Synthesis method and application of betaine-derived carbon quantum dots

Abstract

The present application relates to a kind of synthesis method and application of betaine-derived carbon quantum dots, first, betaine and amaranth red are used to generate carbon quantum dots stock solution by hydrothermal reaction, the stock solution is filtered, dialysis, freeze-drying, and carbon quantum dots powder is obtained.Second, a certain concentration range of carbon quantum dots solution is configured, and the antioxidant enzyme activity of betaine-derived carbon quantum dots is detected.Then, with zebra fish hypercholesterolemia model as an example, betaine-derived carbon quantum dots solution is applied to the treatment of zebra fish hypercholesterolemia model, and the mouse hyperlipidemia model is verified.Finally, in hepatocyte system, it is found that betaine-derived carbon quantum dots are through inhibiting PCSK9 to play the role of lowering blood lipid.The present application is not only an antioxidant enzyme with catalase activity, but also can reduce total cholesterol and total triglyceride in hypercholesterolemia by inhibiting PCSK9, and relieve liver steatosis.It has higher use value and application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, and in particular to a synthesis method and application of betaine-derived carbon quantum dots. BACKGROUND

[0002] Hyperlipidemia is an important pathophysiological mechanism leading to cardiovascular and cerebrovascular diseases. Hyperlipidemia refers to a type of dyslipidemia in which the total cholesterol (T-CHO) and total triglyceride (TG) levels in the blood exceed the normal range, and is considered an important risk factor for coronary heart disease, ischemic myocardial infarction, cerebral infarction, and other diseases, and is also closely related to the occurrence of hypertension and diabetes. Statins are currently the first choice for clinical lipid-lowering drugs, but data analysis has found that some patients are intolerant to statins, increasing the risk of recurrent myocardial infarction and cardiovascular events in patients with previous myocardial infarction. Currently, for patients who are intolerant to statins, the use of statins alone cannot achieve the desired effect. Therefore, new lipid-lowering drugs, PCSK9 inhibitors, have begun to develop, which can significantly increase low-density lipoprotein (LDL) receptors and reduce LDL cholesterol.

[0003] Studies have shown that hypercholesterolemia patients have increased serum T-CHO levels, accompanied by increased production of reactive oxygen species (ROS), leading to increased oxidative stress, vascular endothelial damage and dysfunction, and promoting the occurrence and development of atherosclerosis. Therefore, antioxidant drugs not only have the effect of lowering blood lipids in hypercholesterolemia patients, but also can prevent cardiovascular diseases related to hypercholesterolemia.

[0004] Carbon quantum dots (CDs) are a classic zero-dimensional material with a diameter of less than 10 nm, which have the characteristics of simple synthesis, low cost, good biocompatibility, and environmental friendliness. In addition, the nanoscale enzyme properties of CDs have gradually attracted people's attention. Nanoscale enzymes have emerged and been widely used due to their low cost, high stability, flexible operation, and ease of large-scale production. Among them, nanoscale enzymes that efficiently catalyze the removal of reactive oxygen species (ROS) have been used to alleviate cellular oxidative stress and related antioxidant therapy. The betaine-derived carbon quantum dots of the present application are a highly efficient antioxidant nanoscale enzyme, and are also a potential effective means for inhibiting PCSK9 to treat hypercholesterolemia. SUMMARY

[0005] The present application designs a synthesis method and application of betaine-derived carbon quantum dots, which solves the technical problem of overcoming the difficulty of treating patients who are intolerant to statin drugs.

[0006] To solve the above technical problems, the present application adopts the following scheme:

[0007] The application relates to a synthesis method of betaine-derived carbon quantum dots, which comprises the following steps: step 1, dissolving betaine and amaranth red in a solvent; step 2, generating carbon quantum dot stock solution through a hydrothermal reaction; step 3, filtering the carbon quantum dot stock solution obtained in step 2 through a filter membrane to form a filtrate; and step 4, dialyzing the filtrate, freeze-drying, and finally obtaining betaine-derived carbon quantum dot powder.

[0008] Preferably, the molar ratio of betaine to amaranth red in step 1 is 6:1-12:1, and the solvent is water, ethanol, formamide or dimethyl sulfoxide.

[0009] Preferably, the hydrothermal reaction condition in step 2 is 120-240 DEG C for 2-10 hours.

[0010] Preferably, the filter membrane filtration condition in step 3 is that the filter membrane pore size is 0.22-0.45 mu m.

[0011] Preferably, the dialysis condition in step 4 is that the dialysis bag has a molecular weight of 300-2000 Da, the dialysis time is 1-3 days, and the betaine-derived carbon quantum dot powder is obtained through freeze-drying in a freeze dryer.

[0012] The application also relates to betaine-derived carbon quantum dots, which are prepared by using the above synthesis method.

[0013] The application further relates to the application of betaine-derived carbon quantum dots with antioxidant enzyme activity, which comprises the following steps: preparing a betaine-derived carbon quantum dot solution with a concentration of not more than 0.5 mg / mL by using the above betaine-derived carbon quantum dot powder, and detecting the catalase activity.

[0014] Preferably, the concentration of the betaine-derived carbon quantum dot solution is 0.05 mg / ml, 0.1 mg / ml, 0.2 mg / ml, 0.3 mg / ml or 0.4 mg / ml.

[0015] The application further relates to the application of betaine-derived carbon quantum dots with antioxidant enzyme activity, and the application establishes a zebrafish hypercholesterolemia model, which comprises the following steps:

[0016] 0.04 g of cholesterol is completely dissolved in 20 mL of hot anhydrous ethanol; 9.96 g of egg yolk powder is continuously added into the anhydrous ethanol and uniformly mixed to obtain a mixed solution; the mixed solution is placed in an oven for complete drying to obtain high-cholesterol feed; and the zebrafish larvae are fed with the high-cholesterol feed for 10 days to establish a zebrafish hypercholesterolemia model.

[0017] Preferably, the high-cholesterol feed is a powder obtained by completely dissolving 0.04 g of cholesterol and 9.96 g of egg yolk powder in 20 mL of hot anhydrous ethanol, uniformly mixing and completely drying in an oven.

[0018] Preferably, the zebrafish is a 5 dpf (days post fertilization) juvenile fish.

[0019] Preferably, the high cholesterol diet is fed for 10 days.

[0020] The application of betaine-derived carbon quantum dots with antioxidant enzyme activity is characterized in that: the betaine-derived carbon quantum dot powder is used to configure a betaine-derived carbon quantum dot solution with a concentration not greater than 0.5 mg / mL, which is applied to reduce the total cholesterol and total triglycerides of a zebrafish hypercholesterolemia model, and the concentration range of the betaine-derived carbon quantum dot solution for reducing the total cholesterol and total triglycerides of the zebrafish hypercholesterolemia model is 0.001-0.1 mg / mL.

[0021] Preferably, the concentration of the betaine-derived carbon quantum dot solution for reducing the total cholesterol and total triglycerides of the zebrafish hypercholesterolemia model is 0.01 mg / mL.

[0022] Preferably, the treatment time of the betaine-derived carbon quantum dots for reducing the total cholesterol and total triglycerides of the zebrafish hypercholesterolemia model is 2-24 hours.

[0023] Preferably, the treatment time of the betaine-derived carbon quantum dots for reducing the total cholesterol and total triglycerides of the zebrafish hypercholesterolemia model is 4 hours.

[0024] The application of betaine-derived carbon quantum dots with antioxidant enzyme activity is characterized in that: the betaine-derived carbon quantum dot powder is used to configure a betaine-derived carbon quantum dot solution with a concentration not greater than 0.5 mg / mL, which is applied to reduce the total cholesterol and total triglycerides of a zebrafish hypercholesterolemia model, and the concentration range of the betaine-derived carbon quantum dot solution for reducing the total cholesterol and total triglycerides of the zebrafish hypercholesterolemia model is 0.001-0.1 mg / mL.

[0025] Preferably, the concentration of the betaine-derived carbon quantum dot solution for reducing the total cholesterol and total triglycerides of the zebrafish hypercholesterolemia model is 0.01 mg / mL.

[0026] Preferably, the treatment time of the betaine-derived carbon quantum dots for reducing the total cholesterol and total triglycerides of the zebrafish hypercholesterolemia model is 2-24 hours.

[0027] The application of betaine-derived carbon quantum dots with antioxidant enzyme activity is characterized in that: the betaine-derived carbon quantum dot powder is used to prepare a betaine-derived carbon quantum dot solution with a concentration of not more than 0.5 mg / mL, and the solution is applied to inhibit the expression of PCSK9 of human liver cell line HepG2, and the concentration of the carbon quantum dot solution for inhibiting the expression of PCSK9 of human liver cells ranges from 0.001 mg / mL to 0.1 mg / mL.

[0028] Preferably, the concentration of the betaine-derived carbon quantum dots for inhibiting the expression of PCSK9 is 0.01 mg / mL.

[0029] A medicine for reducing hypercholesterolemia, characterized in that: the medicine comprises the betaine-derived carbon quantum dots, and has antioxidant enzyme activity, blood lipid-lowering function and PCSK9 inhibition.

[0030] Based on the antioxidant and blood lipid-lowering functions, the use right can also be applied to the development of antioxidant-related medicines, the application of oxidative stress-related diseases and the application of hypercholesterolemia-related diseases, including but not limited to high blood pressure, coronary heart disease, fatty liver, thrombosis, cardiomyopathy, diabetes and arteriosclerosis.

[0031] The synthesis method and application of the betaine-derived carbon quantum dots have the following beneficial effects:

[0032] (1) The betaine-derived carbon quantum dots not only have antioxidant enzymes with catalase activity, but also can reduce total cholesterol and total triglycerides in zebrafish hypercholesterolemia, and have high use value and application prospect.

[0033] (2) The application range of the betaine-derived carbon quantum dots can also be applied to the development of antioxidant-related medicines, the application of oxidative stress-related diseases and the application of hypercholesterolemia-related diseases, including but not limited to high blood pressure, coronary heart disease, fatty liver, thrombosis, cardiomyopathy, diabetes, inflammation-related diseases and arteriosclerosis.

[0034] (3) The betaine-derived carbon quantum dot solution is applied to the treatment of zebrafish hypercholesterolemia model, and the mouse hyperlipidemia model is verified.

[0035] (4) The betaine-derived carbon quantum dots are found to reduce blood lipids by inhibiting PCSK9 in liver cell lines. The betaine-derived carbon quantum dots not only have antioxidant enzymes with catalase activity, but also can reduce total cholesterol and total triglycerides in hypercholesterolemia by inhibiting PCSK9, and relieve liver steatosis. The betaine-derived carbon quantum dots have high use value and application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1The fluorescence emission spectrum of the betaine-derived carbon quantum dots (TCJCD) under the system of the application;

[0037] Figure 2 The ultraviolet absorption spectrum of the betaine-derived carbon quantum dots (TCJCD) under the system of the application;

[0038] Figure 3 The catalase activity graph of the betaine-derived carbon quantum dots (TCJCD) of the application;

[0039] Figure 4 The catalase activity graph of the betaine-derived carbon quantum dots (TCJCD) of the application;

[0040] Figure 5 The influence diagram of the betaine-derived carbon quantum dots (TCJCD) of the application on the zebrafish hypercholesterolemia model (HCD);

[0041] Figure 6 The influence diagram of the betaine-derived carbon quantum dots (TCJCD) of the application on the mouse hyperlipidemia model (HFD);

[0042] Figure 7 The influence diagram of the betaine-derived carbon quantum dots (TCJCD) of the application on the mRNA levels of PCSK9 and LDLR of human liver cells HepG2. DETAILED DESCRIPTION

[0043] The application will be further described below: Figures 1 to 5

[0044] Example 1:

[0045] This embodiment 1 provides a preparation method of betaine-derived carbon quantum dots (TCJCD), which specifically comprises the following steps:

[0046] a. Different molar ratios of betaine and amaranth are stirred uniformly in different solvents, and then carbon quantum dots stock solution is generated through hydrothermal reaction;

[0047] The molar ratio of betaine to amaranth is 10:1, 20 ml of deionized water, and the glass rod is stirred uniformly and then transferred to the polytetrafluoroethylene liner and placed in the reaction kettle.

[0048] b. The reaction kettle is placed in an oven and reacted at 120-240 ℃ for 2-10 h, and the hydrothermal reaction condition is preferably 180 ℃ for 5 h.

[0049] ​c. After the reaction is completed, it is naturally cooled to room temperature, the carbon quantum dot stock solution is filtered through a filter membrane to form a filtrate, the filter membrane filtration condition is that the filter membrane pore size is 0.22 μm, 1000 Da dialysis bag is purified by dialysis, and freeze-drying is carried out to obtain TCJCD.

[0050] d. 0.1 mg / mL of TCJCD is prepared in sequence, fluorescence and absorption properties thereof are tested by a fluorescence spectrophotometer and an ultraviolet absorption spectrometer respectively, and a curve graph is recorded and drawn as shown in Figures 1-2 .

[0051] Figure 1 The fluorescence emission spectrum of TCJCD is shown in the figure, it can be seen that TCJCD has excitation dependence, the emission wavelength is red-shifted with the increase of the excitation wavelength, and has two obvious peak values. When 340 nm is excited, TCJCD has the strongest blue fluorescence emission near 470 nm; when 400 nm is excited, TCJCD has the strongest green fluorescence emission near 550 nm.

[0052] Figure 2 The ultraviolet absorption spectrum of MCDs is shown in the figure, and there are obvious absorption peaks at 260 nm and 520 nm, 260 nm corresponds to π−π* of the aromatic structure in the structure, and 520 nm corresponds to pyridinic-N absorption.

[0053] It is concluded that the carbon quantum dots synthesized in the application have fluorescence excitation dependence, and the results show that the carbon quantum dots synthesized in the application can be used for biological imaging.

[0054] Example 2:

[0055] The example 2 provides an application of betaine-derived carbon quantum dots (TCJCD) having antioxidant enzyme activity, and specifically includes the following steps:

[0056] H2O2 is added to PBS with pH=7.4 to obtain an oxidation system with a H2O2 concentration of 2 mM, 10 μL of a sample is added to 5 mL of the oxidation system, and an oxygen concentration in the solution is recorded once every 10 seconds by using a dissolved oxygen meter.

[0057] H2O2 is added to PBS with pH=7.4 to obtain an oxidation system with a H2O2 concentration of 2 mM, 10 μL of a sample with different concentrations (0, 0.05, 0.1, 0.2, 0.3, 0.4 mg / mL) is added to 5 mL of the oxidation system, respectively, and an oxygen concentration in the solution is recorded every 10 seconds by using a dissolved oxygen meter.

[0058] Figure 3Schematic diagram of the characterization of catalase activity of TCJCD in Example 2. The rate of TCJCD decomposing H2O2 to produce oxygen and the substrate concentration conform to the Michaelis-Menten equation curve (R2=0.9951), in which the maximum reaction rate (Vmax) is in the range of 29.51-33.75*10 -7 M·s -1 The Michaelis constant (Km) is in the range of 27.77-43.99.

[0059] Figure 4 The catalase activity of TCJCD of Example 2 was in the range of 0-0.4 mg / mL and increased with increasing TCJCD concentration.

[0060] Example 3:

[0061] This Example 3 provides an application of betaine-derived carbon quantum dots (TCJCD) for reducing hypercholesterolemia, which specifically includes the following steps:

[0062] a. 5 dpf zebrafish were selected and fed with normal diet in the control group, and high cholesterol diet in the model group and treatment group for 10 days to establish the hypercholesterolemia model.

[0063] b. The treatment group was treated with 0.01 mg / ml TCJCD for 4 h, and the therapeutic effect was detected;

[0064] c. The zebrafish were homogenized with PBS, the supernatant was extracted, and total cholesterol (TC) and total triglyceride (TG) were detected using a kit.

[0065] The conclusion is: Figure 5 As shown in the results, TCJCD significantly reduced TC and TG in zebrafish, indicating that TCJCD has a good therapeutic effect on the zebrafish hypercholesterolemia model.

[0066] Example 4:

[0067] This Example 4 provides an application of betaine-derived carbon quantum dots (TCJCD) in reducing hyperlipidemia in mice, specifically comprising the following steps:

[0068] a. Eight-week-old ICR mice were selected and fed with a normal diet for the control group, and a high-fat diet for the model and treatment groups for 14 days to establish a hyperlipidemia model.

[0069] b. Treatment groups added 0.015 mg / ml and 0.06 mg / ml TCJCD to drinking water for 14 days, and then the treatment effects were tested;

[0070] c. Collect mouse blood, centrifuge at 4000 rpm for 5 minutes, and use the serum kit to detect total cholesterol (TC), total triglycerides (TG), LDL, and HDL.

[0071] d. Mouse livers were fixed with 4% neutral formaldehyde, dehydrated with sucrose, and frozen and sectioned for Oil Red O staining.

[0072] The conclusion is: Figure 6 As shown in A, TCJCD significantly reduced TG in mice; Figure 6 As shown in B, TCJCD significantly reduced T-CHO in mice; Figure 6 As shown in C, low concentration of TCJCD showed a trend of lowering LDL in mice; Figure 6 As shown in D, TCJCD caused a trend of increasing HDL in mice; Figure 6 As shown in E, TCJCD made the Oil Red O staining color of mouse liver lighter, indicating that TCJCD has a good therapeutic effect on the mouse hyperlipidemia model.

[0073] Example 5:

[0074] This Example 5 provides an application of betaine-derived carbon quantum dots (TCJCD) for inhibiting human liver PCSK9, specifically comprising the following steps:

[0075] a. Human liver cell line HepG2 was selected, NC was the control group without addition, and the experimental group was added with 0.01 mg / mL TCJCD and cultured for 1 day;

[0076] b. Wash the cells three times with PBS, add Trizol to extract cellular RNA, reverse transcribe it into cDNA, and then detect the expression levels of PCSK9 and LDLR by qPCR.

[0077] The conclusion is: Figure 7 As shown in the results, TCJCD significantly reduced the expression of PCSK9 and increased LDLR in human liver cells, indicating that TCJCD has the function of inhibiting PCSK9 in human liver cells HepG2.

[0078] The present invention is described above by way of example in conjunction with the accompanying drawings. It is obvious that the implementation of the present invention is not limited to the above-mentioned method. As long as various improvements are made using the method concept and technical solution of the present invention, or the concept and technical solution of the present invention are directly applied to other occasions without improvement, they are all within the scope of protection of the present invention.

Claims

1. A use of betaine-derived carbon quantum dots in the preparation of antioxidant drugs, characterized in that: The preparation method of the betaine-derived carbon quantum dots comprises the following steps: Betaine and amaranth are dissolved in water and subjected to a hydrothermal reaction at 120-240° C. for 2-10 hours to prepare a carbon quantum dot stock solution. The carbon quantum dot stock solution is filtered to form a filtrate, which is dialyzed and freeze-dried to obtain the product; the molar ratio of betaine to amaranth is 6:1-12:1; The betaine-derived carbon quantum dots have catalase activity.

2. A drug for reducing hypercholesterolemia, characterized in that: The invention relates to betaine-derived carbon quantum dots, wherein the preparation method of the betaine-derived carbon quantum dots comprises the following steps: Betaine and amaranth are dissolved in water and subjected to a hydrothermal reaction at 120-240° C. for 2-10 hours to prepare a carbon quantum dot stock solution. The carbon quantum dot stock solution is filtered to form a filtrate, which is dialyzed and freeze-dried to obtain the product; the molar ratio of betaine to amaranth is 6:1-12:1; The drug has antioxidant enzyme activity, lipid-lowering function and PSCK9 inhibition.

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