WO2018113686A1 - Use of water-soluble fullerene structure in preparation of drug for treating fatty liver - Google Patents

Abstract

Disclosed is the use of a water-soluble fullerene structure in the preparation of a drug for treating a fatty liver. Also disclosed are a pharmaceutical composition and method for treating a fatty liver. Further disclosed is a healthcare composition for improving a fatty liver. The effective ingredients for treating a fatty liver are a water-soluble fullerene, a water-soluble endohedral metallofullerene, a composition of the water-soluble fullerene and the water-soluble endohedral metallofullerene, pharmaceutically acceptable esters of the three above, and pharmaceutical acceptable salts of the three above. The water-soluble fullerene structure can reduce fat bubbles in the liver, decrease blood lipids, and significantly improve the liver function, such that the content of glutamic-pyruvic transaminase and glutamic-oxaloacetic transaminase remains normal, and can also regulate the oxidation-reduction level in the liver.

Description

Application of water-soluble fullerene structure in preparing medicine for treating fatty liver

cross reference

The present invention claims the priority of Chinese patent application filed on December 19, 2016, filed with the Chinese Patent Office, the application number is CN201611180067.3, and the invention name is "the application of the water-soluble fullerene structure in the preparation of a medicament for treating fatty liver". The entire contents of this application are incorporated herein by reference.

The invention also claims that the Chinese patent application filed on July 11, 2017, with the application number CN201710560609.8 and the invention name "the application of the water-soluble fullerene structure in the preparation of drugs for treating fatty liver" is preferred. The entire contents of this application are incorporated herein by reference.

Technical field

The invention belongs to the field of biomedicine and relates to the application of a water-soluble fullerene structure in preparing a medicament for treating fatty liver.

Background technique

Fatty liver disease (ie, fatty liver) is a disease that can cause excessive accumulation of fat in liver cells caused by various causes. It is also a pathological process in the development of various liver diseases, and is the most common diffuse liver disease. one. The mechanism of fatty liver has not yet been fully clarified. Fatty liver is characterized by excessive accumulation of intrahepatic lipids, especially triglycerides, which exceeds 5% of liver weight; or more than 50% of hepatic parenchymal adenosis in the histology, and more than half of the hepatocytes in the liver. According to the pathogenesis, fatty liver can be divided into alcoholic fatty liver, obesity fatty liver, hyperlipidemia fatty liver, diabetic fatty liver, malnutrition fatty liver, drug-induced fatty liver and gestational fatty liver. It can also be divided into cholesterol fatty liver, phospholipid fatty liver and neutral fatty fatty liver. The most common one is fatty liver formed by abnormal accumulation of neutral fat in the liver.

In recent years, with the changes in the diet and diet of our people and the improvement of medical imaging techniques, the diagnostic rate of fatty liver caused by various incentives has increased year by year in China. Fatty liver is seriously threatening the health of Chinese people and becoming the second largest liver disease after viral hepatitis. If fatty liver is not treated in time, it is likely to develop into steatohepatitis, liver fibrosis, cirrhosis, end-stage liver failure or liver cancer.

Fullerenes are another allotrope of carbon other than graphite, diamond and amorphous carbon. This type of substance refers to a cage structure composed of carbon atoms. The most abundant molecules are C ₆₀ , then C ₇₀ and C ₈₄ , followed by C ₇₆ , C ₇₈ , C _{82 ,} etc. with relatively small contents. In addition, since the inside of the carbon cage of fullerenes is a cavity structure, the internal cavity can be embedded with different atoms, ions or clusters, which is called embedded fullerene, such as La@C ₆₀ , indicating La embedded. In the cage structure of C ₆₀ , @ denotes at, the image expresses the meaning of embedded.

The information disclosed in this Background section is only intended to provide an understanding of the general background of the invention, and should not be construed as an admission

Summary of the invention

The object of the present invention is to provide a water-soluble fullerene, a water-soluble inlaid metal fullerene, a water-soluble fullerene and a water-soluble inlaid metal fullerene composition, and the above three The use of a pharmaceutically acceptable ester or a pharmaceutically acceptable salt of the above three in the preparation of a medicament for the treatment of fatty liver. Another object of the present invention is to provide a pharmaceutical composition and method for treating fatty liver. The water-soluble fullerene structure of the active ingredient of the invention can reduce the liver index, reduce the fat bubble and fat content in the liver, lower the blood lipid, and obviously improve the liver function, so that the alanine aminotransferase and the aspartate aminotransferase content tend to be normal, and the high-efficiency treatment of fatty liver is achieved. The effect; at the same time, the water-soluble fullerene structure of the active ingredient can also regulate the redox level in the liver and reduce the mitochondrial structure damage in the fatty liver subjects.

In order to achieve the object, the present invention provides the following technical solutions:

a water-soluble fullerene, a water-soluble inlaid metal fullerene, the water-soluble fullerene and the water-soluble inlaid metal fullerene composition, and the medicinal of the above three Use of an ester or a pharmaceutically acceptable salt of the above three in the preparation of a medicament for the treatment of fatty liver.

The invention also provides a method of treating fatty liver comprising administering to a subject having fatty liver an effective amount of at least one active ingredient selected from the group consisting of water soluble fullerenes, water soluble inlaid A metal fullerene, a water-soluble fullerene, and a water-soluble inlaid metal fullerene composition, a pharmaceutically acceptable ester of the above three, and a pharmaceutically acceptable salt of the above three.

The present invention also provides a pharmaceutical composition for treating fatty liver, comprising at least one active ingredient selected from the group consisting of water-soluble fullerenes, water-soluble inlaid metal fullerenes, and said water-soluble rich a composition of a olefin and a water-soluble inlaid metal fullerene, a pharmaceutically acceptable ester of the above three, a pharmaceutically acceptable salt of the above three, the pharmaceutical composition further comprising a pharmaceutically acceptable carrier, At least one of a pharmaceutically acceptable diluent and a pharmaceutically acceptable excipient.

The present invention also provides a health care product composition for improving fatty liver, comprising at least one active ingredient selected from the group consisting of water-soluble fullerenes, water-soluble inlaid metal fullerenes, and said water-soluble a composition of fullerene and the water-soluble inlaid metal fullerene, a pharmaceutically acceptable ester of the above three, a pharmaceutically acceptable salt of the above three, the health care product composition further comprising a health care product At least one of a carrier, a diluent useful for a health care product, and an excipient that can be used in a health care product.

In another embodiment, the water-soluble fullerene is selected from one or more of the group consisting of: (1) the outer surface of the carbon cage is modified with a pro a fullerene of a water group; (2) a fullerene surrounded by a hydrophilic biomolecule on the outer surface of the carbon cage; (3) a fullerene supported by a biocompatible carrier material; (4) The formed water-soluble supramolecular system fullerene is assembled.

In another embodiment, the water-soluble inlaid metal fullerene is selected from one or more of the group consisting of: (1) the outer surface of the carbon cage Inlaid metal fullerene modified with a hydrophilic group; (2) embedded metal fullerene surrounded by a hydrophilic biological small molecule on the outer surface of the carbon cage; (3) supported by a biocompatible carrier material Inlaid metal fullerenes; (4) self-assembled water-soluble supramolecular systems embedding metal fullerenes.

In another embodiment, the fullerene comprises one or more cage structures consisting of carbon atoms of the formula C _2m , 30 ≤ m ≤ 60, for example; C ₆₀ , C ₇₀ , C ₈₄ and the like.

In another embodiment, the inlaid metal fullerene comprises M@C _2n , M ₂ @C _2n , MA@C _2n , M ₃ N@ One or more of C _2n , M ₂ C ₂ @C _2n , M ₂ S@C _2n , M ₂ O@C _2n and M _x A _3-x N@C _2n , wherein: M and A represent The metal element and M and A are each selected from any one of a lanthanide metal element, Sc and Y, 30 ≤ n ≤ 60, and optionally n is 30 or 35 or 41; 0 ≤ x ≤ 3, for example: Gd@ C ₈₂ . N represents a nitrogen element, C represents a carbon element, S represents a sulfur element, and lanthanide metal elements include La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb and Lu.

In another embodiment of the above application, method, pharmaceutical composition or nutraceutical composition, the hydrophilic group comprises one or more of a hydroxyl group, a carboxyl group, a thiol group, an amino group and a water-soluble amino acid residue.

In another embodiment, the water-soluble amino acid residue refers to a water-soluble amino acid that is lost when the fullerene and/or the inlaid metal fullerene are modified. The incomplete amino acid remaining after a portion of the amino acid molecule, ie, the amino acid residue is part of the amino acid molecule, which is an incomplete amino acid. Any part of the missing amino acid molecule is considered to be an amino acid residue, such as: loss of hydrogen on the amino group in the amino acid, loss of hydrogen or hydroxyl groups on the carboxyl group in the amino acid, and the like. Optionally, the water-soluble amino acid residue is at least one of an alanine residue, a glycine residue, a serine residue, an arginine residue, a lysine residue, and a tyrosine residue.

In another embodiment, the water-soluble fullerene has the formula C _2a (OH) _b (Amino Acid) _c and the Amino Acid represents water solubility. Amino acid residue; 30 ≤ a ≤ 60, optional a is 30 or 35; 0 < b ≤ 50, optional 0 < b ≤ 30, 10 ≤ b ≤ 30, and optionally b = 13, 20, 22, 24, 26, etc.; 0 ≤ c ≤ 20, optional c = 2-15, and optionally c = 6.

In another embodiment, the water-soluble inlaid metal fullerene has the formula M@C _2d (OH) _e (Amino Acid) _f , Amino Acid represents a water-soluble amino acid residue; M is selected from a rare earth metal, the optional rare earth metal is Gd or La; 30 ≤ d ≤ 60, optionally d is 41 or 30 or 35; 0 < e ≤ 50, optional 0<e≤30, optional 10≤e≤30, and optionally e=13, 20, 22, 24, 26, etc.; 0≤f≤20, optional f=2-15, The selected f=6.

In another embodiment, the water-soluble fullerene comprises C ₇₀ (OH) ₂₄ ; the water-soluble inlaid metal fullerene comprises Gd@C ₈₂ (in other embodiments). OH) ₂₆ , Gd@C ₈₂ (OH) ₁₃ (NHCH ₂ CH ₂ COOH) ₆ .

In another embodiment, the hydrophilic bio-small molecule comprises at least one of an amino acid and a peptide chain.

In another embodiment of the above application, method, pharmaceutical composition or nutraceutical composition, the biocompatible carrier material comprises at least one of a liposome and a cell membrane carrier.

In another embodiment, the water-soluble fullerene is obtained by water-soluble modification of the raw material fullerene; the water-soluble inner The metal-inlaid fullerene is obtained by water-soluble modification of the metal fullerene embedded in the raw material.

In another embodiment of the above application, method, pharmaceutical composition or nutraceutical composition, the method of water-soluble modification is any one of the following methods:

(1) The method of surface-modifying a hydrophilic group is generally achieved by a solid-liquid or liquid-liquid reaction under the action of a base, specifically, at least one of a fullerene and a metal fullerene embedded in a raw material and a hydrogen peroxide. The solution is mixed and reacted, washed with ethanol, and then dialyzed to obtain a water-soluble hydroxy derivative corresponding to the starting material. If it is desired to obtain a water-soluble aminated derivative, the alkali solution in the above step may be replaced with ammonia water. Alternatively, the alkaline solution is a NaOH and/or KOH solution.

(2) Physical coating method at least one of raw material fullerenes and raw material embedded metal fullerenes may be mixed with at least one of polyethylene glycol, polyvinyl pyrrolidone and cyclodextrin and ball-milled or Ultrasonic or the like can obtain a coated water-soluble fullerene structure corresponding to the raw material, such as polyethylene glycol-coated fullerene and/or polyethylene glycol-coated inlaid metal fullerene, polyethylene Pyrrolidone-coated fullerene and/or polyvinylpyrrolidone-coated inlaid metal fullerenes.

(3) When the number of amino acid residues in the water-soluble fullerene structure is not 0, the following steps are included: (a) preparing a water-soluble amino acid alkali solution using a water-soluble amino acid with NaOH and/or KOH (optional) , water-soluble amino acid: NaOH and / or KOH molar ratio of 1:1-10, optionally 1:2 or 1:1-8; optional, water-soluble amino acid alkaline solution in NaOH and / or The mass fraction of KOH may be 10-50%, and optionally 14% or 10-30%); (b) according to the water-soluble amino acid: raw material fullerenes and/or raw material inlaid metal fullerene molar ratio is 1-1000:1, optionally 50-1000:1,100-1000:1,200-1000:1, mixing the amino acid alkali solution with the raw material fullerenes and/or the raw material embedded metal fullerenes; (c) reacting the above mixture at 40-80 ° C (optionally, the reaction is vigorously stirred at 50 ° C for 1-7 hours, further optionally 1.5 hours), and filtering to remove unreacted small amount of solid powder; (d) The filtrate is dialyzed to remove small molecular impurities, and after filtration, the resulting brown-black solution is the amino acid-modified water-soluble fullerene/embedded metal fullerene of the present invention. Optionally, the molecular weight cut-off molecular weight of the dialysis bag used for dialysis is Mw=3500; alternatively, the pore size of the microporous membrane used for filtration after dialysis is 200-220 nm; optionally, the conductance of the liquid dialysis to the outside of the dialysis bag The rate is less than 1 μs/cm.

In another embodiment of the above application, method, pharmaceutical composition or nutraceutical composition, the water-soluble amino acid is at least one of alanine, glycine, serine, arginine, lysine and aspartic acid. Kind.

In another embodiment, the solid-liquid reaction in the water-soluble modified method (1) comprises: weighing 20-200 mg, optionally 30-100 mg C ₆₀ solid Or C ₇₀ solid or Gd@C ₈₂ solid, and 3-15ml 20-30% hydrogen peroxide, 2-10ml 5-20% alkali solution, mixed at 50-100 ° C until the solid is completely dissolved, and then used The ethanol is washed and dialyzed to obtain the corresponding hydroxylated derivative, that is, a water-soluble fullerene structure. Optionally, the conductivity of the liquid dialysis to the outside of the dialysis bag is less than 1 [mu]s/cm. In this description, the proportional relationship between the substances is shown, and in practical applications, it is not limited by the specific reaction scale of 50-200 mg, 3-15 ml, and 2-10 ml, and can be expanded in proportion.

In another embodiment of the above application, method, pharmaceutical composition or nutraceutical composition, the solid-liquid reaction in the water-soluble modified method (1) comprises: weighing 30-100 mg of C ₆₀ solid or C ₇₀ solid or Gd @C ₈₂ solid, and 7 ml of 30% hydrogen peroxide, 3 ml of 10%-15% NaOH solution, mixed at 50 ° C until the solids were all dissolved, then washed with ethanol and dialyzed to obtain the corresponding hydroxylated derivative.

In another embodiment, the raw material fullerene comprises one or more cage structures composed of carbon atoms of the formula C _2m , 30≤ m ≤ ₆₀ , for example; C ₆₀ , C ₇₀ , C ₈₄ and the like.

In another embodiment, the raw material inlaid metal fullerene comprises M@C _2n , M ₂ @C _2n , MA@C _2n , M ₃ N One or more of @C _2n , M ₂ C ₂ @C _2n , M ₂ S@C _2n , M ₂ O@C _{2n ,} and M _x A _3-x N@C _2n , where: M, A, Representing a metal element and M and A are each selected from any one of a lanthanide metal element, Sc and Y, 30 ≤ n ≤ 60, and optionally n is 30 or 35 or 41; 0 ≤ x ≤ 3. N represents a nitrogen element, C represents a carbon element, S represents a sulfur element, and lanthanide metal elements include La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb and Lu.

In another embodiment, the fatty liver is obese fatty liver, hyperlipidemia fatty liver, diabetic fatty liver, alcoholic fatty liver or a drug. Fatty liver.

The above application, method, and pharmaceutical composition In another embodiment, the treating fatty liver comprises: 1) making the content of free fatty acids in the liver normal; 2) reducing diffuse fat vacuoles (may be vacuoles) Reduce the number or decrease the volume); 3) Improve liver function, make liver function indicators (hepatic function indicators include: alanine aminotransferase, aspartate aminotransferase) tend to normal; 4) make liver weight normal; 5) improve liver Redox levels, so that SOD activity, CAT activity and MDA content returned to normal; 6) lowering blood lipids (blood lipid indicators include: total cholesterol, triglycerides, high and low density lipids); 7) reducing hepatocyte mitochondrial damage.

In another embodiment, the improved fatty liver comprises: 1) improving the content of free fatty acids in the liver; 2) reducing diffuse fat vacuoles; 3) improving liver function and improving liver function indicators. (Hepatic function indicators include: alanine aminotransferase, aspartate aminotransferase); 4) reduce liver weight; 5) improve redox levels in the liver, restore SOD activity, CAT activity and MDA content; 6) lower blood lipids (lipid index) Including: total cholesterol, triglycerides, high and low density lipids; 7) reduce liver cell mitochondrial damage.

In another embodiment, the drug or the pharmaceutical composition may be a tablet, a pill, a powder, a lozenge, a sachet, a cachet, an elixir, a suspending agent, Formulations of emulsions, solutions, syrups, aerosols, ointments, soft and hard gelatin capsules, suppositories, sterile injectable solutions or sterile packaging powders. The method of preparing an active ingredient into a pharmaceutical or pharmaceutical composition in the present invention can be prepared by a method known to those skilled in the art to provide an immediate release, sustained release or delayed release of the active ingredient after administration to a subject, for example: The active ingredient can be mixed with the carrier, diluted with the carrier or enclosed in a carrier.

The drug or the above pharmaceutical composition in the above application. In another embodiment, some examples suitable as carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, starch, resins, Acacia, calcium phosphate, alginate, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water syrup, methylcellulose, methylparaben And propyl ester, talc, magnesium stearate and liquid paraffin.

The medicament or the above pharmaceutical composition in the above application. In another embodiment, the medicament or pharmaceutical composition may additionally comprise a lubricant, a wetting agent, an emulsifying and suspending agent, a preservative, a sweetener or a flavoring agent. And other additives.

The drug or the above pharmaceutical composition in the above application. In another embodiment, when the drug or the pharmaceutical composition is present in a liquid form, the concentration of the active ingredient in the drug or the pharmaceutical composition is 0.01-50 mg/mL, optionally 0.01-10 mg/mL, 0.01-20 mg/mL, 0.01-30 mg/mL, 0.01-40 mg/mL; when the drug or the pharmaceutical composition is present in a solid form, The concentration of the active ingredient in the drug or the pharmaceutical composition is from 0.01 to 50 mg/g, alternatively from 0.01 to 10 mg/g, from 0.01 to 20 mg/g, from 0.01 to 30 mg/g, from 0.01 to 40 mg/g.

In another embodiment, the subject is a human or an animal, and the animal can be a mammal such as a mouse, a guinea pig, a rat, a dog, a rabbit, a monkey, or the like.

In another embodiment, the active ingredient is administered at a dose of 1 mg/kg/d to 500 mg/kg/d, optionally 1-100 mg/kg/d, 1-20 mg/kg/d, 1-10mg/kg/d, 10-100mg/kg/d, the course of application can be 5 days to 30 days, depending on the condition, short-term or long-term use; the active ingredient can be administered orally, intravenously or intraperitoneally. .

As used herein, the term "treatment" includes its generally accepted meaning, which includes preventing, preventing, inhibiting, ameliorating, and slowing, halting, or reversing the development of a symptom or a desired condition. As such, the invention encompasses both therapeutic and prophylactic administration.

The term "active ingredient", "active ingredient water-soluble fullerene structure" or "water-soluble fullerene structure" as used herein means both water-soluble fullerenes, water-soluble inlaid metal fullerenes, At least one of a water-soluble fullerene and a water-soluble inlaid metal fullerene composition, a pharmaceutically acceptable ester of the above three, and a pharmaceutically acceptable salt of the above three.

The term "effective amount" as used herein, refers to an amount or dose of the active ingredient that is administered to a patient in a single or multiple administrations to provide a desired effect to the patient being diagnosed or treated. The effective amount can be determined by the participating diagnostician as a result of known techniques by those skilled in the art and in similar circumstances. In determining the effective amount or dose of the active ingredient administered, the participating diagnostician should consider a variety of factors including, but not limited to, the mammalian species; volume, age, and general health; the particular disease involved Degree or severity of involvement of the disease; response of the individual patient; specific compound administered; mode of administration; bioavailability properties of the administered formulation; selected dosing regimen; use with drug therapy; Related situation.

As used herein, the term "raw material fullerene" and "fullerene" without any modifier mean a fullerene which is not water-soluble modified, that is, a fullerene body.

As used herein, the term "inlaid metal fullerene in the raw material" and "inlaid metal fullerene" without any modifier means an inlaid metal fullerene which is not water-soluble modified, that is, an inlaid metal. Fullerene body.

The term "water-soluble fullerene" as used in the present invention means a water-soluble modified fullerene obtained by subjecting a bulk of a fullerene to a water-soluble modification.

The term "water-soluble inlaid metal fullerene" as used in the present invention means a water-soluble modified inlaid metal fullerene obtained by water-soluble modification of an inlaid metal fullerene body.

In order to facilitate the metering, all the quantitative determinations of the specific content, concentration and the like of the water-soluble fullerene and the water-soluble metal fullerene in the present invention are specific to the corresponding fullerene body or embedded metal fullerene body. The content, concentration, etc., for example, the effective dose of the active ingredient is from 1 mg/kg/d to 500 mg/kg/d, which means the corresponding fullerene bulk carbon in the active ingredient to be administered per 1 kg of mice per day. The amount of the cage is from 1 mg to 500 mg.

Compared with the prior art, the beneficial effects of the present invention are:

1. In the present invention, the raw material fullerene and the raw material inlaid metal fullerene are water-soluble, so that they are transported to the liver along the blood circulation in the living body, thereby functioning in the liver, thereby improving liver lipid metabolism. Reduce free fatty acids, reduce fatty vesicles in the liver, reduce the content of triglycerides, significantly improve liver function, so that the content of alanine aminotransferase and aspartate aminotransferase tend to be normal, and achieve the effect of high-efficiency treatment of fatty liver. At the same time, the effective treatment of fatty liver also fundamentally avoids the possibility of fatty liver developing into severe cases such as steatohepatitis, liver fibrosis, cirrhosis, terminal liver failure or liver cancer.

2. In the present invention, the water-soluble fullerene structure of the active ingredient enters the liver, and because of its good free radical scavenging effect, the liver can be "cleaned" to improve the redox level in the liver.

3. The water-soluble fullerene structure of the active ingredient in the present invention can be rapidly metabolized into the body and is not toxic to internal organs.

DRAWINGS

1A is a thermogravimetric analysis and a micro-commercial thermogravimetric curve of a hydroxylated derivative corresponding to C ₇₀ prepared in Example 1; FIG. 1B is a thermogravimetric of a hydroxylated derivative corresponding to Gd@C ₈₂ prepared in Example 1. Analysis and derivative heat weight curve.

Figure 2 is an electron spin nuclear magnetic resonance (ESR) image of a Gd@C ₈₂ (OH) _n material.

3 is a metabolic distribution of the Gd@C ₈₂ (OH)n material obtained in Example 1 in vivo for 24 hours.

Figure 4 is a comparison diagram of H&E staining of mouse livers of each group in Example 5 of the present invention.

Figure 5 is a graph showing the content of triglyceride in mice after administration for 2 weeks in Example 5 of the present invention.

Figure 6 is a graph showing the content of alanine aminotransferase ALT in mice after administration for 2 weeks in Example 5 of the present invention.

Figure 7 is a graph showing the content of aspartate aminotransferase AST in mice after administration for 2 weeks in Example 5 of the present invention.

_{13 (NHCH 2 CH 2 COOH)} 6 micro-thermal analysis and thermogravimetry curve obtained in Example 6 Gd @ C ₈₂ (OH) 8 of the present invention, FIG.

Figure 9 is an infrared spectrum of Gd@C ₈₂ (OH) ₁₃ (NHCH ₂ CH ₂ COOH) ₆ obtained in Example 6 of the present invention.

Figure 10 is a graph showing the proportion of liver in mice after treatment in Example 8 of the present invention, wherein NS represents physiological saline, and GF represents Gd@C ₈₂ (OH) ₁₃ (NHCH ₂ CH ₂ COOH) ₆ , that is, from left to right. They were: blank + saline group, blank + GF group, model group, treatment group - 0.375 mM, treatment group - 0.75 mM, and treatment group - 1.5 mM.

Figure 11 is a diagram showing the pathological section of HE staining of mouse liver after treatment in Example 8 of the present invention.

Figure 12 is a graph showing the staining of liver oil red staining of mice after treatment in Example 8 of the present invention.

Figure 13 is a graph showing the relative contents of liver lipids in mice after treatment in Example 8 of the present invention.

Figure 14 is a graph showing serum ALT and AST contents of mice after treatment in Example 8 of the present invention.

Figure 15 is a transmission electron micrograph of liver tissue in Example 9 of the present invention, the dark gray arrow indicates the mitochondria of the outer membrane rupture; the light gray arrow indicates the mitochondria with intact outer membrane but internal damage; the white arrow indicates normal mitochondria, model Normal mitochondria appeared in the third image of the group, but normal mitochondria appeared in the three images of the treatment group.

Figure 16 is a bar graph showing the ratio of liver to body weight of mice in each group in Example 10, where # indicates P<0.05 compared with group A, and * indicates P<0.05 compared with group C.

Figure 17 is a bar graph of lipid content in the liver of each group of mice in Example 10 of the present invention, ## indicates P<0.01 compared with group A, and * indicates P<0.05 compared with group C.

Figure 18 is a pathological section of HE staining of liver of each group of mice in Example 10 of the present invention.

Figure 19 is a bar graph showing the contents of ALT and AST in mouse serum in Example 10 of the present invention.

Figure 20 is a comparison of the effects of C ₆₀ -OH, C ₇₀ -OH and Gd@C ₈₂ (OH) _{n in the} treatment of fatty liver in the present invention.

detailed description

The specific embodiments of the present invention are described in detail below with reference to the accompanying drawings, but it is understood that the scope of the present invention is not limited by the specific embodiments. The raw material Gd@C ₈₂ solid powder used in the following examples was purchased from Xiamen Funa New Material Technology Co., Ltd., with a molecular weight of 1141 and a purity of 99.1%. The raw material C ₆₀ solid powder used in the following examples was purchased from Xiamen Funa New Material Technology Co., Ltd., with a molecular weight of 720 and a purity of 99%. The raw material C ₇₀ solid powder used in the following examples was purchased from Xiamen Funa New Material Technology Co., Ltd., with a molecular weight of 840 and a purity of 99%.

Example 1. Preparation of water-soluble hydroxylated fullerene structure

Weigh 100mg C ₆₀ solid or 100mg C ₇₀ solid or 100mg Gd@C ₈₂ solid, 7ml 30% hydrogen peroxide, 3ml 10% sodium hydroxide solution, react at 50 ° C, until the powder is completely dissolved, then use ethanol Washing, dialysis, gives the corresponding hydroxylated derivative, the water soluble fullerene structure.

The 3 ml of 10% sodium hydroxide solution was replaced with a certain amount of 30% aqueous ammonia, and the other steps were carried out according to the above methods to obtain the corresponding aminated derivative, that is, a water-soluble fullerene structure.

The hydroxylated derivative or the aminated derivative obtained after dialysis contains more liquid, and it can also be concentrated to obtain a solid, such as by freeze-drying. However, whether or not the concentration is carried out, the water-soluble modification of the fullerene/material embedded fullerene in the raw material has been completed, and whether or not the concentration is performed does not affect the use of the fullerene/embedded metal fullerene after the water-soluble modification. Just adjust it to the right concentration.

Example 2

Hydroxy derivatives corresponding to C ₇₀ obtained in Preparation Example 1 (hereinafter referred to as C ₇₀ -OH) Elemental analysis embodiment (Flash EA 1112), and combined TGA and derivative thermogravimetric analysis results of the number of hydroxyl groups they are attached. In the results of elemental analysis, the C ₇₀ -OH had a C content of 37.85%, an H content of 1.51%, and an N content of <0.3%. It can be seen from the thermogravimetric analysis in Fig. 1A that the C ₇₀ -OH solid powder contains 3.7% water, and the ratio of the H content to the C content in the elemental analysis can be used to estimate the surface of the carbon cage to modify 24 hydroxyl groups. Therefore, the average structural formula of C ₇₀ -OH is C ₇₀ (OH) ₂₄ .

Hydroxy derivative prepared in Example 1 above to give the corresponding Gd @ C ₈₂ (hereinafter referred to as _{Gd @ C 82 (OH) n} ) elemental analysis, and combined TGA and derivative thermogravimetric analysis result of its contact hydroxyl number . In the results of the elemental analysis, in the Gd@C ₈₂ (OH) _n , the C content was 36.95%, the H content was 2.36%, and the N content was 0%. It can be seen from the thermogravimetric analysis in Fig. 1B that the Gd@C ₈₂ (OH) _n solid powder contains 12.6% water, and the ratio of the H content to the C content in the elemental analysis can be used to estimate the 26 hydroxyl groups on the surface of the carbon cage. Therefore, Gd @ C ₈₂ (OH) _n in the average structural formula of _{Gd @ C 82 (OH) 26} . The particle size of the water-soluble derivative is measured by dynamic light scattering (DLS), and the particle diameter of the Gd@C82(OH)n material is about 145.2 nm, which can be injected into a living body.

Example 3: Water-soluble inlaid metal fullerene Gd@C ₈₂ (OH) _n scavenging free radical ability detection

The present invention detects the ability of water-soluble inlaid metal fullerene Gd@C ₈₂ (OH) _{n to} scavenge free radicals by electron spin resonance spectroscopy (ESR).

Detection method: using UV-induced hydroxyl radical generation method, 50 μL of 39% hydrogen peroxide solution, 50 μL PBS buffer solution (pH=7.4) and trace amount (0.133 mM) of lutidine N-oxide (DMPO, free The base capture agent was mixed with the solution, and the control group was directly irradiated with ultraviolet light, and the experimental group immediately added 20 μL of the water-soluble inlaid metal fullerene Gd@C ₈₂ (OH) _n aqueous solution prepared according to the method of Example 1 and used 10 μL, respectively. The 280 nm ultraviolet light was irradiated for 4 min to detect a radical signal. As shown in FIG. 2, the non-smooth line is a blank control without adding the water-soluble inlaid metal fullerene Gd@C ₈₂ (OH)n obtained by the method of Example 1, and the smooth line is added to Gd@C ₈₂ (OH). The experimental group of n, compared with the blank control, the signal added to the Gd@C ₈₂ (OH) _n sample was significantly reduced, indicating that the experimental group added to the Gd@C ₈₂ (OH) _n sample had less free radicals, Gd @C ₈₂ (OH) _n has a strong ability to scavenge free radicals.

Example 4: In vivo metabolism of water-soluble inlaid metal fullerene Gd@C ₈₂ (OH) _n

Gd@C ₈₂ (OH) _n 200 μL prepared according to the method of Example 1 at a concentration of 1 mM was injected into C57 mice by intraperitoneal injection. After normal feeding for 24 hours, the mice were dissected and the organs, liver, spleen and lungs were taken. The kidney and pancreas were weighed and digested with 65% nitric acid overnight at 120 ° C. After 50-fold dilution, the cesium ion concentration was determined by ICP. As in Figure 3, it can be seen that the water-soluble inlaid metal fullerene Gd@C ₈₂ (OH) _{n is} much more enriched in the liver than in the heart, kidney, lung and spleen.

Example 5: Treatment of fatty liver with water-soluble inlaid metal fullerene Gd@C ₈₂ (OH) _n

(1) Experimental method

The experimental fatty liver model mice were db/db mice, which were models of leptin receptor gene deficiency leading to obesity and then developed into more severe fatty liver. Purchased from the Nanjing Animal Model Center, from the Jackson Laboratory in the United States.

The experimental animals were divided into 3 groups of 6 each. Group A, 6 db/m fat-free liver mice were used as the blank group, and the administration was performed by administering the same volume of physiological saline as the drug used in Group C. In group B, 6 db/db mice were used as the model group. The administration treatment was the same volume of physiological saline as that used in the group C; 6 db/db mice in the group C were used as the Gd@C ₈₂ (OH) _n experimental group, and the administration treatment was the method according to the example 1. Prepared Gd@C ₈₂ (OH) _n . The AC group was administered by intraperitoneal administration. The mice of each group started to enter the 10th week and were administered once a day at a dose of 10 mg/kg/d for two weeks.

(2) Experimental results

In the present invention, the mice are sacrificed after the end of the administration, and the liver organs of the mice are subjected to H&E pathological sectioning, and the serum is used for detecting the liver function indexes alanine aminotransferase ALT, aspartate aminotransferase AST, and blood lipid triglyceride detection. The results are as follows.

1) H&E test

The mice were sacrificed 2 weeks after administration, and the liver of the mice was fixed in 4% formaldehyde, then embedded in paraffin, sectioned, and stained. As shown in Fig. 4, the blank group was a normal mouse, and the liver structure was normal, and there was no fatty vesicle. The model group was a mouse with fatty liver but only treated with physiological saline, and the mouse can be seen in the figure. There are a large number of white vacuoles in the liver, that is, fatty vesicles, and the mice in the experimental group treated with Gd@C ₈₂ (OH) _n prepared according to the method of Example 1 have significantly reduced fat vesicles in the liver, demonstrating water solubility. Metal-filled fullerenes have a good effect on the treatment of fatty liver.

2) Triglyceride content

Two weeks after the administration of the mice, blood was taken from the eyelids and whole blood was taken. The blood samples were allowed to stand at room temperature for 1 hour, centrifuged at 3500 rpm for 15 min, and the upper serum was aspirated. The content of triglyceride TG was measured by an automatic blood biochemistry analyzer, as shown in Fig. 5. It is shown that the amount of triglyceride TG in the model group is significantly higher than that in the blank group, which is because the triglyceride content of the fat metabolism caused by the fatty liver is increased, and the results of the treatment group show that, according to the embodiment 1, The Gd@C ₈₂ (OH) _n- treated mice had significantly lower triglyceride levels, which proved to have the effect of treating fatty liver and lowering blood lipids.

3) Liver function test

Two weeks after the administration of the mice, blood was taken from the eyelids, and whole blood was taken. After standing at room temperature for 1 hour, the supernatant was aspirated at 3500 rpm for 15 minutes, and the contents of the liver function index alanine aminotransferase ALT and aspartate aminotransferase AST were measured by an automatic blood biochemistry analyzer. As shown in Figures 6 and 7, the alanine aminotransferase ALT content of the model group was significantly increased compared to the blank group, while the experimental group treated with Gd@C ₈₂ (OH) _n prepared according to the method of Example 1 was small. The alanine aminotransferase ALT content of the rats was significantly reduced and tends to be normal. The aspartate aminotransferase AST also has a corresponding rule, which proves that the liver function of mice is significantly improved after Gd@C ₈₂ (OH) _n treatment.

The above examples of treatment of db/db mice with fatty liver showed that the water-soluble fullerene structure can effectively reduce fatty vesicles, lower blood lipids and improve liver function.

Example 6. Preparation of a water-soluble amino acid modified inlaid metal fullerene structure

Add 10mg Gd@C ₈₂ solid to a single-mouth bottle and add 14% NaOH mass fraction of β-alanine-sodium hydroxide solution (ie, the solution contains alanine and sodium hydroxide, the quality of sodium hydroxide The fraction is 14%), the molar ratio of β-alanine to sodium hydroxide is specifically 1:2; the molar ratio of β-alanine to Gd@C ₈₂ is 1000:1, and the stirring is vigorously 1.5 at 50 °C. h, the black solid gradually dissolves to form a brownish black solution. The unreacted small amount of solid powder was removed by filtration, the filtrate was washed with ethanol, and the small molecular impurities were removed by dialysis on a Mw=3500 dialysis bag. The brown-black solution obtained by filtration using a 220 nm microporous membrane was embedded in the water-soluble amino acid modification of the present invention. Metal fullerene structure.

Example 7

Elemental analysis and thermogravimetric curve analysis were carried out on the water-soluble amino acid-modified inlaid metal fullerene structure prepared in Example 6 using Gd@C _82. The results of the elemental analysis are shown in the following table, and the thermogravimetric curve analysis is shown in Fig. 8. Show:

Elemental analysis results of β-alanine modified inlaid metal fullerene structure

According to the thermogravimetric analysis, it can be concluded that the water-soluble amino acid modified inlaid metal fullerene structure contains 10.9% water, and after calculation, about 13 H ₂ O molecules, combined with elemental analysis, the average molecular formula of the material is further obtained. Gd@C ₈₂ (OH) ₁₃ (NHCH ₂ CH ₂ COOH) ₆ , hereinafter referred to as GF.

Fig. 9 is a stretching vibration in which the strong absorption peak of the infrared spectrum at about 3300 cm ^-1 is assigned to OH, and the intermediate absorption peak at 1740 and 1630 cm ^-1 is assigned to the stretching vibration of C=O and the bending vibration of NH, at 1568, The strong peaks at 1400 and 1300 cm ^-1 are the stretching vibration of C=C, the bending vibration of OH, and the stretching vibration peaks of CO and CN, respectively. As is apparent from Fig. 9, the β-alanine-modified inlaid metal fullerene structure of the present invention contains the above-mentioned hydrophilic group, that is, it is water-soluble.

Example 8, treatment of fatty liver with water-soluble amino acid modified inlaid metal fullerene structure

Non-alcoholic fatty liver disease (NAFLD) is characterized by hepatic steatosis, in which lipids such as triglycerides accumulate in the cytoplasm of hepatocytes in the form of lipid droplets, so the liver becomes hypertrophied, the mass increases, and the liver accounts for body weight. The proportion is increasing.

(1) Treatment experiment

Ob/ob mice were used in this experiment. Ob/ob mice are a kind of leptin-secreting gene-deficient obesity mice, which can not produce leptin (a protein hormone, which can participate in sugars in the body). Fat metabolism), so ob/ob mice lacking leptin secretion will overtake food. Compared with normal mice with no leptin secretion gene, ob/ob mice have significantly increased body weight and become larger. A severe fatty liver model. The mouse was purchased from Nanjing University-Nanjing Biomedical Research Institute and was cited from the Jackson Laboratory in the United States.

The mice in this experiment were divided into 6 groups, namely: blank + saline group, blank + GF group, model group, treatment group -0.375 mM, treatment group -0.75 mM, treatment group -1.5 mM, 6 mice per group. The blank + saline group and the blank + GF group were all background mice without fatty liver. The blank + saline group and the blank + GF group began to be administered at the 6th week after birth. The average body weight at the time of administration was 23g; model group, treatment group -0.375mM, treatment group -0.75mM, treatment group -1.5mM are ob/ob mice, the mice started to be administered at the 6th week after birth, and the average body weight was 33g when administered. . The manner of administration in each of the above groups was carried out as follows.

Blank + saline group: Each time the mice were administered, 150 μl of physiological saline was intraperitoneally injected. It is administered once a day for 16 days.

Blank + GF group: Each mouse was administered 150 μl of a 1.5 mM solution prepared by intraperitoneal injection of the water-soluble metal fullerene GF prepared in Example 6. It is administered once a day for 16 days.

Model group: Each mouse was intraperitoneally injected with 150 μl of physiological saline. It is administered once a day for 16 days.

Treatment group - 0.375 mM: Each dose of the mice was 150 μl of a 0.375 mM solution prepared by intraperitoneal injection of the water-soluble metal fullerene GF prepared in Example 6. It is administered once a day for 16 days.

Treatment group - 0.75 mM: Each dose of the mice was 150 μl of a 0.75 mM solution prepared by intraperitoneal injection of the water-soluble metal fullerene GF prepared in Example 6. It is administered once a day for 16 days.

Treatment group - 1.5 mM: Each dose of the mice was 150 μl of a 1.5 mM solution prepared by intraperitoneal injection of the water-soluble metal fullerene GF prepared in Example 6. It is administered once a day for 16 days.

(2) Pathological and physiological indicators of hepatic steatosis

As shown in Figure 10 (# and ## indicate significant differences from the blank control group, * and ** indicate significant differences from the model group), and liver index of the model group ob/ob mice (liver accounted for body weight) Significantly higher than healthy background mice without fatty liver, but after treatment with water-soluble metal fullerenes, the liver index of the two treatment groups (0.75 mM and 1.5 mM) at higher concentrations decreased significantly, close to normal levels. It confirms that GF has a therapeutic effect on nonalcoholic fatty liver.

Pathological sections are more intuitive to show that GF material can improve liver steatosis in ob/ob mice. Figure 11 shows the results of HE staining pathological sections of mice in each group at different magnifications. It can be found that the liver of the model group ob/ob mice has a large area of hepatocyte swelling, cytoplasm is loose, lipids are accumulated in the white vacuoles in the figure, the nucleus is squeezed to the edge of the cell, and the basic hepatic lobular structure is destroyed. Only the cytoskeleton was visible; the improvement in the low-concentration treatment group (0.375 mM) was not obvious, and only a small part of the improvement was found in the hepatocytes in the portal area, and the rest of the liver tissue was still covered by a large area of fat vacuoles. Occupation; the medium concentration treatment group (0.75 mM) showed a significant improvement. From the HE stained sections, it was found that although there were more lipid vacuoles distributed in the liver tissue, the structure of the hepatic lobule was restored, and not Compared with the treated ob/ob mice, the structural integrity of the medium-concentration treatment group had a substantial increase in the number of hepatocytes, and most of the nucleus was in the normal position; the liver structure recovery in the high-concentration treatment group (1.5 mM) was superior to other treatment groups. The hepatocytes in the hepatic lobular confluence area are completely restored to normal, arranged neatly, forming an ordered liver plate and hepatic sinusoidal structure, and only the hepatocytes of each hepatic lobules away from the portal area However, there will be a number of lipid vacuoles, compared with no treatment of ob / ob mouse liver, hepatic steatosis situation has been improved to a large extent. In addition, the physiological saline solution of the intraperitoneal injection of the sample GF to healthy mice did not adversely affect the liver in a pathological manner compared with the control group in which normal mice were not injected with normal liver.

Oil red staining will specifically stain the lipid into orange-red, so liver oil red staining can directly reflect the presence of lipids in the liver. Figure 12 shows the liver oil red staining of mice in each group at different magnifications. result. Normal mouse hepatocytes do not show lipid droplets, so they are still blue-violet after oil red staining, while large red-orange lipid droplets can be seen on oil red stained sections of ob/ob mice liver, using GF material. After treatment, lipid droplets were preferentially reduced from around the portal area containing the portal vein, hepatic artery, and bile duct, consistent with the results shown by HE stained sections. Densitometric analysis of oil red staining results can more intuitively compare the lipid content in the liver between groups, as shown in Figure 13 (# and ## indicate significant differences from the blank control group, * and ** Significantly different from the model group, normal mice did not show lipid droplets stained with orange-red in the liver, while ob/ob mice were injected intraperitoneally with 0.375 mM, 0.75 mM and 1.5 mM GF physiological saline solution for 16 days. Compared with the ob/ob mice (ie, the model group) that received intraperitoneal injection of normal saline, the lipid content in the liver decreased by 40%, 60%, and 85%, respectively. This proves that GF material can improve fat well by intraperitoneal injection. The symptoms of the liver reduce the lipid content in the liver.

In addition, serum levels of glutamate pyruvate transaminase (ALT) and aspartate aminotransferase (AST) are also important indicators of liver function. Because ALT and AST are mainly present in the cytoplasm of hepatocytes, they are released into the blood only when the liver cells are damaged, and can exist in the blood for a long time, so the content of these two enzymes in the serum can be Reflects the extent of liver damage. As shown in Figure 14 (# and ## indicate significant differences from the blank control group, * and ** indicate significant differences from the model control group), and the left and right sides of each column indicate serum ALT and AST, respectively. The content of ALT and AST in the model group with fatty liver was significantly increased. The serum ALT and AST levels were decreased after treatment with water-soluble metal fullerene GF. The treatment group and model were observed. There was a significant difference in the comparison between the groups, indicating that the damage of the liver cells was alleviated and gradually returned to normal.

Example 9. Water-soluble amino acid modified inlaid metal fullerene structure reduces mitochondrial structure damage in fatty liver mice

Mitochondria play an initial and amplifying role in the apoptotic pathway, and endogenous mitochondrial apoptosis is one of the main pathways of apoptosis. The mitochondrial signals caused by various stress stimuli cause the mitochondrial outer membrane to change, the permeability increases, and the apoptosis-related proteins present in the mitochondrial membrane space under normal conditions diffuse into the cytoplasm. Among them, the released cytochrome c binds to apoptotic protease activator to form apoptotic body of cytochrome c/caspase activating factor and promote apoptosis.

(1) Experimental process

The mouse strain used in this experiment is [N000103]B6.Cg-Lep ^ob /Nju, ie ob/ob mice, which is a leptin-deficient obese mouse, purchased from Nanjing University-Nanjing Biomedical Research The hospital, cited from the Jackson Laboratory in the United States.

The experiment was divided into 3 groups, 6 mice in each group. The blank group was injected with normal saline in ob background mice without fatty liver. The model group was injected with normal saline for ob/ob mice with fatty liver; the treatment group was suffering from A fatty liver ob/ob mouse was injected with a solution of the water-soluble metal fullerene GF prepared in Example 6. All three groups of mice were administered intraperitoneally at a dose of 12 mg/kg/d. The mice were started at 6 weeks after birth and administered once a day for 15 days. After the samples were taken, the liver tissue samples were fixed, sliced and observed by cryo-TEM.

(2) Experimental results

As shown in Figure 15, the mitochondria in the liver of ob/ob mice were severely damaged, and many mitochondrial membrane ruptures lost function; there were also many mitochondrial matrix loss, which produced vacuoles, which seriously affected normal physiological functions; A small percentage of normal mitochondria are present. After 15 days of treatment with water-soluble metal fullerene in the treatment group, the mitochondrial state in the liver tissue was significantly improved, the mitochondria in the normal state increased, and the number of mitochondria in which the outer membrane rupture completely lost function was greatly reduced. This demonstrates that water-soluble metal fullerenes improve mitochondrial structural damage in fatty liver mice.

Example 10C _70- OH treatment of fatty liver

1. Experimental animals

The modeled mice used in this experiment were 5 week old C57BL6/J mice, male, with an average body weight of 23.31 g. The C57BL6/J mice were fed with 30% fructose water and standard feed for 10 weeks to construct model fatty liver mice at 5 weeks old. The average body weight of the mice after 30 weeks of feeding with 30% fructose water and standard feed was 30.26 g. All of them had fatty liver; the remaining 5 weeks old C57BL6/J normal mice were fed with sterile water and standard feed for 10 weeks as control. The average weight of normal control mice after 10 weeks of sterilized water and standard feed was 27.70. g.

2. Experimental grouping

The experimental animals were divided into 4 groups of 6 each, each group being:

Group A was normal mouse + saline group: this group of mice were normal C57BL6/J mice without fatty liver. The experiment lasted for 3 weeks, and the normal saline was injected once a week, three, and five tails, and the injection volume was 5 ml/ Kg;

Group B was normal mice + C ₇₀ -OH physiological saline solution group: this group of mice were normal C57BL6/J mice without fatty liver. The experiment lasted for 3 weeks, and C _{70 was} injected intravenously every week, three, and five tails. a C ₇₀ -OH physiological saline solution having a concentration of 2.5 mg/ml, an injection amount of 5 ml/kg, and C ₇₀ -OH is a hydroxylated derivative corresponding to C ₇₀ prepared in Example 1;

Group C was the model mouse + saline group: the model mice had fatty liver, the experiment lasted for 3 weeks, and the normal saline was injected once a week, three, and five tails, and the injection volume was 5 ml/kg;

Group D was model mice + C ₇₀ -OH physiological saline solution (2.5 mg/ml) group: model mice had fatty liver, the experiment lasted for 3 weeks, and the concentration of C ₇₀ -OH was injected intravenously every week, three, and five tails. 2.5 mg/ml of a C ₇₀ -OH physiological saline solution having an injection amount of 5 ml/kg, and C ₇₀ -OH is a hydroxylated derivative corresponding to C ₇₀ prepared in Example 1.

The above experiment lasted for 3 weeks, during which the body weight was counted, and finally the blood, various organs and tissues were taken and fixed or frozen for further testing.

3. Experimental results

(1) Liver index

The accumulation of lipids in the liver increases the quality of the liver and increases the proportion of body weight. Therefore, the liver index (heal mass as a percentage of body weight) can reflect the accumulation of fat in the liver. As shown in Fig. 16, the liver weight ratio of the mice in group C fed with 30% fructose solution was significantly increased compared with group A, and the liver index decreased in group D after intravenous injection of C ₇₀ -OH physiological saline solution, and group D and C. There was a significant difference between the groups, indicating that C ₇₀ -OH has a certain therapeutic effect on fatty liver by tail vein injection.

(2) Pathological section

As shown in Figure 17, the lipid content in group C increased, which was significantly different from the lipid content in the liver of group A normal mice, and the liver lipid content of group D treated mice decreased significantly. The liver accumulation in the mice was reduced after treatment.

Figure 18 is the results of HE staining pathological sections of livers of each group of mice. The liver of mice fed with 30% fructose water showed watery degeneration, the volume of hepatocytes increased, the cytoplasm was loose and lightly stained, and the damage in the portal area was more serious than other areas, and large vacuoles appeared in the liver cells. The degree of liver lesions in mice was improved after C ₇₀ -OH injection in the tail vein.

(3) serum blood biochemical indicators

Figure 19 is a statistical diagram of serum ALT and AST levels in each group of mice. Serum ALT and AST levels reflect the degree of hepatocyte damage and necrosis. It can be seen that the serum ALT and AST of mice fed with 30% fructose water only slightly increased. There was no significant difference. The ALT/AST levels of the D group mice decreased, which corresponded to the conclusions of liver index and pathology.

In conclusion, the tail vein injection of a C ₇₀ -OH physiological saline solution at a concentration of 2.5 mg/ml has a therapeutic effect on fructose-induced fatty liver.

Example 11C Comparison of therapeutic effects of _60- OH, C ₇₀ -OH and Gd@C ₈₂ (OH) _n : pathological tissue section analysis

The effects of C ₆₀ -OH, C ₇₀ -OH and Gd@C ₈₂ (OH) _n prepared in Example 1 on lipid accumulation in fatty liver were examined as shown in FIG. Pathological sections showed that C ₆₀ -OH, C ₇₀ -OH and Gd@C ₈₂ (OH) _n can treat lipid accumulation in fatty liver, and lipid accumulation on the liver of mice treated with Gd@C ₈₂ (OH) _n Significantly reduced, followed by C ₇₀ -OH, and finally lipid accumulation of C ₆₀ -OH, therefore, in terms of therapeutic effect, C ₆₀ -OH < C ₇₀ -OH < Gd@C ₈₂ (OH) _n .

Industrial applicability

The invention discloses a water-soluble fullerene structure for preparing a medicament for treating fatty liver, and discloses a pharmaceutical composition and a method for treating fatty liver. The active ingredient for treating fatty liver in the present invention is a water-soluble fullerene, a water-soluble inlaid metal fullerene, a water-soluble fullerene, and a water-soluble inlaid metal fullerene composition, the above three A pharmaceutically acceptable ester, a pharmaceutically acceptable salt of the above three. The water-soluble fullerene structure of the active ingredient of the invention can reduce fatty vesicles in the liver, lower blood lipids, and obviously improve liver function, so that the content of alanine aminotransferase and aspartate aminotransferase tends to be normal, and the effect of treating fatty liver is highly effective; The fullerene structure also regulates the level of redox in the liver.

Claims (14)

1. a water-soluble fullerene, a water-soluble inlaid metal fullerene, the water-soluble fullerene and the water-soluble inlaid metal fullerene composition, and the medicinal of the above three Use of an ester or a pharmaceutically acceptable salt of the above three in the preparation of a medicament for the treatment of fatty liver.
2. A method of treating fatty liver comprising administering to a subject having fatty liver an effective amount of at least one active ingredient selected from the group consisting of water soluble fullerenes, water soluble inlaid metal fullerenes, A composition of the water-soluble fullerene and the water-soluble inlaid metal fullerene, a pharmaceutically acceptable ester of the above three, and a pharmaceutically acceptable salt of the above three.
3. A pharmaceutical composition for treating fatty liver, comprising: at least one active ingredient selected from the group consisting of water-soluble fullerenes, water-soluble inlaid metal fullerenes, and said water-soluble fullerene a composition of an alkene and the water-soluble inlaid metal fullerene, a pharmaceutically acceptable ester of the above three, a pharmaceutically acceptable salt of the above three, the pharmaceutical composition further comprising a pharmaceutically acceptable carrier, At least one of a pharmaceutically acceptable diluent and a pharmaceutically acceptable excipient.
4. A health care product composition for improving fatty liver, comprising: at least one active ingredient selected from the group consisting of water-soluble fullerenes, water-soluble inlaid metal fullerenes, and said water-soluble rich a composition of the olefin and the water-soluble inlaid metal fullerene, a pharmaceutically acceptable ester of the above three, a pharmaceutically acceptable salt of the above three, the health care product composition further comprising a health care product At least one of a carrier, a diluent which can be used for a health care product, and an excipient which can be used for a health care product.
5. The application according to claim 1 or the method according to claim 2 or the pharmaceutical composition according to claim 3 or the health care composition according to claim 4, wherein:

   The water-soluble fullerene is selected from one or more of the group consisting of: (1) a fullerene having a hydrophilic group modified on the outer surface of the carbon cage; and (2) a hydrophilic organism on the outer surface of the carbon cage a small molecule-encapsulated fullerene; (3) a fullerene supported by a biocompatible carrier material; (4) a water-soluble supramolecular system fullerene formed by self-assembly;

   The water-soluble inlaid metal fullerene is selected from one or more of the group consisting of: (1) an inlaid metal fullerene having a hydrophilic group modified on the outer surface of the carbon cage; and (2) a carbon cage Inlaid metal fullerenes coated with hydrophilic small biomolecules; (3) embedded metal fullerenes supported by biocompatible carrier materials; (4) water-soluble supramolecular systems formed by self-assembly Embedded metal fullerenes.
6. The use according to claim 5 or the pharmaceutical composition according to claim 5 or the health care composition according to claim 5, wherein the hydrophilic group comprises a hydroxyl group One or more of a carboxyl group, a thiol group, an amino group, and a water-soluble amino acid residue; optionally, the water-soluble amino acid residue is an alanine residue, a glycine residue, a serine residue, an arginine residue At least one of a lysine residue and a tyrosine residue.
7. The application according to claim 6 or the method according to claim 6 or the pharmaceutical composition according to claim 6 or the health care composition according to claim 6, wherein:

   The water-soluble fullerene has the general formula C _2a (OH) _b (Amino Acid) _c , and Amino Acid represents a water-soluble amino acid residue; 30 ≤ a ≤ 60, and optionally a is 30 or 35; b ≤ 50, optional 0 < b ≤ 30, 10 ≤ b ≤ 30, and optionally b = 13, 20, 22, 24 or 26; 0 ≤ c ≤ 20, optional c = 2-15, Also optional c=6;

   The water-soluble inlaid metal fullerene has the formula M@C _2d (OH) _e (Amino Acid) _f , Amino Acid represents a water-soluble amino acid residue; M is selected from a rare earth metal, and the optional rare earth metal is Gd or La; 30 ≤ d ≤ 60, optional d is 41 or 30 or 35; 0 < e ≤ 50, optional 0 < e ≤ 30, optional 10 ≤ e ≤ 30, and optionally e = 13, 20, 22, 24 or 26; 0 ≤ f ≤ 20, optional f = 2-15, and optionally f = 6.
8. The application according to claim 1 or the method according to claim 2 or the pharmaceutical composition according to claim 3 or the health care composition according to claim 4, wherein the water-soluble fullerene It is obtained by water-soluble modification of the raw material fullerene; the water-soluble inlaid metal fullerene is obtained by water-soluble modification of the metal inlaid fullerene in the raw material.
9. The application according to claim 8 or the method according to claim 8 or the pharmaceutical composition according to claim 8 or the health care composition according to claim 8, wherein:

   The raw material fullerenes comprise one or more cage structures consisting of carbon atoms of the formula C _2m , 30 ≤ m ≤ 60, optionally m = 30 or 35;

   The metal inlaid metal fullerenes include M@C _2n , M ₂ @C _2n , MA@C _2n , M ₃ N@C _2n , M ₂ C ₂ @C _2n , M ₂ S@C _2n , M ₂ One or more of O@C _2n and M _x A _3-x N@C _2n , wherein: M and A each represent a metal element and M and A are each selected from the group consisting of a lanthanide metal element, Sc and Y. One type, 30≤n≤60, optional n=30 or 35 or 41; 0≤x≤3; N stands for nitrogen element, C stands for carbon element, S stands for sulfur element, and lanthanide metal element includes La, Ce, Pr , Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, and Lu.
10. The application according to claim 8 or the method according to claim 8 or the pharmaceutical composition according to claim 8 or the health care composition according to claim 8, wherein the water-soluble modification is selected from the group consisting of One of the following methods:

    (1) A method for preparing a water-soluble fullerene having a surface modified with a hydroxyl group comprises: mixing a raw material fullerene with a hydrogen peroxide solution and an alkali solution, and performing a reaction, and then washing the dialysis;

    The preparation method of the water-soluble inlaid metal fullerene having a surface modified with a hydroxyl group comprises: mixing the raw material inlaid metal fullerene with hydrogen peroxide and an alkali solution, reacting, and washing the dialysis;

    (2) A method for preparing a water-soluble fullerene having a surface modified with a hydroxyl group and an amino acid residue comprises: (a) preparing a water-soluble amino acid alkali solution using a water-soluble amino acid and a base; (b) preparing a fullerene molar according to an amino acid and a raw material The ratio of 1-1000:1 is mixed with the raw material fullerene; (c) the above mixture is reacted at 40-80 ° C, filtered to remove a small amount of unreacted solid powder; (d) the filtrate is dialyzed to remove small molecular impurities, filter;

    A method for preparing a water-soluble inlaid metal fullerene having a surface modified with a hydroxyl group and an amino acid residue comprises: (a) preparing a water-soluble amino acid alkali solution using a water-soluble amino acid and a base; (b) enriching the metal according to the amino acid and the raw material embedded metal The molar ratio of the enol is 1-1000:1, and the amino acid alkali solution is mixed with the raw material embedded fullerene; (c) the above mixture is reacted at 40-80 ° C, and the unreacted small amount of solid powder is removed by filtration; (d) the filtrate Diazetes were removed by dialysis and filtered.
11. The application according to claim 10 or the method according to claim 10 or the pharmaceutical composition according to claim 10 or the health care composition according to claim 10, wherein the surface is modified with a water-soluble hydroxyl group A method for preparing fullerenes or water-soluble inlaid metal fullerenes having a hydroxyl group surface modified comprises: weighing 20-200 mg of optional 30-100 mg C ₆₀ solid or C ₇₀ solid or Gd@C ₈₂ solid, and 3 -15ml 20-40% hydrogen peroxide, 2-10ml 5-20% alkali solution, mixed at 50-100 ° C until the solid is completely dissolved, then washed with ethanol, dialyzed to obtain the corresponding hydroxylated derivative, That is, the water-soluble fullerene structure; alternatively, the alkali solution is a NaOH solution or a KOH solution; alternatively, the conductivity of the liquid dialyzed to the outside of the dialysis bag is less than 1 μs/cm.
12. The use according to claim 10 or the method according to claim 10 or the pharmaceutical composition according to claim 10 or the health care composition according to claim 10, wherein the molar ratio of the water-soluble amino acid to the base It is 1:10-10, optionally 1:2 or 1:1-8; alternatively, the alkali content of the water-soluble amino acid alkali solution may be 10-50%, and optionally 14%. or

    10-30%); alternatively, the base is NaOH or KOH; alternatively, the molar ratio of the amino acid to the raw fullerene or the amino acid and the raw material inlaid metal fullerene is 50-1000:1, 100-1000 : 1,200-1000:1.
13. The application according to claim 1 or the method according to claim 2 or the pharmaceutical composition according to claim 3 or the health care composition according to claim 4, wherein the fatty liver comprises obesity fat At least one of liver, hyperlipidemic fatty liver, diabetic fatty liver, alcoholic fatty liver or drug-induced fatty liver.
14. The method according to claim 1 or the method according to claim 2 or the pharmaceutical composition according to claim 3, wherein the treating fatty liver comprises: 1) making the content of free fatty acids in the liver normal 2) reduce diffuse fat vacuoles in the liver; 3) improve liver function changes caused by fatty liver, make liver function indicators (alanine aminotransferase, aspartate aminotransferase) tend to normal; 4) make liver weight normal; 5) Improve the redox level in the liver, restore the SOD activity, CAT activity and MDA content to normal; 6) reduce blood lipids; 7) reduce hepatocyte mitochondrial damage.

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