Copper-cleaning cellulose, preparation method thereof and application of copper-cleaning cellulose in preparation of oral medicine for treating hepatolenticular degeneration

Abstract

The invention provides copper-purifying cellulose, a preparation method thereof and application of the copper-purifying cellulose in preparation of oral medicines for treating hepatolenticular degeneration, and belongs to the technical field of biological medicines. According to the invention, microcrystalline cellulose with good biocompatibility is used as a carrier material, and a functional group is grafted on microcrystalline cellulose through a chemical modification method, so that the affinity of pure copper cellulose to copper ions is greatly improved, and efficient removal of copper ions can be realized. The pure copper cellulose prepared by the invention is suitable for oral maintenance treatment and reduction or removal of redundant copper ions deposited in the body of a hepatolenticular degeneration patient.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to pure copper cellulose, a preparation method thereof, and application thereof in preparing an oral drug for treating hepatolenticular degeneration. Background Art

[0002] Wilson's disease, also known as Wilson's disease, is an autosomal recessive copper metabolism disorder. Wilson's disease is one of the few treatable neurogenetic diseases. Timely diagnosis and active treatment can effectively prevent the development of the disease. Its related gene ATP7B encodes a copper-transporting P-type ATPase (ATP7B protein), which participates in the synthesis of ceruloplasmin and promotes the excretion of bile copper. Defects in the ATP7B gene cause biliary copper excretion disorders, and a large amount of copper is deposited in organs such as the liver, brain, and cornea. Patients experience liver damage, neuropsychiatric symptoms, corneal pigment rings, and other manifestations. Therefore, reducing the accumulation of copper in the body is an effective strategy for treating Wilson's disease.

[0003] Clinically, traditional chelating agents (penicillamine, trientine, etc.), new chelating agents (ammonium tetrathiomolybdate) and zinc preparations (zinc gluconate, etc.) are usually used to treat Wilson's disease. Traditional chelating agents increase urinary copper excretion by complexing copper deposits in the liver. However, the released free copper may enter the brain, causing worsening neuropsychiatric symptoms (such as dystonia, mental and behavioral abnormalities), and long-term use often has adverse reactions (such as nephrotoxicity). Ammonium tetrathiomolybdate is a potent copper excretion drug that can form a stable complex with copper and protein to reduce the body's absorption of copper; it can replace copper on metallothionein and reduce copper deposition in organs; it can cross the blood-brain barrier to exert its effects, and the risk of worsening neurological symptoms is low. However, ammonium tetrathiomolybdate has not yet officially entered the clinic and cannot be used for maintenance treatment because molybdenum is retained in the liver, spleen and bone marrow. Zinc preparations can promote the synthesis of metallothionein by intestinal mucosal cells, bind dietary copper and endogenous copper, and excrete through the intestines after shedding. Zinc preparations have definite efficacy and few adverse reactions, but they are slow to take effect and require a long time (4 to 6 months) to produce a negative balance of copper metabolism in the body. At present, Wilson's disease still requires lifelong maintenance treatment, and interruption of treatment may cause severe liver damage, irreversible aggravation of neuropsychiatric symptoms, and even death. Therefore, the current treatment for Wilson's disease is mostly limited to copper chelators and zinc preparations, but zinc preparations are slow to take effect, and copper chelators are prone to aggravate neuropsychiatric symptoms due to the redistribution of copper. Finding a new copper removal strategy with fast onset, excellent effect, and few adverse reactions for long-term use has good application prospects in the clinical treatment of Wilson's disease.

[0004] With the improvement of the "gastrointestinal dialysis" theory, oral adsorbents have been used in the treatment of acute drug poisoning, chronic kidney disease, etc., such as hyperkalemia and potassium-lowering resin (polystyrene sulfonate sodium powder). Its mechanism of action is to bind to pathogenic factors in the gastrointestinal tract through adsorption and inhibit their absorption; maintain a relative concentration gradient so that pathogenic factors continue to enter the intestine from the blood; some pathogenic factors are excreted through the hepatoenterocirculation, and oral adsorbents can effectively inhibit their reabsorption. However, the application of copper ion oral adsorbents in the intervention study of hepatolenticular degeneration has not been reported. Summary of the invention

[0005] The purpose of the present invention is to provide a pure copper cellulose and a preparation method thereof and an application of the pure copper cellulose in the preparation of an oral drug for treating Wilson's disease. The pure copper cellulose has the characteristics of large copper ion adsorption capacity and good biocompatibility, and thus has a fast onset of action, excellent effect, and few adverse reactions after long-term use. The pure copper cellulose can treat Wilson's disease by regulating the negative balance of copper metabolism, and can be used for oral treatment of Wilson's disease.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a method for preparing pure copper cellulose, comprising the following steps:

[0008] Mixing microcrystalline cellulose, a first dispersant and a modifying agent to chemically modify the microcrystalline cellulose to obtain modified microcrystalline cellulose; the modifying agent includes epichlorohydrin or sodium periodate;

[0009] The modified microcrystalline cellulose, the second dispersant and the amination agent are mixed and subjected to amination modification to obtain pure copper cellulose.

[0010] Preferably, after the amination modification, the method further comprises: mixing the amination-modified cellulose obtained by the amination modification, a third dispersant and a silane coupling agent, and performing coupling modification under alkaline conditions to obtain pure copper cellulose.

[0011] Preferably, when the modification agent is epichlorohydrin, it also includes adding alkali solution; the first dispersant is dimethyl sulfoxide; the solid-liquid ratio of the microcrystalline cellulose to the first dispersant is 4g:35-50mL; the dosage ratio of the microcrystalline cellulose to the modification agent is 4g:25-35mL; the temperature of the chemical modification is 40-60°C, and the time is 6-8h.

[0012] Preferably, when the modification reagent is sodium periodate, the first dispersant is water; the solid-liquid ratio of the microcrystalline cellulose to the first dispersant is 1g:80-150mL; the mass ratio of the microcrystalline cellulose to the modification reagent is 1:1-2; the temperature of the chemical modification is 45-55°C, and the time is 6-8h.

[0013] Preferably, the amination agent includes polyethyleneimine, triethylenetetramine, ethylenediamine or methylamine; the second dispersant includes water or anhydrous ethanol; and the dosage ratio of the modified microcrystalline cellulose to the second dispersant is 1 g:25-50 mL.

[0014] Preferably, the mass ratio of the modified microcrystalline cellulose to the amination agent is 1:0.5-2; the temperature of the amination modification is 50-60° C., and the time is 5-6 hours.

[0015] Preferably, the silane coupling agent includes KH-580 or KH-590; and the third dispersant is anhydrous ethanol.

[0016] Preferably, the mass ratio of the amino cellulose to the silane coupling agent is 1:0.25-0.45; the dosage ratio of the amino cellulose to the third dispersant is 1g:60-80mL; the coupling modification temperature is 50-60°C, and the time is 2-3h.

[0017] The present invention provides pure copper cellulose prepared by the preparation method described in the above technical scheme.

[0018] The present invention provides the use of the pure copper cellulose described in the above technical solution in the preparation of an oral drug for treating Wilson's disease.

[0019] The present invention provides a method for preparing pure copper cellulose, which uses microcrystalline cellulose with good biocompatibility as a carrier material, and grafts functional groups (such as one or two of amino and thiol groups) on the microcrystalline cellulose through a chemical modification method, which greatly improves the affinity of pure copper cellulose for copper ions and can achieve efficient removal of copper ions. Based on the theory of gastrointestinal dialysis, pure copper cellulose can treat hepatolenticular degeneration by regulating the negative balance of copper metabolism. Oral pure copper cellulose treatment can treat hepatolenticular degeneration by regulating the body's copper metabolism; it can effectively reduce the absorption of dietary copper and bile copper; through the exchange of substances between the rich capillaries in the intestine, endogenous copper is removed to achieve the body's negative balance of copper metabolism. This negative balance of copper metabolism is slow and dynamic, and avoids the deterioration of neuropsychiatric symptoms caused by a sharp increase in plasma copper. Therefore, the pure copper cellulose prepared by the present invention is suitable for oral maintenance treatment to reduce or remove excess copper ions deposited in patients with hepatolenticular degeneration.

[0020] In the clean copper cellulose prepared by the present invention, abundant functional groups are modified on the microcrystalline cellulose carrier, and the copper cellulose is almost insoluble in water and dilute acid solution, but can be dispersed in water.

[0021] The preparation process of the clean copper cellulose provided by the present invention is simple, and the epoxy group can be modified on the microcrystalline cellulose carrier by an epichlorohydrin reaction, or the aldehyde group can be modified on the microcrystalline cellulose carrier by a sodium periodate reaction, polyethyleneimine is added to the reaction system, and the amino group is modified on the microcrystalline cellulose by the reaction of the amino group with the epoxy group or the aldehyde group.

[0022] Furthermore, the present invention can modify the thiol groups on the microcrystalline cellulose using a silane coupling agent on the basis of the amino-treated pure copper cellulose to obtain bifunctional pure copper cellulose.

[0023] The preparation method of the copper-free cellulose provided by the present invention is simple, has low manufacturing cost, is non-toxic and has no side effects, and can regulate the body's copper metabolism through material exchange in the intestine, thereby treating Wilson's disease.

[0024] The microcrystalline cellulose of the present invention is milky white amorphous particles, that is, the particle size is 75-150 μm (100-200 mesh), and the specific surface area is about 35 m 2 After chemical modification of one or both of the amino and thiol functional groups, the resulting pure copper cellulose is still milky white amorphous particles, but the particle size is significantly reduced to 10-100 μm, and the specific surface area is about 58 m 2 / g.

[0025] The present invention selects microcrystalline cellulose as the carrier material. The microcrystalline cellulose is produced by acid hydrolysis of natural cellulose, has abundant yield and good biocompatibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The following are scanning electron microscope images of raw material microcrystalline cellulose, where A is a 50 μm electron microscope image and B is a 4 μm electron microscope image;

[0027] Figure 2 The scanning electron microscope images of the bifunctional clean copper cellulose prepared in Example 2, wherein A is a 50 μm electron microscope image and B is a 4 μm electron microscope image;

[0028] Figure 3 This is the XPS graph of the bifunctional clean copper cellulose prepared in Example 2, where A represents N1s and B represents S2p;

[0029] Figure 4 The static adsorption results of copper ions in vitro by the microcrystalline cellulose raw material and the pure copper cellulose prepared from Examples 1 to 3 and Comparative Examples 1 to 2. DETAILED DESCRIPTION

[0030] In the present invention, unless otherwise specified, the required raw materials or reagents are commercially available products well known to those skilled in the art.

[0031] The present invention provides a method for preparing pure copper cellulose, comprising the following steps:

[0032] Mixing microcrystalline cellulose, a first dispersant and a modifying agent to chemically modify the microcrystalline cellulose to obtain modified microcrystalline cellulose; the modifying agent includes epichlorohydrin or sodium periodate;

[0033] The modified microcrystalline cellulose, the second dispersant and the amination agent are mixed and subjected to amination modification to obtain pure copper cellulose.

[0034] The invention mixes microcrystalline cellulose, a first dispersant and a modifying agent, and performs chemical modification to obtain modified microcrystalline cellulose; the modifying agent comprises epichlorohydrin or sodium periodate.

[0035] In the present invention, when the modifying agent is epichlorohydrin, it is preferred that an alkali solution is also added; the alkali solution is preferably a 3 mol / L NaOH aqueous solution; the dosage ratio of the microcrystalline cellulose to the alkali solution is preferably 4 g: 4-10 mL, more preferably 4 g: 4-5 mL.

[0036] In the present invention, the first dispersant is preferably dimethyl sulfoxide; the solid-liquid ratio of the microcrystalline cellulose to the first dispersant is 4g:35-50mL, more preferably 4g:40-50mL; the dosage ratio of the microcrystalline cellulose to the modification agent is preferably 4g:25-35mL, more preferably 4g:25-30mL; the temperature of the chemical modification is preferably 40-60°C, more preferably 45-50°C, and the time is preferably 6-8h, more preferably 6h.

[0037] The present invention preferably adds microcrystalline cellulose to the first dispersant, swells for 20 to 30 minutes under the chemical modification temperature, adds epichlorohydrin and NaOH aqueous solution, magnetically stirs for chemical modification, centrifuges, and washes to obtain epoxidized microcrystalline cellulose. The washing is preferably performed three times with distilled water and ethanol alternately; the present invention has no special limitation on the centrifugal separation, and it can be performed according to the process well known in the art.

[0038] In the present invention, when the modification agent is sodium periodate, the first dispersant is preferably water; the solid-liquid ratio of the microcrystalline cellulose to the first dispersant is preferably 1g:80-150mL, more preferably 1g:80-100mL; the mass ratio of the microcrystalline cellulose to the modification agent is preferably 1:1-2, more preferably 1:1.3-1.5; the temperature of the chemical modification is preferably 45-55°C, more preferably 45-50°C, and the time is preferably 6-8h, more preferably 6h.

[0039] The present invention preferably disperses microcrystalline cellulose in distilled water by ultrasonication for 30 minutes, adds sodium periodate, and performs chemical modification by magnetic stirring in a light-proof environment. After the reaction is completed, ethylene glycol is added to neutralize the unreacted sodium periodate to obtain aldehyde-modified microcrystalline cellulose. The present invention has no particular limitation on the amount of ethylene glycol used, and complete neutralization can be achieved according to requirements.

[0040] After obtaining the modified microcrystalline cellulose, the present invention mixes the modified microcrystalline cellulose, a second dispersant and an amination agent to perform amination modification to obtain pure copper cellulose.

[0041] In the present invention, the aminating agent preferably includes polyethyleneimine, triethylenetetramine, ethylenediamine or methylamine, and more preferably polyethyleneimine; the second dispersant preferably includes water or anhydrous ethanol; the dosage ratio of the modified microcrystalline cellulose to the second dispersant is preferably 1g:25-50mL, and more preferably 1g:30-50mL.

[0042] In the present invention, the mass ratio of the modified microcrystalline cellulose to the amination agent is preferably 1:0.5-2, more preferably 1:1-2; the temperature of the amination modification is preferably 50-60°C, more preferably 50-55°C, and the time is preferably 5-6h, more preferably 5.5-6h.

[0043] In the present invention, the modified microcrystalline cellulose is preferably ultrasonically dispersed in the second dispersant for 10 minutes, an amination agent is added, and the amination modification is performed by magnetic stirring, and the amination modification is performed by centrifugation and washing to obtain the amination cellulose, i.e., the amination clean copper cellulose. The washing is preferably performed three times by alternating distilled water and ethanol; the centrifugation is not particularly limited in the present invention, and can be performed according to a process well known in the art.

[0044] As a further preferred embodiment of the present invention, after the amination modification, the process further comprises: mixing the amination-modified cellulose obtained by the amination modification, a third dispersant and a silane coupling agent, and performing coupling modification under alkaline conditions to obtain pure copper cellulose.

[0045] In the present invention, the silane coupling agent preferably includes KH-580 (3-mercaptopropyltriethoxysilane) or KH-590 (3-mercaptopropyltrimethoxysilane); and the third dispersant is preferably anhydrous ethanol.

[0046] In the present invention, the mass ratio of the amino cellulose to the silane coupling agent is preferably 1:0.25-0.45, more preferably 1:0.3; the amount ratio of the amino cellulose to the third dispersant is preferably 1g:60-80mL, more preferably 1g:60-70mL.

[0047] In the present invention, the reagent used to provide the alkaline condition is preferably ammonia water, and the mass concentration of the ammonia water is preferably 25%; the mass ratio of the ammonia water to the silane coupling agent is preferably 0.2-0.35:0.3, more preferably 0.25:0.3.

[0048] In the present invention, the coupling modification temperature is preferably 50-60° C., more preferably 55-60° C., and the time is preferably 2-3 h, more preferably 2 h.

[0049] The present invention preferably disperses the amino cellulose in a third dispersant by ultrasonic for 30 minutes, adds a silane coupling agent, stirs magnetically to dissolve, then drops ammonia water, stirs magnetically under alkaline conditions to carry out coupling modification, centrifuges, and washes to obtain bifunctionalized clean copper cellulose. The washing is preferably carried out three times by alternating distilled water and ethanol; the present invention has no special limitation on the centrifugal separation, and it can be carried out according to a process well known in the art.

[0050] The present invention provides pure copper cellulose prepared by the preparation method described in the above technical scheme.

[0051] The present invention provides the use of the pure copper cellulose in the above technical solution in the preparation of an oral drug for treating Wilson's disease. The present invention preferably prepares the pure copper cellulose into a gastric soluble capsule by conventional process, and releases the capsule under the action of gastric acid to exert its effect.

[0052] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0053] Example 1

[0054] Weigh 4 g of dry microcrystalline cellulose into a 100 mL three-necked round-bottom flask, add 40 mL of dimethyl sulfoxide, swell under magnetic stirring at 40°C for 30 min, add 25 mL of epichlorohydrin and 4 mL of 3 mol / L NaOH solution, and stir magnetically at 40°C for 6 h. Separate the product after the reaction by centrifugation at 4000 rpm for 10 min, wash it alternately with distilled water and ethanol three times to obtain epoxidized microcrystalline cellulose, and store it at 4°C for later use;

[0055] 2 g of epoxidized microcrystalline cellulose was ultrasonically treated in 100 mL of distilled water for 10 min, 2 g of polyethyleneimine was added, and magnetic stirring was performed at 60° C. for 6 h. The product after the reaction was separated by centrifugation at 4000 rpm for 10 min, and washed alternately with distilled water and ethanol three times to obtain amino-cleaned copper cellulose, which was freeze-dried for later use and numbered MCC-1.

[0056] Comparative Example 1

[0057] Weigh 1 g of dry microcrystalline cellulose into a 100 mL three-necked round-bottom flask, add 70 mL of anhydrous ethanol, and ultrasonically disperse for 30 min. Add 0.3 g of silane coupling agent KH-580 (3-mercaptopropyltriethoxysilane), stir magnetically to dissolve, then drop 0.25 g of ammonia water (mass concentration is 25%), stir magnetically at 60 ° C for 2 h, separate the reaction product by centrifugation at 4000 rpm for 10 min, wash it alternately with distilled water and ethanol three times to obtain thiolated clean copper cellulose, freeze-dried for later use, and numbered MCC-2.

[0058] Comparative Example 2

[0059] The only difference from Comparative Example 1 is that the silane coupling agent used is KH-590 (3-mercaptopropyltrimethoxysilane), and mercaptolated pure copper cellulose is obtained, which is numbered MCC-3.

[0060] Example 2

[0061] Weigh 1 g of dry microcrystalline cellulose in a 200 mL three-necked round-bottom flask, add 100 mL of distilled water, ultrasonically disperse for 30 min, add 1.3 g of sodium periodate, and stir magnetically for 6 h at 50 ° C in a dark environment. After the reaction is completed, add 2 mL of ethylene glycol to neutralize the unreacted sodium periodate, separate the product after the reaction by centrifugation at 4000 rpm for 10 min, wash it three times with distilled water and ethanol alternately, and obtain aldehyde-modified microcrystalline cellulose, which is stored at 4 ° C for future use;

[0062] 1 g of aldehyde-modified microcrystalline cellulose was ultrasonically treated in 50 mL of anhydrous ethanol for 10 min, 2 g of polyethyleneimine was added, and magnetic stirring was performed at 50° C. for 6 h. The product after the reaction was separated by centrifugation at 4000 rpm for 10 min, and washed alternately with distilled water and ethanol three times to obtain amino-modified microcrystalline cellulose, which was freeze-dried for later use;

[0063] Weigh 1 g of dry amino-modified microcrystalline cellulose into a 100 mL three-necked round-bottom flask, add 70 mL of anhydrous ethanol, and ultrasonically disperse for 30 min. Add 0.3 g of silane coupling agent KH-580 (3-mercaptopropyltriethoxysilane), stir magnetically to dissolve, then drop 0.25 g of ammonia water (mass concentration is 25%), stir magnetically at 60 ° C for 2 h, separate the reaction product by centrifugation at 4000 rpm for 10 min, wash it alternately with distilled water and ethanol three times, and freeze-dry it for later use to obtain bifunctional clean copper cellulose, which is numbered MCC-4.

[0064] Example 3

[0065] The only difference from Example 2 is that the silane coupling agent used in this example is KH-590 (3-mercaptopropyltrimethoxysilane), and the bifunctional pure copper cellulose is obtained, which is numbered as MCC-5.

[0066] Structural characterization

[0067] Figure 1 The following are scanning electron microscope images of raw material microcrystalline cellulose, where A is a 50 μm electron microscope image and B is a 4 μm electron microscope image; Figure 2 The scanning electron micrographs of the bifunctional clean copper cellulose prepared in Example 2, wherein A is a 50 μm electron micrograph and B is a 4 μm electron micrograph; Figure 1 and Figure 2 By comparison, it can be seen that the microcrystalline cellulose used in the present invention is an amorphous particle with a mesh size of 100 to 200, that is, a particle size of 75 to 150 μm. After chemical modification of the amino group and the thiol group, the particle size of the bifunctional clean copper cellulose is significantly reduced to 10 to 100 μm. This is because during the chemical modification process, multiple ultrasonic dispersions are used to make the particles of the microcrystalline cellulose smaller. The loading of nano-scale silica particles can be observed on the surface of the bifunctional clean copper cellulose.

[0068] Figure 3 This is the XPS graph of the bifunctional clean copper cellulose prepared in Example 2, where A represents N1s and B represents S2p; Figure 3 The results showed that the bifunctional pure copper cellulose was successfully modified with abundant amino and thiol groups.

[0069] Test Example 1

[0070] Determination of static adsorption of copper ions in aqueous solution in vitro

[0071] Use copper sulfate pentahydrate to prepare a 200 mg / L copper ion aqueous solution (calculated based on the copper ion content).

[0072] Take 40 mg of pure copper cellulose powder of microcrystalline cellulose raw material (MCC-0), MCC-1, MCC-2, MCC-3, MCC-4, and MCC-5 prepared in Examples 1 to 3 and Comparative Examples 1 to 2, respectively, in a 10 mL centrifuge tube, add 5 mL of copper ion aqueous solution, and adsorb at 37 ° C, 160 rpm for 2 hours. Filter and collect the filtrate after adsorption. The copper ion content in the filtrate is reacted by a colorimetric reaction of a bis(cyclohexanone) oxalyl dihydrazone reagent under alkaline conditions, detected by an enzyme reader or a spectrophotometer at a wavelength of 600 nm, and its adsorption rate (%) is calculated.

[0073] Adsorption rate AP = (C0-C1) / C0×100%;

[0074] Adsorption capacity AC = (C0-C1) × V / m;

[0075] Wherein, C0 and C1 are the copper ion concentrations of the solution before adsorption and at adsorption equilibrium, respectively (mg / L); V is the volume of the copper ion aqueous solution (mL); and m is the mass of the adsorbent (i.e., pure copper cellulose powder) used (mg).

[0076] Figure 4 The static adsorption results of copper ions in vitro by the microcrystalline cellulose raw material and the pure copper cellulose prepared by Examples 1 to 3 and Comparative Examples 1 to 2; Figure 4 The results show that the pure copper cellulose of the present invention has good copper ion adsorption performance, among which MCC-4 has the best copper ion adsorption performance, with an adsorption rate of up to 85.52% and an adsorption amount of 21.38 mg / g.

[0077] It is well known in the art that the serum copper content of a normal person is 0.70-1.55 mg / L, which is much lower than the copper ion concentration (200 mg / L) used in the test in Test Example 1. Therefore, the MCC-1, MCC-4 and MCC-5 pure copper cellulose in Examples 1-3 are more effective in preparing oral drugs for treating Wilson's disease.

[0078] Test Example 2

[0079] Maintenance therapy with copper-free cellulose affects copper clearance in mice with hepatolenticular degeneration

[0080] The transgenic mouse model (ATP7B) was purchased from Jackson Laboratory tx-J ), breeding and expansion, to obtain two-month-old male Wilson's disease transgenic mice. Add the MCC-1, MCC-2, MCC-3, MCC-4 and MCC-5 pure copper cellulose powders in Examples 1 to 3 and Comparative Examples 1 to 2 to purified feed to obtain a compound feed, and the content of pure copper cellulose in the feed is 1wt%. Take 30 two-month-old male transgenic mice and randomly divide them into 6 groups, 5 in each group. Among them, the model group was fed with purified feed, and the intervention group was fed with compound feed added with corresponding pure copper cellulose. After feeding for 4 months, the mice were euthanized, the liver was taken, freeze-dried, and the copper content in the liver was detected by ICP-OES. The results are shown in Table 1.

[0081] Table 1 Copper content in liver after treatment in different groups

[0082]

[0083]

[0084] Two-month-old transgenic mice were selected for research. At this time, the liver copper content of the mice was 896±174μg / g, and the liver copper deposition basically reached the highest. When the transgenic mice were six months old, the liver copper content was 855±94μg / g, and the liver copper deposition did not increase, which was basically equivalent to that of two months old. From the results in Table 1, it can be seen that using MCC-1, MCC-2, MCC-3, MCC-4 and MCC-5 pure copper cellulose to intervene in transgenic mice for 4 months can well reduce the copper deposition in the body of hepatolenticular degeneration mice, but the MCC-1, MCC-4 and MCC-5 pure copper cellulose in Examples 1 to 3 have better effects.

[0085] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing pure copper cellulose, characterized in that: The following steps are involved: Mixing microcrystalline cellulose, a first dispersant and a modifying agent to chemically modify the microcrystalline cellulose to obtain modified microcrystalline cellulose; the modifying agent includes epichlorohydrin or sodium periodate; The modified microcrystalline cellulose, the second dispersant and the amination agent are mixed and subjected to amination modification to obtain pure copper cellulose.

2. The preparation method according to claim 1, characterized in that: After the amination modification, the method further comprises: mixing the amination cellulose obtained by the amination modification, a third dispersant and a silane coupling agent, and performing coupling modification under alkaline conditions to obtain pure copper cellulose.

3. The preparation method according to claim 1 or 2, characterized in that: When the modification agent is epichlorohydrin, it also includes adding alkali solution; the first dispersant is dimethyl sulfoxide; the solid-liquid ratio of the microcrystalline cellulose and the first dispersant is 4g:35-50mL; the dosage ratio of the microcrystalline cellulose and the modification agent is 4g:25-35mL; the temperature of the chemical modification is 40-60°C, and the time is 6-8h.

4. The preparation method according to claim 1 or 2, characterized in that: When the modification agent is sodium periodate, the first dispersant is water; the solid-liquid ratio of the microcrystalline cellulose to the first dispersant is 1g:80-150mL; the mass ratio of the microcrystalline cellulose to the modification agent is 1:1-2; the temperature of the chemical modification is 45-55°C, and the time is 6-8h.

5. The preparation method according to claim 1 or 2, characterized in that: The amination agent includes polyethyleneimine, triethylenetetramine, ethylenediamine or methylamine; the second dispersant includes water or anhydrous ethanol; and the dosage ratio of the modified microcrystalline cellulose to the second dispersant is 1g:25-50mL.

6. The preparation method according to claim 5, characterized in that: The mass ratio of the modified microcrystalline cellulose to the amination agent is 1:0.5-2; the temperature of the amination modification is 50-60° C., and the time is 5-6 hours.

7. The preparation method according to claim 2, characterized in that: The silane coupling agent includes KH-580 or KH-590; and the third dispersant is anhydrous ethanol.

8. The preparation method according to claim 7, characterized in that: The mass ratio of the amino cellulose to the silane coupling agent is 1:0.25-0.45; the dosage ratio of the amino cellulose to the third dispersant is 1g:60-80mL; the coupling modification temperature is 50-60°C and the time is 2-3h.

9. Clean copper cellulose prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the pure copper cellulose according to claim 9 in preparing an oral drug for treating Wilson's disease.

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