Chitosan-glycine-copper chelate and its preparation method and application in treating leukoplakia and vitiligo

Abstract

The application discloses a chitosan-glycine-copper chelate and a preparation method and application thereof in treating leukoplakia and vitiligo. In the preparation, chitosan is first placed in an acid aqueous phase system, and is fully dissolved and forms a uniform transparent or translucent solution under stirring; then glycine is added, and the chitosan and the glycine are fully mixed under stirring to build an organic ligand environment containing multiple coordination groups; subsequently, a copper source is introduced to perform a chemical reaction, and finally the chitosan-glycine-copper chelate is obtained. The chitosan-glycine-copper chelate shows a strong promoting activity completely different from all other substances (including simple mixtures of components thereof), the chelate can continuously and significantly activate tyrosinase activity, and promote the formation of melanin, so that the purpose of treating leukoplakia and vitiligo is achieved.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to the treatment of vitiligo, specifically to a chitosan-glycine-copper chelate, its preparation method, and its application in the treatment of vitiligo. Background Technology

[0002] The pathogenesis of vitiligo and leukoderma is related to impaired melanin synthesis, and melanin is mainly formed from tyrosine through a series of chemical reactions. Tyrosinase (TYR) is a 75kD copper-containing enzyme derived from embryonic nerve sheath cells. It is a key enzyme in melanin metabolism and catecholamine production, possessing two sites: copper A and copper B. Binding to one site promotes the binding of copper to the other site. Tyrosinase is a crucial enzyme in skin melanin synthesis; its activity is related to the amount of melanin synthesized by melanocytes, and controlling its activity controls the amount of melanin produced.

[0003] Chinese patent CN102274359A discloses a symptomatic and root-cause-treating vitiligo concealing liquid and its preparation method. The preparation is made from the following raw materials in parts by weight: 50-100 parts fig leaves, 50-100 parts duckweed, 50-100 parts orange peel, 200-300 parts 75% ethanol, 10-20 parts copper chloride, and 700-800 parts loofah water. The preparation steps are as follows: Soak the fig leaves, duckweed, and orange peel separately in water, dry them, and cut them into thin strips. Then, thoroughly mix the three types of strips. Add 75% ethanol solution and soak for 48 hours. Filter out the residue to obtain a pigment solution. Add copper chloride to the pigment solution and mix thoroughly. Then add loofah water and mix thoroughly to obtain the final product. This vitiligo concealing liquid has the effect of naturally concealing white patches on the skin with pigment, and also has the effect of activating the tyrosinase activity of melanocytes and promoting melanin production through natural plant extracts. Therefore, it has a symptomatic and root-cause-treating effect on vitiligo. However, this concealing liquid is mainly used to cover up vitiligo, and its therapeutic effect is not ideal. Therefore, there is an urgent need for a safe and effective topical therapeutic product that can promote melanin production in a highly efficient, safe, and stable manner. Summary of the Invention

[0004] The first objective of this invention is to provide a chitosan-glycine-copper chelate, which has a strong activating effect on tyrosinase, can significantly increase the activity of tyrosinase in melanocytes, promote melanin formation, and thus achieve the purpose of treating vitiligo. It has good therapeutic effect. Through experimental comparison, it has been found that the chitosan-glycine-copper chelate exhibits a strong promoting activity that is completely different from all other substances (including simple mixtures of their components), and can continuously and significantly promote the activity of tyrosinase.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: A chitosan-glycine-copper chelate is prepared by first dissolving chitosan, then mixing it with glycine to construct an organic ligand environment containing multiple coordination groups, introducing a copper source, and carrying out a chemical reaction to allow copper ions to be fully dispersed in the system and gradually participate in the coordination reaction, ultimately obtaining the chitosan-glycine-copper chelate.

[0006] As a preferred embodiment of the present invention, in the chitosan-glycine-copper chelate, the chitosan mass fraction is 0.1%-2%, and the chitosan is chitosan or its derivative with a degree of deacetylation of 50%-99%, including carboxymethyl chitosan, quaternized chitosan, and low molecular weight chitosan; the glycine mass fraction is 0.05%-0.8%, and the copper content is 0.05%-0.5%.

[0007] A second objective of this invention is to provide a method for preparing the chitosan-glycine-copper chelate described above, wherein the method comprises: As a preferred embodiment of the present invention, the preparation method specifically includes the following steps: Step 1. Take chitosan powder, add 1%-5% glacial acetic acid solution, and stir at 200-300 rpm for 0.2-1 hour at 20-50℃ until the chitosan is completely dissolved to form a transparent chitosan glacial acetic acid solution; Step 2. Weigh glycine and slowly add it to the transparent chitosan-glacial acetic acid solution from Step 1. Maintain a 20-50°C water bath and stir at 200-500 rpm for 0.2-4 hours. Then adjust the pH to 4.5-7.5 with 0.1-1 mol / L sodium hydroxide. Step 3. At 20-50℃, add the copper source in steps and dropwise to make the copper content 0.05%-0.5%, and continuously stir the system for 0.2-4 h to allow copper ions to gradually enter the coordination environment formed by chitosan-glycine, and be fully dispersed in the system and participate in the coordination reaction.

[0008] As a preferred embodiment of the present invention, the copper content in step 3 is 0.2%, and the pH of the system is adjusted during the reaction to maintain it within the range of 5.5-6.0.

[0009] As a preferred embodiment of the present invention, the copper source is selected from copper ions or copper-containing compounds or a combination of both, and the copper-containing compounds include inorganic copper salts, organic copper salts, copper complexes, and copper nanomaterials.

[0010] As a further preferred embodiment of the present invention, the inorganic copper salt is selected from any one or a mixture of two or more of copper sulfate, copper chloride, copper oxide or copper hydroxide, the organic copper salt is selected from any one or a mixture of two or more of copper acetate or copper gluconate, and the copper complex is selected from any one or a mixture of two or more of copper chlorophyll, copper pyrrolidone carboxylate or copper aspartate.

[0011] This invention provides the first preparation of a chitosan-glycine-copper chelate and discloses for the first time the effect of the chitosan-glycine-copper chelate on the in vitro activity of tyrosinase. Experiments have demonstrated that the chitosan-glycine-copper chelate activates tyrosinase and promotes melanin formation. Clinical trials have shown that it can significantly reduce the area of ​​vitiligo or leukoderma. Therefore, the chitosan-glycine-copper chelate provided by this invention can be used in the preparation of drugs for treating vitiligo or leukoderma.

[0012] The present invention also provides a drug for treating vitiligo, wherein the active ingredient in the drug is chitosan-glycine-copper chelate.

[0013] As a preferred embodiment of the present invention, the dosage form of the drug is a gel or ointment.

[0014] Advantages and beneficial effects of the present invention: (1) This invention first constructs a chitosan-glycine complex system, then adds a copper source step by step, and forms a stable ternary coordination structure by reasonably controlling the pH. This structure can stably complex copper ions, prevent local high-concentration precipitation, and improve the solubility stability of copper ions, thus preparing a chitosan-glycine-copper chelate that can continuously and significantly activate tyrosinase activity.

[0015] (2) The chitosan-glycine-copper chelate prepared in this invention achieves the slow release and stable supply of copper ions through the chelation structure, making it easier for copper ions to participate in the activation process of the catalytic center of tyrosinase. It can show a significant activation effect on tyrosinase under in vitro conditions, which is beneficial to promoting the conversion of tyrosine to dopa and dopaquinone, thereby promoting the formation of melanin and showing biological regulatory characteristics superior to free copper ions.

[0016] (3) Through comparative experiments, the present invention found that, compared with control systems using chitosan, amino acids or copper sulfate alone, the chitosan-glycine-copper chelate exhibited a more significant and stable activation effect in the in vitro activity test of tyrosinase, indicating that the structure formed by chitosan-glycine synergistic chelation of copper ions has obvious advantages in functional performance.

[0017] (4) The present invention further verified the effect of the chitosan-glycine-copper chelate through clinical trials. The results showed that it can be applied to the skin affected by vitiligo and white spots, and can significantly reduce the area of ​​white spots or white spots, and has a significant improvement effect. In addition, the stable coordination network formed by chitosan and glycine can control the release rate of copper ions, reduce the peak concentration of free copper ions, reduce the risk of irritation, and improve the safety of local use. Attached Figure Description

[0018] Figure 1 A comparative diagram showing the effects of chitosan-glycine-copper chelate and other non-chelates on tyrosinase activity; Figure 2 This is a before-and-after comparison of vitiligo treated with a gel for three months in clinical practice. Figure 1 ; Figure 3 This is a before-and-after comparison of vitiligo treated with a gel for three months in clinical practice. Figure 2 ; Figure 4 This is a before-and-after comparison of vitiligo treated with ointment for three months in clinical practice. Figure 1 . Detailed Implementation

[0019] This invention is not limited to the following embodiments, and specific implementation methods can be determined according to the technical solutions and actual conditions of this invention. Unless otherwise specified, all chemical reagents and chemical products mentioned in this invention are well-known and commonly used chemical reagents and chemical products in the prior art; unless otherwise specified, all percentages in this invention are mass percentages; unless otherwise specified, all solutions in this invention are aqueous solutions with water as the solvent; room temperature in this invention generally refers to a temperature between 15°C and 25°C, generally defined as 25°C.

[0020] This invention places chitosan in a weakly acidic aqueous system and, under stirring, allows it to fully dissolve and form a homogeneous transparent or semi-transparent solution. Glycine is then added, and the mixture is further stirred to ensure thorough mixing of chitosan and glycine, constructing an organic ligand environment (composite system) containing multiple coordinating groups. Based on this composite system, a copper source is introduced. The copper source is selected from copper ions, copper-containing compounds, or a combination of both. The copper-containing compounds include inorganic copper salts, organic copper salts, copper complexes, and copper nanomaterials. Inorganic copper salts include, but are not limited to, copper sulfate, copper chloride, copper oxide, and copper hydroxide. Organic copper salts include, but are not limited to, copper acetate and copper gluconate. Copper complexes include, but are not limited to, copper chlorophyll, copper PCA (copper pyrrolidone carboxylate), and copper aspartate, preferably copper sulfate. This allows copper ions to be fully dispersed in the system and gradually participate in the coordination reaction, ultimately preparing a water-soluble chitosan-glycine-copper chelate. Using L-tyrosine as a substrate and mushroom tyrosinase as a model enzyme, a colorimetric assay system for tyrosinase activity was established in a 96-well plate. This demonstrated that the chitosan-glycine-copper chelate of the present invention activates tyrosinase and promotes melanin formation. Simultaneously, the effects of the chitosan-glycine-copper chelate on the in vitro activity of tyrosinase were compared with those of chitosan, glycine, and copper sulfate alone, demonstrating the unique advantages of the chitosan-glycine-copper chelate. The present invention will be further elaborated below with specific embodiments.

[0021] In this embodiment, the raw materials required for preparing the chitosan-glycine-copper chelate include: chitosan, copper sulfate, glycine, sodium hydroxide, and glacial acetic acid. The specific preparation method is as follows: Step A1. Preparation of chitosan-glacial acetic acid solution: Take chitosan powder, add a 1%-5% glacial acetic acid solution, and stir at 200-300 rpm for 0.2-1 hour at 20-50℃ until the chitosan is completely dissolved to form a transparent chitosan glacial acetic acid solution; Specifically, in this embodiment, 0.6g of chitosan powder was added to 350g of 1.45% glacial acetic acid solution, and stirred at 200-300 rpm for 40 minutes at 30°C until the chitosan was completely dissolved.

[0022] It should be noted that in this embodiment, the chitosan is chitosan or its derivatives with a degree of deacetylation of 50%-99%, including but not limited to carboxymethyl chitosan, quaternized chitosan, and low molecular weight chitosan.

[0023] Step A2. Preparation of chitosan-glycine solution: Weigh out glycine and slowly add it to the transparent chitosan-glacial acetic acid solution from step A1. Maintain a water bath at 20-50°C and stir at 200-500 rpm for 0.2-4 hours. Then adjust the pH to 4.5-7.5 with 0.1-1 mol / L sodium hydroxide.

[0024] Specifically, in this embodiment, 0.36g of glycine was weighed, dissolved in an appropriate amount of water, and slowly added to the transparent chitosan glacial acetic acid solution in step A1. The process was carried out in a 30°C water bath, and stirred at 200-500 rpm for 2 hours. After that, the pH was adjusted to 6.0.

[0025] Step A3. Preparation of chitosan-glycine-copper chelate: Copper sulfate, copper chloride, copper gluconate, copper aspartate, or copper PCA are added in steps and slowly dropwise at 20-50℃ to achieve a final copper content of 0.2%. The system is continuously stirred and reacted for 0.2–4 h to allow copper ions to gradually enter the coordination environment formed by chitosan-glycine, fully disperse in the system, and participate in the coordination reaction.

[0026] Specifically, in this embodiment, copper sulfate was added stepwise and slowly at 30°C to achieve a final copper content of 0.2%, and the system was continuously stirred for 2 hours.

[0027] Furthermore, in this embodiment, the pH of the system is controlled by adding 0.1–1 mol / L sodium hydroxide as a regulator during the reaction process, maintaining it within the range of 5.5–6.0, so as to promote the full chelation reaction between metal ions and organic ligands and form a structurally stable chitosan-glycine-copper chelate.

[0028] The chitosan-glycine-copper chelate prepared in this embodiment is uniformly dispersed in the aqueous system, and no obvious precipitation or exudation was observed. It is stable after standing for 24 hours, indicating that the copper ions exist in a stable chelated state. Moreover, this technical solution has good adaptability to different copper sources.

[0029] Referring to the above method, by controlling the amounts of chitosan, glycine, and copper sulfate, this invention also prepared a chitosan-glycine-copper chelate with a copper content of 0.2%, a chitosan mass fraction of 0.12%, and a glycine mass fraction of 0.05%. The above embodiments are not intended to limit this invention. This invention can also adjust the mass fractions of chitosan and glycine to control the chitosan mass fraction to 0.1%-2%, the glycine mass fraction to 0.05%-0.8%, and the copper content to 0.05%-0.5%.

[0030] Before preparing the structurally stable chitosan-glycine-copper chelate, other methods were also explored. Through multiple rounds of comparison and optimization of process parameters, the following key technical points that significantly affect the formation of the chelation system were summarized: (1) Before introducing the copper source, it is necessary to ensure that chitosan is fully dissolved under weakly acidic conditions and forms a highly homogeneous complex system with amino acids (glycine). In a weakly acidic to near-neutral pH environment, the free amino groups (-NH2) on the chitosan molecular backbone react with the amino groups (-NH2) and carboxyl groups (-COO) in the amino acid molecules. - It can work together as a coordination site to provide a stable multi-coordination group environment for the subsequent complexation of copper ions.

[0031] (2) The method of adding the copper source has a significant impact on the formation of the chelate structure. It is preferable to introduce the copper source in a stepwise, slow, dropwise manner, so that Cu... 2+ It can gradually participate in the complexation reaction in the coordination environment constructed by chitosan and amino acids, thereby avoiding non-specific precipitation or side reactions caused by the instantaneous high concentration of local copper ions.

[0032] (3) Precise control of the pH of the reaction system is one of the key factors in obtaining a stable chelate structure. Experimental results show that when the pH of the system is controlled within the range of approximately 5.5–6.0, chitosan can be maintained in a basically dissolved or semi-dissolved state, and the deprotonation of amino acid carboxyl groups is also beneficial, thereby enhancing its affinity for Cu. 2+ Its coordination ability significantly improves the stability of the chelation system.

[0033] To verify the effect of the chitosan-glycine-copper chelate prepared in this embodiment on tyrosinase activity, this invention uses an in vitro tyrosinase activity testing model to compare the effects of the prepared chitosan-glycine-copper chelate with solutions of chitosan, glycine, and copper sulfate on tyrosinase activity. The specific experimental procedure is as follows: I. Experimental Procedure: Step B1. Prepare PBS (0.2 M, pH 6.8): (1) Prepare a NaH2PO4·2H2O stock solution, for example: weigh an appropriate amount of NaH2PO4·2H2O and dissolve it in water to prepare a 0.2 M solution.

[0034] (2) Prepare Na2HPO4·12H2O stock solution (0.2 M).

[0035] (3) Mix the two stock solutions in a certain volume ratio and monitor the pH in real time, and adjust it to pH 6.8 (a small amount of HCl or NaOH can be used for fine adjustment).

[0036] (4) Dilute to 0.05 M PBS as needed for preparing enzyme solutions, substrate solutions, etc.

[0037] Step B2. Preparation of L-tyrosine solution (1 mg / mL): (1) Weigh 100 mg of L-tyrosine, add a small amount of water, and dissolve by sonication or gentle heating.

[0038] (2) After dissolving, dilute with water to 100 mL to obtain a 1 mg / mL L-tyrosine solution.

[0039] (3) Prepare and use immediately or store at 4 ℃ for a short period away from light.

[0040] Step B3. Preparation of tyrosinase solution: (1) Weigh a certain amount of tyrosinase powder, dissolve it in 0.05 M PBS, and prepare an enzyme stock solution of 1 mg / mL.

[0041] (2) Dilute to 0.01 mg / mL working solution as needed before use.

[0042] (3) Keep on ice throughout the process to reduce activity loss.

[0043] It should be noted that the specific concentrations in the above experiments can be optimized through preliminary experiments. If the OD... 475 If the increase is too rapid or saturation occurs within a short time, the enzyme concentration should be reduced; if the change is too small, the enzyme concentration should be appropriately increased or the measurement time extended. Therefore, an enzyme that shows good linear growth within 5-10 minutes and has a high OD value should be selected. 475 Unsaturated enzyme concentration is used as a formal experimental condition. This ensures the reaction is in the initial rate phase, preventing substrate depletion or enzyme inactivation that could cause curve distortion.

[0044] Step B4. Sample solution: Dilute the samples to different concentrations with PBS; Step 5.96: Enzyme activity assay in 6-well plates (using 180 µL per well as an example): (1) Grouping and control settings: Different concentration gradients were set for each sample (chitosan, glycine, copper sulfate, chitosan-glycine-copper chelate, etc.), and the following were set: control wells containing only enzyme and no sample (A1 type); system blank wells containing neither enzyme nor sample (A2 type); and sample blank wells containing only sample and no enzyme (A4 type).

[0045] (2) Sampling order: The total reaction volume per well is 180 µL, and the composition is as follows: Add 50 µL of sample solution. Add an equal volume of PBS (without sample) to the control wells.

[0046] Add 30 µL of tyrosinase working solution. Do not add enzyme to the sample blank wells (A4), but instead add the same volume of PBS. Gently vortex the 96-well plate to mix, and pre-incubate at the set temperature for 5 min (e.g., 25–30 °C).

[0047] Add 100 µL of L-tyrosine substrate solution. The total volume per well is now 180 µL. After adding the substrate, immediately tap the plate to mix thoroughly and quickly place it into the microplate reader.

[0048] (3) Microplate reader readings: Wavelength setting: 475 nm; Reading mode: Kinetic; Reading interval: once every 1 min; Total time: set to 10–20 min, depending on the linear range of the pre-experimental curve. The microplate reader automatically records the OD of each well. 475 Changes over time.

[0049] II. Data Processing: OD Definitions: A1: Enzyme + Substrate, no sample (standard control); A2: No enzyme, no sample, only system background (PBS + substrate); A3: Sample + Enzyme + Substrate (experimental group); A4: Sample + Substrate, no enzyme (sample background).

[0050] Background subtraction: 1. Standard line: Astandard(t) = A1(t) - A2(t); where A1(t) and A2(t) represent the absorbance of the enzyme-containing reaction system and the absorbance of the enzyme-free blank control, respectively; 2. Sample line: Asample(t) = A3(t) - A4(t); where A3(t) and A4(t) represent the absorbance of the enzyme-containing sample group and the absorbance of the enzyme-free control group, respectively. The above calculations are performed at each time point to obtain the net reaction curve after subtracting the background and sample self-absorption.

[0051] Experimental results: like Figure 1As shown, chitosan alone significantly inhibits tyrosinase activity; copper sulfate alone exhibits weak promotion, with activity continuously decreasing; glycine alone, while relatively stable, shows weak promotion; a simple mixture of chitosan and glycine also shows strong inhibition; a mixture of copper sulfate and glycine shows weak promotion, with overall activity far lower than that of the chelate; although a simple mixture of chitosan and copper ions (copper sulfate) shows promotion, with short-term effects approaching those of the chelate, long-term activity decreases and stability is insufficient; only when the three are chemically combined to form a stable chelate (chitosan-glycine-copper chelate) can it exhibit a strong promoting activity completely different from all other substances (including simple mixtures of their components), and can continuously and significantly activate tyrosinase activity (significantly improving the catalytic activity of tyrosinase), showing better stability and reproducibility.

[0052] In the chitosan-glycine-copper chelate system of this invention, the amino groups on the chitosan backbone synergistically participate in the multi-site coordination of copper ions with the amino and carboxyl groups in the glycine molecule, enabling the copper ions to exist stably in a non-free state. This chelate structure reduces the irritation of free copper ions while providing a suitable metal cofactor microenvironment for tyrosinase, thereby enhancing its catalytic function and promoting melanin formation.

[0053] Example 2: Preparation of Topical Ointment Formulation Ointment formulation composition: 20% chitosan-glycine-copper chelate solution (copper 0.2%) from Example 1, 6% xanthan gum, 1% trehalose, 10% emulsifier, and purified water to make up to 100%. Mix evenly in a 70°C water bath and cool to form an ointment.

[0054] Example 3: Preparation of topical gel formulation The gel formulation consists of: 30% chitosan-glycine-copper chelate solution (copper 0.2%) from Example 1, 1% xanthan gum, 10% glycerol, purified water to 100%, and triethanolamine to adjust the pH to 6.5 to form a gel.

[0055] External application observation: Fifty patients with vitiligo or leukoderma were selected and treated with topical application twice daily (25 cases used ointment, 25 cases used gel) for 12 consecutive weeks. Observations included changes in affected area, pigmentation changes, and local irritation reactions.

[0056] Results: Comparison of before-and-after photos of the affected areas showed increased pigmentation and a significant reduction in the area of ​​vitiligo in all 50 patients. Some patients recovered completely, and no significant adverse irritation reactions were observed. (See details...) Figures 2 to 4 .

[0057] The above technical features constitute the embodiments of the present invention, which have strong adaptability and implementation effect. Unnecessary technical features can be added or removed according to actual needs to meet the needs of different situations.

Claims

1. A chitosan-glycine-copper chelate, characterized in that, In the preparation of the chitosan-glycine-copper chelate, chitosan is first dissolved, then mixed with glycine to construct an organic ligand environment containing multiple coordination groups. A copper source is then introduced to carry out a chemical reaction, and finally the chitosan-glycine-copper chelate is obtained.

2. The chitosan-glycine-copper chelate according to claim 1, characterized in that, In the chitosan-glycine-copper chelate, the chitosan mass fraction is 0.1%-2%, and the chitosan is chitosan or its derivatives with a degree of deacetylation of 50%-99%, including carboxymethyl chitosan, quaternized chitosan, and low molecular weight chitosan; the glycine mass fraction is 0.05%-0.8%, and the copper content is 0.05%-0.5%.

3. The method for preparing the chitosan-glycine-copper chelate according to claim 1 or 2, characterized in that, The preparation method is as follows: first, chitosan is placed in an acidic aqueous system and fully dissolved under stirring to form a homogeneous transparent or semi-transparent solution. Then, glycine is added and the mixture is further processed under stirring to ensure that the chitosan and glycine are fully mixed and to construct an organic ligand environment containing multiple coordination groups. After that, a copper source is introduced to carry out a chemical reaction, and finally, a chitosan-glycine-copper chelate is obtained.

4. The method for preparing the chitosan-glycine-copper chelate according to claim 3, characterized in that, The preparation method specifically includes the following steps: Step 1. Take chitosan powder, add 1%-5% glacial acetic acid solution, and stir at 200-300 rpm for 0.2-1 hour at 20-50℃ until the chitosan is completely dissolved to form a transparent chitosan glacial acetic acid solution; Step 2. Weigh glycine and slowly add it to the transparent chitosan-glacial acetic acid solution from Step 1. Maintain a 20-50°C water bath and stir at 200-500 rpm for 0.2-4 hours. Then adjust the pH to 4.5-7.5 with 0.1-1 mol / L sodium hydroxide. Step 3. At 20-50℃, add the copper source in steps and dropwise to make the copper content 0.05%-0.5%, and continuously stir the system for 0.2-4 h to allow copper ions to gradually enter the coordination environment formed by chitosan-glycine, and be fully dispersed in the system and participate in the coordination reaction.

5. The method for preparing the chitosan-glycine-copper chelate according to claim 4, characterized in that, In step 3, the copper content is 0.2%, and the pH of the system is adjusted during the reaction to maintain it within the range of 5.5-6.

0.

6. The method for preparing the chitosan-glycine-copper chelate according to claim 4, characterized in that, The copper source is selected from copper ions or copper-containing compounds or a combination of both, and the copper-containing compounds include inorganic copper salts, organic copper salts, copper complexes, and copper nanomaterials.

7. The method for preparing the chitosan-glycine-copper chelate according to claim 6, characterized in that, The inorganic copper salt is selected from any one or a mixture of two or more of copper sulfate, copper chloride, copper oxide, or copper hydroxide; the organic copper salt is selected from any one or a mixture of two or more of copper acetate or copper gluconate; and the copper complex is selected from any one or a mixture of two or more of chlorophyll copper, pyrrolidone carboxylic acid copper, or aspartic acid copper.

8. The chitosan-glycine-copper chelate prepared by any one of the preparation methods of claims 4 to 7 is used in the preparation of a medicament for treating vitiligo or leukoderma.

9. A drug for treating vitiligo or leukoderma, characterized in that, The active ingredient in the drug is the chitosan-glycine-copper chelate prepared by any one of the preparation methods described in claims 4 to 7.

10. The medicament for treating leukoderma or vitiligo according to claim 9, characterized in that, The drug is in the form of a gel or ointment.

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