance Test:

[0075] (1) PBS solutions of EGCG-Cu complex (Example 1, concentration of 15 mg / L), xanthine (concentration of 100 mg / L), and xanthine oxidase (concentration of 20 mg / L) were prepared respectively. The EGCG-Cu complex and xanthine oxidase were mixed for a period of time in advance to allow the EGCG-Cu complex to fully bind to the xanthine oxidase, and then xanthine was added to start the reaction, and the change of the absorbance intensity at 293 nm over time was measured to reflect the generation rate of uric acid. The smaller the change in the absorption intensity, the stronger the inhibitory effect. The control group was added with an equal amount of PBS solution, a 10 mg / L EGCG PBS solution, a 3.5 mg / L cupric chloride dihydrate PBS solution, and a 3 mg / L AP (allopurinol) PBS solution (the molar amount of the EGCG-Cu complex was calculated according to the EGCG-Cu-10H2O molecular formula).

[0076] Compare the inhibitory effects of different samples with the same molar concentration on xanthine oxidase, refer to Figure 4a At the same concentration, the EGCG-Cu complex has the best effect in inhibiting xanthine oxidase.

[0077] (2) Prepare PBS solutions of EGCG-Cu complex (Examples 1, 3, 4, 5, concentrations of 10 mg / L, respectively), xanthine (concentration of 400 mg / L), and xanthine oxidase (concentration of 40 mg / L). Mix the EGCG-Cu complex and xanthine oxidase for a period of time in advance to allow the EGCG-Cu complex to fully bind to the xanthine oxidase, then add xanthine to start the reaction, and measure the change in absorbance intensity at 293 nm over time to reflect the generation rate of uric acid. The smaller the change in absorbance intensity, the stronger the inhibitory effect. An equal amount of PBS solution was added to the control group.

[0078] Comparison of the inhibitory properties of EGCG-Cu complexes prepared under different pH conditions Figure 4bThe EGCG-Cu complex obtained at pH = 4 has the best effect, followed by the EGCG-Cu complex obtained at pH = 7.

[0079] (3) Different ratios (EGCG / Cu 2+ ) obtained (EGCG-Cu complexes of Examples 1, 6, and 7) had no significant difference in inhibitory effect.

[0080] (4) Prepare an anhydrous ethanol solution of DPPH (100 mg / L), then add the aqueous solution of the EGCG-Cu complex obtained in Example 1 (100 mg / L EGCG-Cu aqueous solution), and add equal amounts of pure deionized water, 100 mg / L EGCG aqueous solution, 100 mg / L copper chloride dihydrate aqueous solution, and 100 mg / L AP aqueous solution to the control group. After standing for half an hour, scan the absorption spectrum at 300-600 nm. The reduction in the number of DPPH free radicals can be reflected in the absorbance at around 517 nm. Figure 5a and Figure 5b , the DPPH free radical scavenging performance test of the EGCG-Cu complex obtained in Example 1 shows that the reduction in the number of DPPH free radicals can be reflected in the absorbance at around 517nm. EGCG has a strong scavenging ability for DPPH free radicals, and the scavenging effect of the EGCG-Cu complex is slightly lower than that of EGCG, while pure copper chloride has no free radical scavenging ability. This phenomenon also proves that the hydroxyl group of EGCG successfully undergoes a coordination reaction with copper ions.

[0081] (5) The coated sample EGCG-Cu-Cs nanoparticles of Example 1 containing an equal amount of EGCG-Cu complex and the uncoated sample EGCG-Cu complex were weighed, dissolved in a dialysis bag, placed in simulated gastric fluid, and the EGCG-Cu complex content in the supernatant was measured at 0.5 h, 1 h, 1.5 h and 2 h, and the relevant release curve was plotted, as shown in FIG. Figure 6 As shown, the release amount of EGCG-Cu-Cs nanoparticles after encapsulation with chitosan in gastric fluid was lower than that of the unencapsulated pure EGCG-Cu complex solution, which is beneficial to the oral delivery of EGCG-Cu complex nanoparticles.

[0082] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement modes and shall be included in the protection scope of the present invention.

Claims

1. A method for preparing an EGCG-Cu complex, characterized in that: The following steps are involved: The EGCG aqueous solution and the aqueous solution containing copper ions are mixed, the pH is adjusted, the reaction is stirred, dialyzed or centrifuged, and freeze-dried to obtain the EGCG-Cu complex.

2. The preparation method of the EGCG-Cu complex according to claim 1, characterized in that, The aqueous solution containing copper ions is an aqueous solution of copper chloride and / or copper sulfate; The molar ratio of copper ion to EGCG is 1 to 3:

1.

3. The preparation method of the EGCG-Cu complex according to claim 1, characterized in that, The pH is adjusted by adding an alkaline solution; Adjust pH to 3.5-7.

5.

4. The preparation method of the EGCG-Cu complex according to claim 1, characterized in that: The stirring reaction time is greater than or equal to 2 hours, and the temperature is 20-50°C.

5. The EGCG-Cu complex prepared by the preparation method according to any one of claims 1 to 4.

6. A method for preparing EGCG-Cu-Cs nanoparticles, characterized in that: The following steps are involved: The EGCG-Cu complex of claim 5 is dissolved in water, chitosan solution is added, stirred for reaction, centrifuged for washing, and freeze-dried to obtain EGCG-Cu-Cs nanoparticles.

7. The method for preparing EGCG-Cu-Cs nanoparticles according to claim 6, characterized in that: Chitosan is at least one of carboxymethyl chitosan and chitosan hydrochloride; The mass ratio of chitosan to EGCG-Cu complex is 1:1 to 1.5:1; The stirring reaction time is 2 to 5 hours, and the temperature is 25 to 40°C.

8. The method for preparing EGCG-Cu-Cs nanoparticles according to claim 6, characterized in that: The speed of centrifugal washing is 8000-10000r, the temperature is 25-40°C, and the time is 4-10min; The freeze-drying time is 24 to 48 hours, and the temperature is -60°C to -80°C.

9. EGCG-Cu-Cs nanoparticles prepared by the preparation method according to any one of claims 6 to 8.

10. Use of the EGCG-Cu complex according to claim 5 or the EGCG-Cu-Cs nanoparticles according to claim 9 in the preparation of uric acid-lowering, antioxidant drugs or foods.

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