RU2801902C1 - Composition and a method of treatment of diabetes - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to the use of a gold cluster (AuC) to obtain a medicinal product for the treatment of diabetes in a subject. Use of a gold cluster (AuC) to produce a medicinal product for the treatment of diabetes in a subject, while the said AuC comprises the following: a gold core and a gold core modifying ligand, where the ligand is selected from the group consisting of L-cysteine and its derivatives, D-cysteine and its derivatives, cysteine-containing oligopeptides and their derivatives and other thiol-containing compounds, the gold core has a diameter in the range of 0.5–2.6 nm.

EFFECT: solution described above makes it possible to use gold clusters to produce a medicinal product for the treatment of diabetes.

7 cl, 23 dwg, 7 tbl, 4 ex

Description

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

1. The present invention relates to the field of diabetes technology, in particular to compositions and methods for the treatment of diabetes.

BACKGROUND OF THE INVENTION

2. Diabetes (also called diabetes mellitus (DM)) affects hundreds of millions of people worldwide and is a group of metabolic disorders characterized by high blood sugar levels over a long period of time. Symptoms of high blood sugar include increased urination (polyuria), thirst (polydipsia), and feeling hungry (polyphagia). If left untreated, diabetes causes acute and chronic complications. Acute complications include diabetic ketoacidosis, hyperosmolar hyperglycemia, or death. Chronic complications include cardiovascular disease, stroke, chronic kidney disease, leg ulcers, and eye damage.

3. Diabetes occurs either due to insulin deficiency (i.e. the pancreas does not produce insulin or does not produce enough insulin) or insulin resistance (i.e. cells within the body do not respond properly to insulin) . Diabetes is divided into three main types. Type 1 diabetes results from the inability of the pancreas to produce enough insulin due to the loss of beta cells. Type 1 diabetes is treated with insulin injections. Type 2 diabetes begins with insulin resistance, a condition in which cells do not respond properly to insulin. Type 2 diabetes can be treated with drugs, with or without insulin. The progression of the disease can lead to insulin deficiency. Gestational diabetes is the third major form and occurs when pregnant women without a previous history of diabetes have elevated blood sugar levels.

4. Although available drugs help lower blood sugar levels, there is still an urgent need for new drugs and treatments for diabetes.

DISCLOSURE OF THE INVENTION

5. The purpose of the present invention is to provide a pharmaceutical composition and method for the treatment of diabetes.

6. In some embodiments, a method for producing ligand-modified gold clusters (AuCs) comprises providing a solution of HAuCl ₄ ; sequential addition of an acidic solution and a solution of the first ligand in a solution of HAuCl ₄ with the formation of a solution of the first mixture; where the molar ratio between the first ligand and HAuCl ₄ is in the range from 1:1 to 20:1; adding the NaBH ₄ solution to the first mixture solution to form a second mixture solution; where the molar ratio between NaBH ₄ and HAuCl ₄ is in the range from 1:1 to 10:1; adding an aprotic polar solvent to the second mixture solution to terminate the reaction and obtain a third mixture solution; centrifuging the solution of the third mixture to collect the bottom solid sediment; dissolving the collected lower solid precipitate in the second ligand solution to obtain a fourth mixture solution and keeping the fourth mixture solution for a certain period of time; where the molar ratio between the second ligand and HAuCl ₄ is in the range from 1:1 to 20:1; centrifuging the fourth solution of the mixture to obtain a supernatant; and dialysis of the supernatant in a dialysis bag with a predetermined molecular size limit.

7. In some embodiments of the method, the HAuCl ₄ solution consists of a neutral or acidic solvent selected from the group consisting of water, methanol, ethanol, n-propanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, pentanol , pentane, formic acid, acetic acid, diethyl ether, acetone, anisole, ethyl formate, methyl acetate, ethyl acetate, propyl acetate, isopropyl acetate, butyl acetate, isobutyl acetate, tributyl methyl ether, dimethyl sulfoxide, 2-methyl-1-propanol, and mixtures of two or more of them.

8. In some embodiments of the method, the acidic solution consists of an acid selected from the group consisting of hydrochloric acid, phosphoric acid, sulfuric acid, acetic acid, propionic acid, butyric acid, and a mixture of two or more of them.

9. In some embodiments of the method, the first ligand and the second ligand are selected from L-cysteine, D-cysteine, and other cysteine derivatives; cysteine-containing oligopeptides and their derivatives; other thiol-containing compounds; and mixtures of two or more of them.

10. In some embodiments of the method, the first ligand solution and the second ligand solution consist of a solvent selected from the group consisting of water, methanol, ethanol, n-propanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, pentanol, ethyl formate, methyl acetate, ethyl acetate, propyl acetate, isopropyl acetate, butyl acetate, isobutyl acetate, 2-methyl-1-propanol, and mixtures of two or more of them.

11. In some embodiments of the method, the NaBH ₄ solution consists of a solvent selected from the group consisting of water, methanol, ethanol, n-propanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, pentanol, ethyl formate, methyl acetate, ethyl acetate, propyl acetate, isopropyl acetate, butyl acetate, isobutyl acetate, 2-methyl-1-propanol, and mixtures of two or more of them.

12. In some embodiments of the method, the aprotic polar solvent is selected from the group consisting of dimethyl sulfoxide (DMSO), acetone, acetonitrile, dimethylformamide (DMF), dimethylacetamide (DMAC), hexamethylphosphoramide (HMP), chloroform, carbon tetrachloride, pyridine, and a mixture of two or more of them.

13. In some embodiments of the method, the residence time of the fourth solution of the mixture is from 3 to 24 hours.

14. In some embodiments, the method further comprises lyophilizing the dialyzed supernatant to obtain an AuCs powder.

15. In some embodiments, the present invention provides an AuCs powder produced by the method of the present invention.

16. In some embodiments, the implementation of the present invention uses a gold cluster (AuC) to obtain a drug for the treatment of diabetes in a subject, where the specified AuC contains a gold core; and a ligand that modifies the gold core.

17. In some applications, the gold core is less than 3 nm in diameter. In some embodiments, the implementation of the gold core has a diameter in the range of 0.5-2.6 nm.

18. In some applications, the ligand is selected from the group consisting of L-cysteine and its derivatives, D-cysteine and its derivatives, cysteine-containing oligopeptides and their derivatives, and other thiol-containing compounds.

19. In some applications, L-cysteine and its derivatives are selected from the group consisting of L-cysteine, N-isobutyryl-L-cysteine (L-NIBC) and N-acetyl-L-cysteine (L-NAC), and D -cysteine and its derivatives are selected from the group consisting of D-cysteine, N-isobutyryl-D-cysteine (D-NIBC) and N-acetyl-D-cysteine (D-NAC).

20. In some applications, the cysteine-containing oligopeptides and derivatives thereof are cysteine-containing dipeptides, cysteine-containing tripeptides, or cysteine-containing tetrapeptides.

21. In some applications, the cysteine-containing dipeptides are selected from the group consisting of L-cysteine-L-arginine (CR) dipeptide, L-arginine-L-cysteine (RC) dipeptide, L-histidine-L-cysteine (HC) dipeptide, and dipeptide L-cysteine-L-histidine (CH).

22. In some applications, the cysteine containing tripeptides are selected from the group consisting of glycine-L-cysteine-L-arginine (GCR) tripeptide, L-proline-L-cysteine-L-arginine (PCR) tripeptide, L-lysine-L tripeptide -cysteine-L-proline (CSR) and L-glutathione (GSH).

23. In some applications, the cysteine-containing tetrapeptides are selected from the group consisting of glycine-L-serine-L-cysteine-L-arginine (GSCR) tetrapeptide and glycine-L-cysteine-L-serine-L-arginine (GCSR) tetrapeptide.

24. In some applications, other thiol-containing compounds are selected from the group consisting of 1-[(2S)-2-methyl-3-thiol-1-oxopropyl]-L-proline, thioglycolic acid, mercaptoethanol, thiophenol, D-3- trololol, N-(2-mercaptopropionyl)-glycine and dodecylmercaptan.

25. The objects and advantages of the present invention will become apparent from the following detailed description of the preferred embodiments thereof in connection with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

26. Next, preferred embodiments of the present invention will be described with reference to the Figures, in which like reference numerals denote like elements.

27. In FIG. 1 shows ultraviolet and visible (UV) spectra, transmission electron microscope (TEM) images, and size distribution diagrams of L-NIBC ligand-modified gold nanoparticles (L-NIBC-AuNP) with different particle sizes.

28. In FIG. 2 shows ultraviolet (UV) and visible spectra, TEM images, and particle size distribution diagrams of L-NIBC ligand-modified gold clusters (L-NIBC-AuC) with various particle sizes.

29. In FIG. 3 shows infrared spectra of L-NIBC-AuCs with different particle sizes.

30. In FIG. 4 shows UV, IR, TEM and particle size distribution plots of CR ligand modified gold clusters (CR-AuCs).

31. In FIG. 5 shows UV, IR, TEM and particle size distribution plots of RC ligand modified gold clusters (RC-AuCs).

32. In FIG. 6 shows UV, IR, TEM and particle size distribution plots of gold clusters modified with ligand 1-[(2S)-2-methyl-3-thiol-1-oxopropyl]-L-proline (i.e. Cap) (Cap-AuCs).

33. In FIG. 7 shows UV, IR, TEM and particle size distribution plots of GSH ligand modified gold clusters (GSH-AuCs).

34. In FIG. 8 shows UV, IR, TEM and particle size distribution plots of D-NIBC ligand modified gold clusters (D-NIBC-AuCs).

35. In FIG. 9 shows the results of HE staining of the pancreas; (a) a negative control group; no abnormal changes; (b) a model control group; a sharp decrease in the number of islets, islet volumes and the number of islet cells, as well as an obvious infiltration of lymphocytes into the islets; (c) positive control group; a moderate decrease in the number of islets, islet volumes and number of islet cells, as well as an obvious infiltration of lymphocytes into the islets; (d) sample group; without a clear decrease in the number of islets, volumes of islets and the number of islet cells, as well as without infiltration of lymphocytes into the islets.

36. In FIG. 10 shows the results of immunohistochemistry of insulin in the pancreas; (a) a negative control group; normal insulin-positive islets; (b) a model control group; a sharp decrease in insulin-positive islets; (c) positive control group; a significant decrease in insulin-positive islets; (d) sample group; there is no apparent decrease in insulin-positive islets.

37. In FIG. 11 shows the results of insulin immunohistochemistry in islets; (a) a negative control group; normal number of insulin-positive cells; (b) a model control group; a sharp decrease in the number of insulin-positive cells; (c) positive control group; a significant decrease in the number of insulin-positive cells; (d) sample group; there is no apparent decrease in the number of insulin-positive cells.

38. In FIG. 12 shows a flow diagram of a method for producing AuCs in accordance with some embodiments of the present invention.

39. In FIG. 13 shows a comparison of a normal control group with a model control group: (a) differences in body weight, fat weight and lean meat of mice; (b) fasting blood glucose index and hypoglycemic capacity of insulin; (c) glucose tolerance index.

40. In FIG. 14 shows the acute hypoglycemic effect of drug A-03 in: (a) normal mice; (b) model mice.

41. In FIG. 15 shows the effect of drug A-03 on model mice: (a) body weight; (b) fat, where (1) is a normal control group, (2) is a model control group, (3) is an A-03 medium dose administration group; (c) muscle tissue, where (1) is the normal control group, (2) is the model control group, and (3) is the A-03 medium dose administration group.

42. In FIG. 16 shows the hypoglycemic effect of drug A-03 one month after administration, where (1) is a model control group; (2) is a high dose administration group; (3) is a medium dose group; and (4) is a low dose group.

43. In FIG. 17 shows the hypoglycemic effect of drug A-03 two months after administration, where (1) is a model control group, (2) is a high dose group, (3) is a medium dose group, and (4) is is a low dose group.

44. In FIG. 18 shows the effect of drug A-03 on (a) glucose tolerance and (b) the hypoglycemic effect of insulin (* means P<0.05).

45. In FIG. 19 shows the effect of medium and high doses of drug A-03 on blood lipids in model mice: (a) Plasma non-esterified fatty acids (NEFA plasma); (b) serum triglycerides (plasma TG); (c) total serum cholesterol (plasma cholesterol), where (1) represents the normal control group; (2) is a model control group; (3) represents the A-03 medium dose administration group; (4) represents the A-03 high dose administration group.

46. In FIG. 20 shows the effect of administering an average dose of A-03 on: (a) fasting blood glucose and (b) blood insulin, where (1) is a normal control group; (2) is a model control group; and (3) is the A-03 medium dose administration group.

47. In FIG. 21 shows the effect of administration of a medium dose of A-03 on the liver of mice: (a) expression of glucose-6-phosphate dehydrogenase (G6Pase), where (1) is a normal control group, (2) is a model control group, (3) is group introduction of the average dose of A-03; (b) the amount of glycogen in the liver, where (1) is the normal control group, (2) is the model control group, (3) is the A-03 medium dose administration group; (c) Expression of key molecules in oxidative phosphorylation during glucose metabolism, where in each gene the columns from left to right represent the normal control group, the model control group, and the A-03 mid-dose administration group.

48. In FIG. 22 shows the effect of drug A-03 on the liver: (a) Western blotting showed representative bands of pS473 PKB, PKB, pT389-S6k and S6K proteins; (b) and (c) are the results of the corresponding statistical analysis of expression, where 1-3 represent the normal control group, model control group and the average dose group A-03.

49. In FIG. 23 shows the effect of A-03 administration on (a) glutamine oxaloacetic transaminase and (b) alanine aminotransferase in mice on a high fat diet, where 1-3 represent the normal control group, the model control group, and the A-03 medium dose administration group.

IMPLEMENTATION OF THE INVENTION

50. The present invention may be better understood with reference to the following detailed description of some embodiments of the invention.

51. In this application, where there are references to publications, the disclosure of these publications is hereby incorporated by reference in its entirety into the present application in order to more fully describe the prior art to which the present invention pertains.

52. Gold clusters (AuCs) are a special form of gold that exists between gold atoms and gold nanoparticles. AuCs are less than 3 nm in size and consist of several to several hundreds of gold atoms, which leads to the collapse of the face-centered cubic packing of the gold nanoparticle structure. As a result, AuCs exhibit molecular-like discrete electronic structures with a distinct HOMO-LUMO gap, in contrast to the continuous or quasi-continuous energy levels of gold nanoparticles. This leads to the disappearance of the surface plasmon resonance effect and the corresponding plasmon resonance absorption band (520 ± 20 nm) in the UV-visible spectrum, which ordinary gold nanoparticles have.

53. The present invention relates to AuC modified with a ligand.

54. In some embodiments, the ligand-modified AuC contains a ligand and a gold core in the range of 0.5-3 nm in diameter. In some embodiments, the gold core diameter is in the range of 0.5-2.6 nm.

55. In some embodiments, the ligand of the ligand-modified AuC is a thiol-containing compound or an oligopeptide. The ligand modifies the gold core to form ligand-modified AuC via an Au-S bond.

56. In some embodiments, the implementation of the ligand is, among other things, L-cysteine, D-cysteine or a cysteine derivative. In some embodiments, the cysteine derivative is N-isobutyryl-L-cysteine (L-NIBC), N-isobutyryl-D-cysteine (D-NIBC), N-acetyl-L-cysteine (L-NAC), or N-acetyl -D-cysteine (D-NAC).

57. In some embodiments, the implementation of the ligand is, among other things, a cysteine-containing oligopeptide and its derivatives. In some embodiments, the cysteine-containing oligopeptide is a cysteine-containing dipeptide. In some embodiments, the cysteine-containing dipeptide is L-cysteine-L-arginine (CR) dipeptide, L-arginine-L-cysteine (RC) dipeptide, or L-cysteine-L-histidine (CH) dipeptide. In some embodiments, the cysteine-containing oligopeptide is a cysteine-containing tripeptide. In some embodiments, the cysteine-containing tripeptide is a glycine-L-cysteine-L-arginine (GCR) tripeptide, an L-proline-L-cysteine-L-arginine (PCR) tripeptide, or L-glutathione (GSH). In some embodiments, the cysteine-containing oligopeptide is a cysteine-containing tetrapeptide. In some embodiments, the cysteine-containing tetrapeptide is a glycine-L-serine-L-cysteine-L-arginine (GSCR) tetrapeptide or a glycine-L-cysteine-L-serine-L-arginine (GCSR) tetrapeptide.

58. In some embodiments, the implementation of the ligand is a thiol-containing compound. In some embodiments, the thiol-containing compound is 1-[(2S)-2-methyl-3-thiol-1-oxopropyl]-L-proline, thioglycolic acid, mercaptoethanol, thiophenol, D-3-trololol, or dodecylmercaptan.

59. The present invention provides a pharmaceutical composition for the treatment of diabetes in a subject. In some embodiments, the subject is a human. In some embodiments, the subject is a pet, such as a dog.

60. In some embodiments, the pharmaceutical composition comprises AuC modified with a ligand as described above and a pharmaceutically acceptable excipient. In some embodiments, the excipient is a phosphate buffer solution or saline.

61. The present invention provides the use of the AuCs described above for the preparation of a medicament for the treatment of diabetes in a subject.

62. The present invention provides the use of the AuCs described above for the treatment of diabetes in a subject, or a method of treating diabetes in a subject using the AuCs disclosed above. In some embodiments, the method of treatment comprises administering to the subject a pharmaceutically effective amount of AuCs. A pharmaceutically effective amount can be determined by routine in vivo studies.

63. The present invention provides a method for producing ligand-modified gold clusters (AuCs). Referring now to FIG. 12 is a flow diagram of a method in accordance with some embodiments of the present invention. Method 1200 includes:

64. providing a solution of HAuCl ₄ 1210, wherein the HAuCl ₄ solution consists of a neutral or acidic solvent selected from the group consisting of water, methanol, ethanol, n-propanol, 1-propanol, 2-propanol, 1-butanol, 2- butanol, pentanol, pentane, formic acid, acetic acid, diethyl ether, acetone, anisole, ethyl formate, methyl acetate, ethyl acetate, propyl acetate, isopropyl acetate, butyl acetate, isobutyl acetate, tributyl methyl ether, dimethyl sulfoxide, 2-methyl-1-propanol, and mixtures of two or more of them. In some embodiments, the solvent is methanol. In some embodiments, the HAuCl ₄ solution is pre-cooled to 0° C. or below without light with slow stirring. In some embodiments, the concentration of HAuCl ₄ in the HAuCl ₄ solution is in the range of 1-20 g/L, preferably 2-10 g/L. In some embodiments, when the total volume of the complete reaction is indicated as 1 volume, the HAuCl ₄ solution is in the range of 0.1-0.7 volume, preferably in the range of 0.2-0.5 volume.

65. Sequential addition of the acidic solution and the first ligand solution to the NAuSC solution to form the first mixture solution 1220. In some embodiments, the acidic solution consists of an acid selected from the group consisting of hydrochloric acid, phosphoric acid, sulfuric acid, acetic acid, propionic acid , butyric acid and mixtures of two or more of them. In some embodiments, the implementation of the acidic solution is in the range of 0.01-0.1 volume. In some embodiments, the acidic solution allows the reaction to proceed at pH<5, preferably at a pH in the range of 1-3, most preferably at a pH in the range of 1-2. In some embodiments, the reaction temperature is below 4°C. In some embodiments, the first ligand is selected from L-cysteine, D-cysteine, and other cysteine derivatives such as N-isobutyryl-L-cysteine (L-NIBC), N-isobutyryl-D-cysteine (D-NIBC), N- acetyl-L-cysteine (L-NAC) and N-acetyl-D-cysteine (D-NAC), cysteine-containing oligopeptides and their derivatives, including, but not limited to, dipeptides, tripeptides, tetrapeptides and other cysteine-containing peptides, such as dipeptide L-cysteine-L-arginine (CR), dipeptide L-arginine-L-cysteine (RC), L-cysteine-L-histidine (CH), tripeptide glycine-L-cysteine-L-arginine (GCR), tripeptide L-proline-L-cysteine-L-arginine (PCR), L-glutathione (GSH), tetrapeptide glycine-L-serine-L-cysteine-L-arginine (GSCR) and tetrapeptide glycine-L-cysteine-L- serine-L-arginine (GCSR), and other thiol-containing compounds such as one or more of 1-[(2S)-2-methyl-3-thiol-1-oxopropyl]-L-proline, thioglycolic acid, mercaptoethanol, thiophenol , D-3-trololol, dodecylmercaptan and mixtures of two or more of them. In some embodiments, the first ligand solution consists of a solvent selected from the group consisting of water, methanol, ethanol, n-propanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, pentanol, ethyl formate, methyl acetate, ethyl acetate , propyl acetate, isopropyl acetate, butyl acetate, isobutyl acetate, 2-methyl-1-propanol, and mixtures of two or more of them. In some embodiments, the concentration of the first ligand in the first ligand solution is in the range of 5-150 g/L, preferably 20-80 g/L. In some embodiments, the solution of the first ligand is in the range of 0.01-0.6 volume. In some embodiments, the implementation of the molar ratio between the first ligand and HAuCL is in the range from 1:1 to 20:1, preferably from 2:1 to 5:1. In some embodiments, the reaction time of the solution of the first mixture is 0.5-2 hours.

66. adding the NaBH ₄ solution to the first mixture solution to form the second mixture solution 1230. In some embodiments, the NaBH ₄ solution consists of a solvent selected from the group consisting of water, methanol, ethanol, n-propanol, 1-propanol, 2- propanol, 1-butanol, 2-butanol, pentanol, ethyl formate, methyl acetate, ethyl acetate, propyl acetate, isopropyl acetate, butyl acetate, isobutyl acetate, 2-methyl-1-propanol, and mixtures of two or more of them. In some embodiments, the NaBH ₄ concentration in the NaBH ₄ solution is in the range of 1-100 g/L, preferably 10-50 g/L. In some embodiments, the volume of the NaBH ₄ solution is in the range of 0.01-0.6. In some embodiments, the implementation of the molar ratio between NaBH ₄ and HAuCl ₄ is in the range from 1:1 to 10:1, preferably from 1:1 to 5:1. In some embodiments, the reaction period of the solution of the second mixture is in the range of 5-100 minutes.

67. adding an aprotic polar solvent to the second mixture solution to terminate the reaction and obtain a third mixture solution 1240. In some embodiments, the aprotic polar solvent is selected from the group consisting of dimethyl sulfoxide (DMSO), acetone, acetonitrile, dimethylformamide (DMF), dimethylacetamide (DMAC) ), hexamethylphosphoramide (HMP), chloroform, carbon tetrachloride, pyridine, and a mixture of two or more of them.

68. centrifugation of the solution of the third mixture to collect solids 1250. In some embodiments, the centrifugation is carried out in the range of 1500-6000g, preferably 2000-5000g.

69. dissolving the solid precipitate from step 1250 in a solution of the second ligand to obtain a solution of the fourth mixture and maintaining the solution of the fourth mixture for a period of time 1260. In some embodiments, the implementation of the second ligand is selected from L-cysteine, D-cysteine and other cysteine derivatives, such as N-isobutyryl-L-cysteine (L-NIBC), N-isobutyryl-D-cysteine (D-NIBC), N-acetyl-L-cysteine (L-NAC) and N-acetyl-D-cysteine (D-NAC ), cysteine-containing oligopeptides and their derivatives, including, but not limited to, dipeptides, tripeptides, tetrapeptides, and other cysteine-containing peptides such as L-cysteine-L-arginine dipeptide (CR), L-arginine-L-cysteine dipeptide ( RC), L-cysteine-L-histidine (CH), tripeptide glycine-L-cysteine-L-arginine (GCR), tripeptide L-proline-L-cysteine-L-arginine (PCR), L-glutathione (GSH) , tetrapeptide glycine-L-serine-L-cysteine-L-arginine (GSCR) and tetrapeptide glycine-L-cysteine-L-serine-L-arginine (GCSR), and other thiol-containing compounds such as one or more of 1- [(2S)-2-methyl-3-thiol-1-oxopropyl]-L-proline, thioglycolic acid, mercaptoethanol, thiophenol, D-3-trololol, dodecylmercaptan, and mixtures of two or more of them. In some embodiments, the second ligand solution consists of a solvent selected from the group consisting of water, methanol, ethanol, n-propanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, pentanol, ethyl formate, methyl acetate, ethyl acetate , propyl acetate, isopropyl acetate, butyl acetate, isobutyl acetate, 2-methyl-1-propanol, and mixtures of two or more of them. In some embodiments, the first ligand and the second ligand are the same. In some embodiments, the solvents in the first ligand solution and the second ligand solution are the same. In some embodiments, the concentration of the second ligand in the second ligand solution is in the range of 5-100 g/L, preferably 20-50 g/L. In some embodiments, the solution of the second ligand is in the range of 0.1-10 volumes, preferably 1-2 volumes. It should be noted that "volume" in this document refers to the same designation as indicated above. In some embodiments, the implementation of the molar ratio between the second ligand and HAuCl ₄ is in the range from 1:1 to 20:1, preferably from 2:1 to 5:1. In some embodiments, the implementation of the time period is in the range from 3 to 24 hours.

70. centrifugation of the fourth mixture solution to obtain supernatant 1270. In some embodiments, the centrifugation is in the range of 2000-8000g, preferably 3000-5000g. In some embodiments, the centrifugation time is in the range of 3-50 minutes, preferably 5-15 minutes.

71. Dialysis of the supernatant in a dialysis bag with a target molecular size threshold of 1280. In some embodiments, the threshold molecular size is in the range of 500-5000 Daltons. In some embodiments, the dialysis is carried out in ultrapure water and the water is changed 3-10 times.

72. Lyophilization of dialyzed supernatant to obtain AuCs 1290 powder.

73. The following examples are given for the sole purpose of illustrating the principles of the present invention; they are not intended to limit the scope of the present invention in any way.

74. Examples

75. Embodiment 1: Obtaining Ligand Modified AuCs

76. 1.1 HAuCl ₄ was dissolved in methanol, water, ethanol, n-propanol or ethyl acetate to obtain solution A, in which the concentration of HAuCl ₄ is 0.01-0.03 M;

77. 1.2 The ligand was dissolved in the solvent to obtain solution B, in which the concentration of the ligand is 0.01~0.18 M; the ligand includes, but is not limited to, L-cysteine, D-cysteine and other cysteine derivatives such as N-isobutyryl-L-cysteine (L-NIBC), N-isobutyryl-D-cysteine (D-NIBC), N-acetyl-L-cysteine (L-NAC) and N-acetyl-D-cysteine (D-NAC), cysteine-containing oligopeptides and their derivatives, including, but not limited to, dipeptides, tripeptides, tetrapeptides, and other cysteine-containing peptides such as L-cysteine-L-arginine dipeptide (CR), L-arginine-L-cysteine dipeptide (RC), L-cysteine-L-histidine (CH), glycine-L-cysteine-L-arginine tripeptide (GCR ), tripeptide L-proline-L-cysteine-L-arginine (PCR), L-glutathione (GSH), tetrapeptide glycine-L-serine-L-cysteine-L-arginine (GSCR) and tetrapeptide glycine-L-cysteine- L-serine-L-arginine (GCSR), and other thiol-containing compounds such as one or more of 1-[(2S)-2-methyl-3-thiol-1-oxopropyl]-L-proline, thioglycolic acid, mercaptoethanol , thiophenol, D-3-trololol and dodecylmercaptan; wherein the solvent is one or more of methanol, ethyl acetate, water, ethanol, n-propanol, pentane, formic acid, acetic acid, diethyl ether, acetone, anisole, 1-propanol, 2-propanol, 1-butanol, 2- butanol, pentanol, butyl acetate, tributyl methyl ether, isopropyl acetate, dimethyl sulfoxide, ethyl formate, isobutyl acetate, methyl acetate, 2-methyl-1-propanol and propyl acetate;

78. 1.3 Solution A and solution B were mixed so that the molar ratio between HAuCl ₄ and ligand was 1:(0.01~100) by stirring them in an ice bath for 0.1~48 hours, adding 0.025~0 ,8 M solution of NaBH ₄ in water, ethanol or methanol, continuing to stir in a bath of ice water and carry out the reaction for 0.1-12 hours. The molar ratio between NaBH ₄ and the ligand is 1:(0.01~100);

79. 1.4 MWCO 3K~30K ultrafiltration tubes were used to centrifuge the reaction solution at 8000~17500 rpm in a gradient for 10~100 minutes after the end of the reaction to obtain a ligand-modified AuCs pellet with various average particle sizes. The opening of filtration membranes for ultrafiltration tubes with various MWCOs directly determines the size of the ligand-modified AuCs that can pass through the membranes. This step can optionally be omitted;

80. 1.5 The precipitate of ligand-modified AuCs with different average particle sizes obtained in step (1.4) was dissolved in water, placed in a dialysis bag, and dialyzed in water at room temperature for 1~7 days;

81. 1.6 Ligand-modified AuCs were lyophilized within 12~24 hours after dialysis to obtain a substance in the form of a powder or flocculant, i.e. AuCs modified with a ligand.

82. It has been found that the particle size of the substance in the form of a powder or flocculant obtained by the above method is less than 3 nm (0.5-2.6 nm in general). There is no clear absorption peak at 520 nm. It was determined that the resulting powder or flakes are ligand-modified AuCs.

83. Option 2. Preparation and characterization of AuCs modified with different ligands

84.2.1 Preparation of L-NIBC-AuCs

85. Taking L-NIBC ligand as an example, the production and confirmation of L-NIBC ligand modified AuCs is described in detail.

86. 2.1.1 Weigh 1.00 g of HAuCl ₄ and dissolve it in 100 ml of methanol to obtain a 0.03 M solution A;

87. 2.1.2 Weigh out 0.57 g of L-NIBC and dissolve it in 100 ml of glacial acetic acid (acetic acid) to obtain a 0.03 M solution B;

88. 2.1.3 Measure 1 ml of solution A, mix it with 0.5 ml, 1 ml, 2 ml, 3 ml, 4 ml or 5 ml of solution B, respectively (i.e. the molar ratio between HAuCl ₄ and L-NIBC is 1:0.5, 1:1, 1:2, 1:3, 1:4, 1:5, respectively), the reaction was carried out in an ice bath with stirring for 2 hours, 1 ml of freshly prepared 0.03 M (prepared by weighing 11.3 mg of NaBH ₄ and dissolving it in 10 ml of ethanol) a solution of NaBH ₄ in ethanol when the solution turned colorless from bright yellow, continue the reaction for 30 minutes after the solution turned dark brown, and 10 ml of acetone was added to stop the reaction.

89. 2.1.4 After the reaction, the reaction solution was subjected to gradient centrifugation to obtain L-NIBC-AuCs powder with particles of different sizes. Specific Method: After completion of the reaction, the reaction solution was transferred to a 50 ml MWCO 30K ultrafiltration tube and centrifuged at 10,000 rpm for 20 minutes, and the retentate in the inner tube was dissolved in ultrapure water to obtain a powder with a particle size of about 2. 6 nm. Then, the mixed solution in the outer tube was transferred to a 50 ml ultrafiltration tube with MWCO 10K and centrifuged at 13,000 rpm for 30 minutes. The retentate in the inner tube was dissolved in ultrapure water to obtain a powder with a particle size of about 1.8 nm. Then, the mixed solution in the outer tube was transferred to a 50 ml ultrafiltration tube with MWCO 3K and centrifuged at 17500 rpm for 40 minutes. The retentate in the inner tube was dissolved in ultrapure water to obtain a powder with a particle size of about 1.1 nm.

90. 2.1.5 Powder in the form of particles of three different sizes obtained by gradient centrifugation was precipitated, the solvent was removed respectively, the crude product was dried with N ₂ , it was dissolved in 5 ml of ultrapure water, placed in a dialysis bag (MWCO is 3 kDa), the dialysis bag was placed in 2 L of ultrapure water, the water changed every other day, dialyzed for 7 days, lyophilized and stored for future use.

91. 2.2 Characterization of L-NIBC-AuCs

92. A characterization experiment was performed on the powder prepared above (L-NIBC-AuCs). Meanwhile, L-NIBC ligand-modified gold nanoparticles (L-NIBC-AuNP) were used as a control. A method for producing gold nanoparticles with a ligand representing L-NIBC refers to the reference (W. Yan, L. Xu, C. Xu, W. Ma, H. Kuang, L. Wang and N. A. Kotov, Journal of the American Chemical Society 2012, 134, 15114; X. Yuan, B. Zhang, Z. Luo, Q. Yao, D. T. Leong, N. Yan and J. Xie, Angewandte Chemie International Edition 2014, 53, 4623).

93. 2.2.1 Observation of morphology with a transmission electron microscope (TEM)

94. Test powders (sample L-NIBC-AuCs and sample L-NIBC-AuNPs) were dissolved in ultrapure water to 2 mg/l as samples, and then test samples were prepared by the hanging drop method. In particular, 5 μl of the samples were dropped onto an ultrathin carbon film, evaporated naturally until the water drop disappeared, and then the morphology of the samples was observed using a JEM-2100F STEM/EDS high resolution field emission TEM.

95. Four TEM images of L-NIBC-AuNPs are shown in panels B, E, H and J in FIG. 1; three TEM images of L-NIBC-AuCs are shown in panels B, E, and H in FIG. 2.

96. Images in FIG. 2 shows that each of the L-NIBC-AuCs samples has the same particle size and good dispersibility, and the average diameter of L-NIBC-AuCs (referring to the diameter of the gold core) is 1.1 nm, 1.8 nm and 2.6 nm respectively, which is in good agreement with the results in panels C, F and I of FIG. 2. In comparison, L-NIBC-AuNPs samples have a larger particle size. Their average diameter (referring to the diameter of the gold core) is 3.6 nm, 6.0 nm, 10.1 nm, and 18.2 nm, respectively, in good agreement with the results in panels C, F, I, and L in FIG. 1.

97. 2.2.2 Absorption spectrum in the ultraviolet (UV)-visible (View) region

98. Test powders (sample L-NIBC-AuCs and sample L-NIBC-AuNPs) were dissolved in ultrapure water to a concentration of 10 mg⋅l ¹ and UV-Vis absorption spectra were measured at room temperature. The scanning range was 190-1100 nm, the sample cuvette was a standard quartz cuvette with an optical path of 1 cm, the reference cell was filled with ultrapure water.

99. UV-Vis absorption spectra of four L-NIBC-AuNPs of different sizes are shown in panels A, D, G, and J of FIG. 1, and the statistical particle size distribution is shown in panels C, F, I, and L in FIG. 1; the absorption spectra in the UV-Visible region of three samples of L-NIBC-AuCs of different sizes are shown in panels A, D and G in FIG. 2, and the statistical particle size distribution is shown in panels C, F, and I of FIG. 2.

100. In FIG. 1 shows that, due to the surface plasmon effect, L-NIBC-AuNPs had an absorption peak at about 520 nm. The position of the absorption peak depends on the particle size. At a particle size of 3.6 nm, the UV absorption peak appears at 516 nm; at a particle size of 6.0 nm, the UV absorption peak appears at 517 nm; at a particle size of 10.1 nm, the UV absorption peak appears at 520 nm, and at a particle size of 18.2 nm, the absorption peak appears at 523 nm. None of the four samples has an absorption peak above 560 nm.

101. In FIG. Figure 2 shows that in the UV absorption spectra of three L-NIBC-AuCs samples with different particle sizes, the absorption peak of the surface plasmon effect at 520 nm disappeared, and two obvious absorption peaks appeared above 560 nm, and the position of the absorption peaks varied slightly depending on the particle sizes. AuCs. This is due to the fact that AuCs exhibit molecular-like properties due to the collapse of the face-centered cubic structure, which leads to a break in the density of states of AuCs, splitting of energy levels, disappearance of the plasmon resonance effect, and the appearance of a new absorption peak in the long-wavelength direction. It can be concluded that all three powder samples with different particle sizes obtained above are ligand-modified AuCs.

102. 2.2.3 Fourier transform infrared spectroscopy

103. Infrared spectra were measured on a Bruker VERTEX80V FTIR spectrometer in total reflection mode with solid powder under high vacuum. The scan range was 4000-400 cm ^-1 and the number of scans was 64. For example, for samples of L-NIBC-AuCs, the test samples were dry powder L-NIBC-AuCs with three different particle sizes, and the control sample was pure L-NIBC powder. The results are shown in FIG. 3.

104. In FIG. 3 shows the infrared spectrum of L-NIBC-AuCs with different particle sizes. Compared to pure L-NIBC (bottom curve), the SH stretching vibrations in L-NIBC-AuCs with different particle sizes completely disappeared at 2500-2600 cm ^-1 , while other characteristic peaks of L-NIBC were still observed, proving that that L-NIBC molecules were successfully anchored to the surface of AuCs via the Au-S bond. The figure also shows that the infrared spectrum of ligand-modified AuCs is independent of their size.

105. AuCs modified with other ligands were prepared in a manner similar to that described above, except that the solvent of solution B, the feed ratio between HAuCl ₄ and ligand, the reaction time, and the amount of NaBH ₄ added were slightly adjusted. For example: when L-cysteine, D-cysteine, N-isobutyryl-L-cysteine (L-NIBC) or N-isobutyryl-D-cysteine (D-NIBC) was used as the ligand, acetic acid was chosen as the solvent; when CR dipeptide, RC dipeptide or 1-[(2S)-2-methyl-3-thiol-1-oxopropyl]-L-proline was used as a ligand, water was chosen as a solvent, and so on and so forth; the other steps are similar, so no further details are given in this document.

106. Within the framework of the present invention, a series of ligand-modified AuCs was prepared and obtained in the manner described above. Ligands and production method parameters are shown in Table 1.

107.

108. The samples listed in Table 1 are confirmed by the above methods. Characteristics of five different ligand-modified AuCs are shown in FIG. 4 (CR-AuCs), in FIG. 5 (RC-AuCs), in FIG. 6 (Cap-AuCs) (Cap is 1-[(2S)-2-methyl-3-thiol-1-oxopropyl]-L-proline), in FIG. 7 (GSH-AuCs) and FIG. 8 (D-NIBC-AuCs). FIG. 4-8 show UV spectra (panel A), infrared spectra (panel B), TEM images (panel C) and particle size distribution (panel D).

109. The results show that all diameters of AuCs modified with various ligands obtained from Table 1 are less than 3 nm. In the ultraviolet spectra, the disappearance of the peak at 520±20 nm and the appearance of an absorption peak above 560 nm are also observed. Only the position of this absorption peak varies slightly depending on the ligand and particle size. Meanwhile, the Fourier transform infrared spectra also show the disappearance of the ligand thiol infrared absorption peak (between the dotted lines in panel B of FIGS. 4-8), while all other infrared characteristic peaks are preserved, suggesting that all ligand molecules were successfully attached to the surface of AuCs, and within the framework of the present invention, AuCs modified with the ligands listed in Table 1 were successfully obtained.

110. Embodiment 3

111. 3.1 Materials and animals

112. 3.1.1 Test specimen

113. 3.1.1.1 L-NIBC-AuCs with a core diameter of 1.5 nm were synthesized according to the protocol below.

114. 0.5 volume of a methanol solution of HAuCl ₄ with a concentration of 5 g/l was added to the reaction vessel for pre-cooling to 0°C without light. Then the methanolic solution of HAuCl ₄ was kept at 0°C and slowly stirred. 0.05 volumes of acetic acid (above analytical grade) and 0.1 volumes of 40 g/L methanolic NIBC were added successively. After 1 hour of reaction, the rotation speed was increased and 0.3 volumes of 15 g/l NaBH ₄ ethanol solution were added. The solution continued to react for 30 minutes, then the reaction was stopped by adding enough acetone. The solution was then centrifuged at 4000 rpm for 10 minutes. The supernatant was removed to collect the bottom solid. Then, 1 volume of 30 g/l NIBC aqueous solution was added to the solid precipitate, which was dissolved and sealed for aging. After standing for 24 hours, the solution was centrifuged at 4000 rpm for 10 minutes to remove the precipitate, and the supernatant was placed in a dialysis bag with a molecular weight of 3000, which was placed in ultrapure dialysis water. After 5 times dialysis, the dialysis bag solution was removed and lyophilized to obtain a powder for use as a test sample. The test piece had a diameter of 1.5 nm and a spherical shape as shown in the TEM image (FIG. 2K). The absorption spectrum of the UV-Vis test sample is shown in FIG. 2J, and the statistical particle size distribution is shown in FIG. 2L.

115. 3.1.1.2 Specification: 25 mg/vial;

116. 3.1.1.3 Characteristics: Grey-brown powder;

117. 3.1.1.4 Storage conditions: 2~8°C Preservation indoors;

118. 3.1.2 Positive control sample

119. 3.1.2.1 Name: Pioglitazone hydrochloride tablets;

120. 3.1.2.2 Batch number: 1809001;

121. 3.1.2.3 Features: White or white-like object;

122. 3.1.2.4 Specification/purity: pioglitazone 15 mg;

123. 3.1.2.5 Packaging: aluminum plastic packaging, 7 pieces per box;

124. 3.1.2.6 Production date: 20180901;

125. 3.1.2.7 Valid until: 202108;

126. 3.1.2.8 Manufacturer: Yantai Zhengfang Pharmaceutical Co., Ltd.;

127. 3.1.2.9 Clinical dose or recommended human dose: The initial dose may be 15 mg or 30 mg once daily. If the initial dose did not give a good response, up to 45 mg was added once a day.

128. 3.1.2.10 Storage conditions: in a closed hermetic state.

129. 3.1.3 Tools

130. 3.1.3.1 Electronic analytical balance (Shanghai Yousheng Weighing Apparatus Co., Ltd.; Sartorius Scientific Instruments (Beijing) Technology Co., Ltd.);

131. 3.1.3.2 Glucometer and blood glucose test strip (Johnson &Johnson);

132. 3.1.3.3 Automatic dehydrator LEICA® ASP300S;

133. 3.1.3.4 Machine for filling biological tissues LEICA® EG1150;

134. 3.1.3.4 LEICA® Rotary Slicer Model RM2235;

135. 3.1.3.5 Distributor LEICA® HI1210;

136. 3.1.3.6 Dryer LEICA® HI1220;

137. 3.1.3.7 LEICA® AutoStainer-XL;

138. 3.1.3.8 Biological microscope OLYMPUS® BX43;

139. 3.1.3.9 Microscopic imaging software OLYMPUS® CellSens Dimension;

140. 3.1.3.10 Image-Pro® Plus Image Analysis Software Version 6.0.

141. 3.1.4 Reagents

142. 3.1.4.1 Sodium chloride solution (Guangdong Kelun Pharmaceutical Co., Ltd.);

143. 3.1.4.2 Sodium carboxymethylcellulose (Tianjin Fuchen Chemical Reagent Factory); accurately weighed 5.0 g of CMC-Na, slowly added to 800 ml of purified water in a beaker with stirring with a magnetic stirrer until dissolved; 2-8°C, during the night; diluted to 1000 ml the next day; and stored at 2-8°C for later use;

144. 3.1.4.3 Glucose (Guangdong Guanghua Technology Co., Ltd.); a 0.25 g/ml glucose solution was prepared by adding 7.5 g glucose to purified water to a final volume of 30 ml; a 0.125 g/ml glucose solution was prepared by adding 7.5 g of glucose to purified water to a final volume of 60 ml;

145. 3.1.4.4 Streptozotocin (STZ) (MP Bio company); a solution for applying STZ with a concentration of 5 mg/ml was prepared by dissolving 0.15 g of STZ in 30 ml of nitrate buffer with a concentration of 0.1 mol/l;

146. 3.1.4.5 Citric acid monohydrate, anhydrous ethanol, 95% ethanol (Guangdong Guanghua Technology Co., Ltd.); sodium citrate (Guangzhou Zhongnan Chemical Reagent Co., Ltd.); 0.1 mol/L citrate buffer was prepared as follows: add 2.1 g of citric acid monohydrate to deionized water to a final volume of 100 ml, which is liquid A; by adding 2.94 g of sodium citrate to deionized water to a final volume of 100 ml, which is liquid B; when used, mixed A and B in a ratio of 1:1~1.2, adjusted the pH to 4.2~4.5; stored at 2~8°C.

147. 3.1.4.6 Wax (Maoming Dachuan Special Wax Factory Co., Ltd.);

148. 3.1.4.7 Transparent agent (Shanghai Hongzi Industrial Co., Ltd.);

149. 3.1.4.8 Pink dye (water soluble) (Shanghai Aladdin Biochemical Technology Co., Ltd.); And

150. 3.1.4.9 Su Musu (Shanghai Aladdin Biochemical Technology Co., Ltd).

151. 3.2 Animal experiment

152. 3.2.1 C57BL/6 mice are the most commonly used in diabetes research. 90 male C57BL/6 mice (SPF level) with a body weight of 18.8-24.2 g at the beginning of the experiment were purchased from the Guangdong Provincial Animal Medical Laboratory Center (Experimental Animal Production License No. SCXK (Yue) 2018-0002; animal certificate No. 44007200059759);

153. 3.2.2 The content and procedures associated with animal testing comply with applicable laws and regulations governing the use and management of laboratory animals and the relevant provisions of the Institutional Animal Welfare Ethics Committee for Laboratory Animals;

154. 3.2.3 At the end of the experiments, mice were intraperitoneally injected with a 3% sodium pentobarbital solution in an amount of 1 ml/kg of body weight, eyeballs and blood were collected, and then sacrificed by dislocation of the cervical vertebrae;

155. 3.2.4 Mice were quarantined for 4 days with daily inspection;

156. 3.2.5 Mice were housed in a single cage with 12 hours of: 12 hours day/night light; the condition of the room was always stable to ensure the validity of the test results. The mice were free to eat and drink during the experiments.

157. 3.2.6 Dose design and grouping

158. 3.2.6.1 Dosing schedule: The dose of the test sample is 10 mg/kg body weight. The clinical dose of pioglitazone hydrochloride tablets is 45 mg/day in humans, i.e. 0.75 mg/kg of body weight, based on an average adult body weight of 60 kg; the test dose is 30 times the human clinical dose; thus, the positive control group was administered a dose of 22.5 mg/kg of body weight;

159. 3.2.6.2 Assignment to groups: after the end of quarantine, animals that had not been fed for approximately 5 hours were bled from the tail, their blood glucose levels were measured (0 hours), and a glucose solution of 0.25 g/ml was administered from calculation of 10 ml/kg of body weight, and the level of glucose in the blood was measured after 0.5 hours. Six mice with too high or too low blood glucose levels were excluded. The remaining mice were divided into a negative control group, a model control group, a positive control group, and a sample group of 12 mice per group (Table 2).

160. Table 2. Distribution of mice into groups 161.

161.

162. 3.2.7 Methods

163. 3.2.7.1 High energy nutrition

164. Animal fat 10%, sucrose 15%, egg yolk powder 15%, casein 5%, cholesterol 1.2%, sodium cholate 0.2%, calcium hydrogen phosphate 0.6%, stone meal 0.4%, supporting material for mice 52.6%.

165. 3.2.7.2 Sample preparation: The test sample of the present invention was prepared by adding sodium chloride solution for injection to the sample vial to obtain a concentration of 0.5 mg/ml; then stored at 2~8°C in a place protected from light. A positive control sample was prepared by mixing 1 pioglitazone hydrochloride tablet (specification 15 mg/tablet) with 0.5% CMC-Na solution and grinding evenly, then adding 0.5% CMC-Na solution to a final volume of 13.3 ml and shaking well before use.

166. 3.2.7.3 Building a diabetic model

167. After each group received the maintenance diet for 1 week, except for the negative control group, the other groups were changed to the high energy diet from 3.1.7.1. After 3 weeks of feeding, each group was provided with fasting for 24 hours. Except for the negative control group, the other groups were intraperitoneally injected with 5 mg/ml of STZ solution at a dose of 20 ml/kg of body weight. All groups were fed for 4 days. Fasting blood glucose and glucose tolerance were measured on day 5 and the test was terminated the next day.

168. 3.2.7.4 Introduction of test specimen and positive control specimen

169. After feeding the maintenance diet, the sample group was intraperitoneally injected with the test sample solution once a day at 20 ml/kg body weight, the negative control group and the model control group were intraperitoneally injected with sodium chloride solution, and the positive control group was intragastrically injected with pioglitazone hydrochloride solution at a dose of 20 ml/kg body weight for 34 days before the end of the trial. On the day of STZ administration, each group was administered 4 hours after STZ administration.

170. 3.2.7.5 Measurement of last fasting blood glucose and glucose tolerance and retrieval

171. On day 5 after intraperitoneal injection of STZ, mice in each group were fasted for approximately 5 hours with water provided. After measuring fasting blood glucose (0 h), the sample group and the positive control group were intraperitoneally administered with the respective drugs. The negative control group and the model control group were intraperitoneally injected with sodium chloride solution. After 20 minutes, each group was intragastrically injected with 0.125 g/ml of glucose solution at the rate of 20 ml/kg of body weight, and the level of glucose in the blood was measured 0.5, 1, 1.5, 2 hours after the administration of glucose solution. The next day, mice in each group were allowed to fast for 5 hours after administration. Mice were anesthetized with 3% sodium pentobarbital at 10 ml/kg body weight and sacrificed after eyeball and blood sampling. Blood samples were centrifuged at 3000 rpm for 10 minutes and serum was collected for serum insulin, triglyceride and total cholesterol determinations. The pancreas of each animal was collected at the same time. Six animals in each group were fixed with 10% paraformaldehyde for HE staining and immunohistochemical staining with insulin in the pancreas. The remaining 6 animals were kept at -80°C to determine the content of insulin.

172. 3.2.8 Detection indicators

173. 3.2.8.1 General clinical observation

174. The general clinical condition of the animals was observed once a day until the end of the study.

175.3.2.8.2 Body weight

176. Weighing was performed at the beginning of the study, at the end of the study, and once a week during the study.

177. 3.2.8.3 Feed intake

178. Each week, the feed intake of the animals for two days was measured once by weighing the added feed and the remaining feed the following day.

179. 3.2.8.4 Rate of decline in blood glucose

180. Blood glucose decline rate = (pre-experiment blood glucose value - post-experiment blood glucose value) / (pre-experiment blood glucose value) × 100%.

181. 3.2.8.5 Area under the blood glucose tolerance curve

182. The area under the blood glucose level curve was calculated 0, 0.5, 1, 1.5, 2 hours after glucose administration. The formula looks like this:

183. Area under the blood glucose curve: [(0 h blood glucose + 0.5 h blood glucose) × 0.5] / 2+[(0.5 h blood glucose + 1 h blood glucose) × 0.5] / 2+[(1 h blood glucose + 1.5 h) blood glucose) × 0.5] / 2 + [(1.5 h blood glucose + 2 h blood glucose) blood) × 0.5] / 2.

184. 3.2.8.6 Serum insulin, cholesterol and triglycerides

185. At the end of the experiment, serum was collected for analysis of serum insulin, triglycerides and total cholesterol. The index of insulin resistance was calculated.

186. Index of insulin resistance = insulin / 22.5e ^{-glucose in the blood}

187. 3.2.8.7 Staining with HE

188. After pancreas from 6 animals in each group were fixed with 10% paraformaldehyde, islet inflammation was observed using HE staining and semiquantitatively assessed on a scale of 0~4.

189.0: No pathological changes;

190. 1: Peripheral inflammation of the islets: signs of pathological infiltration, pathological changes limited to the periphery of the islets;

191.2: mild inflammation of the islets: less than 25% of the islets have pathological infiltration changes;

192. 3: Moderate islet inflammation: 25% to 75% of islets show pathological infiltrating changes;

193. 4: severe inflammation of the islets: more than 75% of the islets have pathological infiltration changes.

194. 3.2.8.8 Immunohistochemistry

195. Pancreatic tissues fixed with 10% paraformaldehyde were used for immunohistochemistry to determine insulin expression.

196. 3.2.8.9 Determination of insulin content in pancreatic tissue

197. Pancreatic tissue of the remaining 6 animals in each group was stored at 80°C for ELISA to determine the content of insulin in the pancreas.

198. 3.2.9 Evaluation of results

199. 3.2.9.1 Conditions for building a model of impaired glucose metabolism

200. The model control group had a blood glucose level of ≥10 mmol/L at 0.5 h, or the blood glucose level or area under the glucose curve at any time point at 0.5, 1, 1.5, and 2 h were statistically a significant increase compared to the negative control group.

201.3.2.9.2 Fasting blood glucose index

202. Under the condition of creating a model of impaired glucose metabolism, compared with the model control group, a decrease in fasting blood glucose or a decrease in the percentage of blood glucose was statistically significant, and the fasting blood glucose index in the test sample was positive.

203. 3.2.9.3 Glucose tolerance index

204. Under the condition of creating a model of impaired glucose metabolism, compared with the model control group, the group of test samples was statistically significant at 0, 1, 1.5 and 2 hours after the administration of glucose solution, or the area under the blood glucose curve 0, 0.5 , 1, 1.5 and 2 hours was statistically significant, so the glucose tolerance index of the test sample was positive.

205. 3.2.10 Statistical analysis

206. All data has been expressed using SPSS 21.0 software for statistical analysis; body weight, food intake, post-test fasting blood glucose, glucose tolerance using repeated measures analysis of variance; preliminary study of fasting blood glucose, cholesterol levels, serum insulin, pancreatic insulin using analysis of variance after logarithmic transformation. The mean optical density of pancreatic insulin was measured using a T-test; percentage drop in blood glucose, area under the curve of blood glucose, TG content was analyzed by the sum of ranks; and statistical analysis of the assessment of islet inflammation using a chi-square test. The test level is α=0.05 and the chi-square test level is adjusted to α=0.0024.

207.3.3 Results

208. 3.3.1 Surveillance

209. Prior to intraperitoneal administration of STZ, no deviations were found in the general clinical situation and the second stool of animals. After intraperitoneal administration of STZ, body weight decreased.

210.3.3.2 Body weight

211. During the study, there were no statistical differences in body weight between the groups (Table 3).

212.

213. 3.3.3 Food intake

214. Compared to the control group, food intake increased in the positive control group at week 1, and food intake increased in the sample group at week 1 but decreased at weeks 2 and 5 (Table 4).

215.

216. 3.3.4 Fasting blood glucose index

217. Compared to the negative control group, the model control group had elevated fasting blood glucose levels. The value of the elevated percentage of fasting blood glucose was negative and statistically significant compared to the negative control group (P<0.05). Compared with the model control group, fasting blood glucose levels in the sample group and the positive control group decreased significantly (P<0.05).

218.

219. 3.3.5 Glucose tolerance index

220. Compared with the negative control group, the blood glucose levels in the model control group at 0.5 h, 1 h, 1.5 h, 2 h and the area under the blood glucose curve were statistically significant (P<0.01) , and a model of impaired glucose metabolism was created. Compared to the model control group, blood glucose levels in the control group at 0.5 h, 1 h, 1.5 h and 2 h were statistically significant (P<0.05 or P<0.01).

221.

222. 3.3.6 Serum insulin

223. Compared with the negative control group, the serum insulin content in the model control group decreased statistically (P<0.01). Compared with the model control group, the serum insulin content in the positive control group increased statistically (P<0.05).

224.3.3.7 Pancreatic insulin content

225. Compared with the negative control group, the content of pancreatic insulin in the model control group decreased statistically (P<0.01). Compared with the model control group, the content of pancreatic insulin in the positive control group increased, but slightly (P>0.05).

226.3.3.8 Cholesterol (CS) content

227. Compared with the negative control group, the content of cholesterol in the model control group increased statistically (P<0.01); compared with the model control group, the content of cholesterol in the sample group decreased statistically (P<0.01).

228. 3.3.9 Triglycerides (TG)

229. Compared with the negative control group, the content of TG in the model control group increased, but there was no statistical significance (P>0.05). The content of TG in the group with a positive drug and in the sample group decreased, but not significantly (P>0.05).

230.

231. 3.3.10 HE stain results

232. Compared to the negative control group (FIG. 9(a)), the model control group showed a sharp decrease in the number of islets, islet volume and number of islet cells, as well as a pronounced infiltration of lymphocytes into the islets (FIG. 9(b)), the positive control group showed a modest decrease in islet number, islet volume, and islet cell number, and obvious lymphocyte infiltration into the islets (FIG. 9(c)), but no apparent decrease in islet number, islet volume, and islet cell number was found in the sample group. , and no lymphocyte infiltration into the islets (FIG. 9(d)).

233. 3.3.11 Insulin immunohistochemistry results

234. Compared with the negative control group (FIG. 10(a) and FIG. 11(a)), a significant decrease in the number of insulin-positive islets and pancreatic cells was observed in the model control group (FIG. 10(b) and FIG. 11( b)), there was a significant decrease in the number of insulin-positive islets and pancreatic cells in the positive control group (FIG. 10(c) and FIG. 11(c)), but no clear decrease in the number of insulin-positive islets and pancreatic cells was observed in the sample group ( FIG 10(d) and FIG 11(d)).

235. Embodiment 4

236.4.1 Reagents

237. Cholesterol test kit, LabAssay™ Cholestro, Baobai Bio/Wako

238. Triglyceride detection kit, LabAssay Triglyceride, Baobai Bio/Wako.

239 Free Fatty Acid Kit, LabAssay NEFA, Baobai Bio/Wako

240. Glucose test kit, LabAssay Glucose, Baobai/Wako

241. Insulin test kit, rat/mouse insulin ELISA, Shanghai Jifeng/Millipore

242. GOT, GPT kit, Nanjing Jiancheng Biology Co., Ltd.

243. Reverse transcription reagent, Novozan

244. HieffTM qPCR SBYR Green Mastey Mix, Eason Biotechnology (Shanghai) Co., Ltd.

245. High Fat Diet, Research Diet, P/N D12492

246. 4.2 Test product

247. A-03: L-NIBC-AuCs with a size of 0.5-2.6 nm

248.4.3 Test methods

249. Mouse Model: High fat B6 mouse model.

250. B6 mice were divided into five groups: normal control group (B6 mice received their usual diet throughout the experiment); drug control group A-03 (B6 mice received their normal diet throughout the experiment and were intraperitoneally injected with a high dose of A-03 (10 mg/kg mouse body weight) from 5 months of age); model control group (B6 mice received a high-fat diet from 2 months of age, and from 5 months of age they were injected intraperitoneally with saline); low dose group (B6 mice received a high-fat diet from 2 months of age and received a low dose of A-03 (1 mg/kg mouse body weight) intraperitoneally from 5 months of age; middle dose group (B6 mice received a high fat diet from 2 months of age and received a medium dose of A-03 (5 mg/kg mouse body weight) intraperitoneally from 5 months of age; and a high dose group (B6 mice received a high fat diet from 2 months of age, and were intraperitoneally injected with a high dose of A-03 (10 mg/kg mouse body weight) from 5 months of age.

251.4.4 Results

252. 4.4.1 Development of a diabetic mouse model

253. A high-fat diet-induced mouse model of obesity was used to study the therapeutic effect of drug A-03 on diabetes.

254. In FIG. 13a shows a comparison of body weight, fat mass and muscle tissue between a normal control group and a model control group. Compared with the normal control group, body weight and fat mass in the model control group were significantly increased (P<0.05, *), but the muscle tissue content did not change significantly.

255. In FIG. 13b shows a comparison of fasting blood glucose index and insulin hypoglycemic capacity between a normal control group and a model control group (all with significant differences; P<0.05, *).

256. In FIG. 13c shows a comparison of glucose tolerance indices between a normal control group and a model control group (all with significant differences, P<0.05, *).

257. All treatment groups were the same as the model control group. The results showed that the model was successfully created.

258. 4.4.2 Effect of drug A-03 on blood glucose levels in diabetic mice

259. Two administration schemes have been developed. The first was rapid drug administration, ie. the test was performed immediately after 30 minutes of drug administration. The second was long-term drug use, ie. the drug was administered once a day for two consecutive months, and the test was carried out 1 month and 2 months after administration.

260. 4.4.2.1 Outcomes of rapid drug administration

261. For normal mice and model mice, 30 minutes after a single injection of drug A-03 (10 mg/kg mouse body weight), the effect of drug A-03d on glucose tolerance was found (injection of saline as a control as for normal , and for model mice). After 30 minutes of administration of high doses of A-03, glucose tolerance in model mice on a high fat diet improved significantly (FIG. 14b, after 15 and 30 minutes, blood glucose levels in the group receiving high doses of A-03 are significantly lower, than the normal saline control group, P<0.05, *), but there were no significant changes in normal mice (FIG. 14a).

262. 4.4.2.2 Results of long-term drug administration

263. In FIG. 15a shows changes in body weight in the low, medium and high dose groups of A-03 compared to the model control group.

264. In FIG. 15b and 15c show the effect of an approximate mean dose of drug A-03 on fat and muscle, respectively.

265. In conclusion, compared with model mice, long-term administration of three doses of A-03 does not significantly affect the body weight and body fat ratio of model mice. Therefore, drug toxicity is excluded in the first place, and it can be explained that the use of drugs does not significantly affect the diet and metabolic requirements of mice.

266. One month after administration, the blood glucose levels of the model control group and each drug-treated group were measured under random diet conditions (no fasting). FIG. 16 shows that three concentrations of drug A-03 have a significant hypoglycemic effect (P<0.05, *).

267. Two months after administration, fasting blood glucose was measured in the model control group and in each drug group after an overnight fast. FIG. 17 shows that drug A-03 has a significant regulatory effect on blood glucose levels (P<0.05, *).

268. These results show that drug A-03 has a significant inhibitory effect on blood glucose levels.

269. Further experiments have shown that low, medium and high doses of drug A-03 can significantly improve glucose tolerance and insulin resistance induced by a high fat diet. FIG. 18 shows the effect of an approximate mean dose of drug A-03 on (a) glucose tolerance and (b) the hypoglycemic effect of insulin (* means P<0.05).

270. 4.4.3 Effect of drug A-03 on plasma total cholesterol in model mice

271. In addition to the hypoglycemic effect, the inventors of the present invention also found that blood lipid levels were significantly improved after drug treatment. Here blood lipids refer to total serum cholesterol, triglycerides and non-esterified fatty acids. FIG. 19 shows the effect of medium and high dose drug A-03 on serum non-esterified fatty acids (FIG. 19a), serum triglycerides (FIG. 19b) and serum total cholesterol (FIG. 19c), where 1-4 represents a normal control group, a model control group, an A-03 medium dose administration group, and an A-03 high dose administration group, respectively. Medium and high doses of A-03 had no significant effect on NEFA and TG, but reduced cholesterol from 243.2±9.7 mg/dl (model control group) to 188.4±9.4 mg/dl and 197.1 ±10.4 mg/dl, respectively, and the differences were significant (P<0.05, *).

272. 4.4.4 Effect of drug A-03 on insulin levels in the blood of model mice

273. In FIG. 20 shows the effect of drug A-03 after administration of an average dose of drug for 2 months on blood glucose levels (FIG. 20a) and blood insulin levels (FIG. 20b) in model mice under fasting conditions, where 1-3 in the column of the graph represents the normal control group, the model control group, and the A-03 mid-dose administration group, respectively. Fasting blood glucose decreased from 10.38±0.39 mm to 8.55±0.45 mm (P<0.05, *). The corresponding level of insulin in the blood decreased from 6.03±0.85 ng/ml to 3.86±0.38 ng/ml (P<0.05, *).

274. 4.4.5 Effect of drug A-03 on the liver

275. Mice were fed and administered drug A-03 according to the long-term drug administration protocol described above, the drug administration period was 2.5 months.

276. 4.4.5.1 Influence on G6Pase, a key enzyme that limits the rate of gluconeogenesis

277. Gluconeogenesis is a process primarily occurring in the liver, in which non-glucose precursors are converted to glucose. Glucose-6-phosphate dehydrogenase (G6Pase) is the key rate-limiting enzyme in gluconeogenesis. G6Pase expression is directly related to the rate of glucose production. FIG. 21a shows that drug A-03 at an approximate average dose can significantly inhibit G6Pase gene expression (P<0.05, *), while FIG. 21b shows that liver glycogen content is significantly reduced (P<0.05, *). FIG. 21C shows the effect on the expression of other key molecules of oxidative phosphorylation in glucose metabolism, such as cytochrome c oxidase IV (Cox4), cytochrome c oxidase VII (Cox7), alpha peptide of the mitochondrial complex F1 of the hydrogen ion transporting ATPase (Atp5a). , cytochrome c (Cycs), cytochrome b gene (Cytb) in mitochondrial DNA, and carnitine palmitoyltransferase 1A (Ctp1a). It can be seen that oxidative phosphorylation did not increase, but Cox 4, Cox 7 and Atp5a decreased significantly (P<0.05, *), indicating that the decrease in blood glucose levels may be associated with effective drug control of gluconeogenesis in the liver, not an increase in consumption.

278.4.4.5.2 Effects on intrahepatic insulin sensitivity

279. Tissue processing, electrophoretic separation of proteins and Western blotting are well known methods and will not be described in this application. FIG. 22 shows the effect of drug A-03 at an approximate average dose on phosphorylation of key insulin sensitivity regulatory proteins, including protein kinase B (PKB) and phosphorylated ribosomal protein kinase S6 (S6K). FIG. 22a shows representative bands of pS473-PKB, PKB, pT389-S6K and S6K proteins, and FIG. 22b and 22c show the results of the respective statistical analysis of expression (among them, 1-3 represent the normal control group, the model control group, and the A-03 medium dose group sequentially). It can be seen that a high fat diet significantly reduces the proportion of phosphorylated protein kinase B in total protein (pS473-PKB/PKB). Compared with the model control group, the administration of drug A-03 significantly increased PKB protein phosphorylation (P<0.05, *) and significantly increased the protein ratio of phosphorylated ribosomal protein kinase ip/ribosomal protein kinase S6 (pT389-S6K/S6K) (P<0 .05, *). The results showed that drug A-03 significantly activated the PKB-S6K signaling pathway, which increased the sensitivity of target cells to insulin and improved insulin resistance.

280. 4.4.5.3 Protective effect in hepatic impairment caused by diabetes mellitus with obesity

281. Methods for determining glutamine oxaloacetic transaminase and alanine aminotransferase in blood are well known and will not be repeated in this application. The detection of glutamine oxaloacetic transaminase (GOT or AST) and alanine aminotransferase (ALT or GPT) in the blood of mice showed that both AST and ALT were significantly elevated in mice fed the model high fat diet. After treatment with drug A-03, a very effective control was achieved. FIG. 23a and 23b show the effect of the average dose of drug A-03 on AST or GOT and ALT or GPT, respectively, where 1-3 in the histogram represent the normal control group, the model control group, and the average dose administration group A-03, respectively. It can be seen that after the administration of the drug A-03, AST and ALT significantly decreased (P<0.05, *). This indicates that drug A-03 has a significant therapeutic effect on liver dysfunction caused by obesity in diabetes mellitus.

282. Other ligand-modified AuCs of various sizes also have similar effects. They will not be described in detail in this application.

283. Industrial Applicability

284. AuCs can be used to treat diabetes. They are suitable for industrial applications.

Claims (11)

1. The use of a gold cluster (AuC) to obtain a drug for the treatment of diabetes in a subject, while the specified AuC contains: golden core and a ligand that modifies the gold core; where the ligand is selected from the group consisting of L-cysteine and its derivatives, D-cysteine and its derivatives, cysteine-containing oligopeptides and their derivatives and other thiol-containing compounds; the gold core has a diameter in the range of 0.5-2.6 nm. 2. Use according to claim 1, characterized in that L-cysteine and its derivatives are selected from the group consisting of L-cysteine, N-isobutyryl-L-cysteine (L-NIBC) and N-acetyl-L-cysteine (L -NAC), and wherein D-cysteine and its derivatives are selected from the group consisting of D-cysteine, N-isobutyryl-D-cysteine (D-NIBC) and N-acetyl-D-cysteine (D-NAC). 3. Use according to claim 1, characterized in that the cysteine-containing oligopeptides and their derivatives are cysteine-containing dipeptides, cysteine-containing tripeptides or cysteine-containing tetrapeptides. 4. Use according to claim 3, characterized in that the cysteine-containing dipeptides are selected from the group consisting of L-cysteine-L-arginine (CR) dipeptide, L-arginine-L-cysteine (RC) dipeptide, L-histidine-L dipeptide -cysteine (HC) and the dipeptide L-cysteine-L-histidine (CH). 5. Use according to claim 3, characterized in that the cysteine-containing tripeptides are selected from the group consisting of glycine-L-cysteine-L-arginine (GCR) tripeptide, L-proline-L-cysteine-L-arginine (PCR) tripeptide, tripeptide L-lysine-L-cysteine-L-proline (KCP) and L-glutathione (GSH). 6. Use according to claim 3, characterized in that the cysteine-containing tetrapeptides are selected from the group consisting of glycine-L-serine-L-cysteine-L-arginine (GSCR) tetrapeptide and glycine-L-cysteine-L-serine-L tetrapeptide -arginine (GCSR). 7. Use according to claim 1, characterized in that the other thiol-containing compounds are selected from the group consisting of 1-[(2S)-2-methyl-3-thiol-1-oxopropyl]-L-proline, thioglycolic acid, mercaptoethanol, thiophenol, D-3-trolovol, N-(2-mercaptopropionyl)-glycine and dodecylmercaptan.