Synthesis method and application of theobromine-derived carbon quantum dots

Abstract

The invention discloses a synthesis method and application of theobromine-derived carbon quantum dots, belongs to the technical field of biological material preparation and application, and discloses a synthesis method and application of theobromine-derived carbon quantum dots. The theobromine-derived carbon quantum dots are prepared by performing centrifugal precipitation, dialysis and freeze-drying on raw materials theobromine, citric acid and ethylenediamine under a hydrothermal reaction condition. The carbon dots can significantly inhibit aging of zebrafish and show an anti-aging effect. The compound has good activity of removing active oxygen, and can reduce MDA (malondialdehyde) and inflammatory factors. Therefore, the compound has great application potential in the aspects of skin care product repair, anti-oxidation and anti-aging development.

Description

Technical Field

[0001] The invention belongs to the technical field of biomaterial preparation and application, and in particular to a theobromine-derived carbon quantum dot and a preparation method and application thereof. Background Art

[0002] Aging is a gradual and irreversible physiological and pathological process of organisms in nature. In biological processes, aging is a spontaneous and inevitable process of organisms over time. As a complex natural phenomenon, aging is mainly manifested in the decline of physiological structure and function, and the decline of adaptability and resistance. In pathology, aging is the result of the continuous accumulation of inflammatory stress, injury infection, immune response decline, malnutrition, metabolic disorders, etc. With the aging of the world's population, the demand for extending human healthy life span to achieve healthy aging is becoming increasingly strong, and the exploration of functional drugs that delay aging has also become an effective means of intervening in aging.

[0003] With the rapid development of nanomedicine, various nanomaterials with antioxidant properties and the ability to scavenge multiple reactive oxygen species (ROS) have been developed, which have attracted widespread attention in the biomedical field, such as metal oxide nanoparticles, metal organic frameworks (MOFs), antimicrobial peptides, and functionalized hydrogels, but these materials usually have certain cytotoxicity. Therefore, the development of a nanomaterial with high safety and broad-spectrum free radical scavenging ability can reduce oxidative stress damage and inhibit inflammation, thereby delaying aging.

[0004] Carbon quantum dots (CDs), as a new type of carbon nanomaterial, have been widely used in the biomedical field due to their simple preparation process, extremely small size, excellent optical properties, low toxicity and outstanding biocompatibility. Such as biosensing, cell imaging, drug delivery, antibacterial and tumor treatment. Recent studies have shown that many CDs have antioxidant capacity, and CDs with surface defects and unpaired electrons have more active sites, which can provide rapid electron transfer effects to achieve chemical adsorption and ROS scavenging. At present, CDs have broad application prospects in scavenging free radicals, anti-oxidation and reducing oxidative stress damage.

[0005] Azobisisobutylamidine hydrochloride (AAPH) can induce zebrafish aging model. β-galactosidase is the most widely used biomarker in aging and good cells because it is quite reliable and easy to detect. β-galactosidase staining can be used to find out whether the drug has the function of delaying aging. Summary of the invention

[0006] The present invention designs a synthesis method and application of carbon quantum dots derived from theobromine, which solves the following technical problems: (1) the existing theobromine does not have the function of delaying aging, but after being synthesized into nanomaterials, it has the functions of delaying aging, anti-inflammatory and antioxidant; (2) there has been no attempt to use carbon quantum dots derived from theobromine in existing anti-aging treatment methods.

[0007] In order to solve the above-mentioned technical problems, the present invention adopts the following solutions: A method for synthesizing theobromine-derived carbon quantum dots comprises the following steps: Step 1, theobromine, citric acid and ethylenediamine are dissolved in water; Step 2, generating a carbon quantum dot stock solution through a hydrothermal reaction; Step 3, centrifuging the carbon quantum dot stock solution obtained in step 2 to obtain a supernatant; Step 4: dialyze the supernatant in step 3, and freeze-dry to finally obtain theobromine-derived carbon quantum dot powder.

[0008] Preferably, in step 1, the molar ratio of theobromine, citric acid and ethylenediamine is 2:4:15.

[0009] Preferably, the hydrothermal reaction conditions in step 2 are 120-240° C. for 2-10 hours.

[0010] Preferably, the centrifugal precipitation conditions in step 3 are 8000-15000 rpm, and the centrifugation time is 5-30 min.

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

[0012] A theobromine-derived carbon quantum dot, characterized in that it is prepared using the above-mentioned synthesis method.

[0013] A medicine for treating and / or preventing aging, characterized by comprising the above-mentioned theobromine-derived carbon quantum dots.

[0014] A skin care product with anti-inflammatory and antioxidant functions, characterized by comprising the above-mentioned theobromine-derived carbon quantum dots.

[0015] An application of the biosafety of theobromine-derived carbon quantum dots, characterized in that: the biosafety concentration of the theobromine-derived carbon quantum dots is determined using zebrafish embryos, the half-lethal concentration of the theobromine-derived carbon quantum dots and the therapeutic concentration used in an aging model are lower than the critical value of the safety concentration, indicating that the theobromine-derived carbon quantum dots have excellent biosafety.

[0016] An application of theobromine-derived carbon quantum dots for delaying aging, characterized in that: a zebrafish aging model is used to determine the therapeutic effect of the theobromine-derived carbon quantum dots, the theobromine-derived carbon quantum dots reduce the staining area on the back of the zebrafish, indicating that the content of β-galactosidase is reduced, indicating that theobromine-derived carbon quantum dots have the property of delaying aging.

[0017] A biological imaging material, characterized in that: it includes the above-mentioned carbon quantum dots derived from theobromine, and its fluorescence emission and ultraviolet absorption characteristics are detected; through the analysis of the fluorescence emission spectrum and the ultraviolet absorption diagram, it has excitation wavelength-dependent fluorescence emission characteristics and can be used for biological imaging.

[0018] An application of carbon quantum dots derived from theobromine, characterized by: preparing a 0.1 mg / ml carbon quantum dot solution, detecting its fluorescence emission and ultraviolet absorption characteristics; through fluorescence emission spectrum and ultraviolet absorption diagram analysis, the results show that there are fluorescent carbon quantum dots and can be used for biological imaging.

[0019] An application of carbon quantum dots derived from theobromine, characterized in that: a 0-0.2 mg / ml carbon quantum dot solution is prepared, and its biosafety concentration is determined using zebrafish embryos, and the half-lethal concentration of the carbon quantum dots derived from theobromine and the therapeutic concentration used in an aging model are lower than the critical value of the safety concentration, indicating that the carbon quantum dots derived from theobromine have excellent biosafety. Preferably, 0.01, 0.02, 0.05, 0.1, 0.2 mg / ml carbon quantum dot solutions are prepared.

[0020] An application of carbon quantum dots derived from theobromine, characterized in that: 0.01-0.1 mg / ml of the carbon quantum dot solution is prepared, and its therapeutic effect is determined using a zebrafish aging model, and the carbon quantum dots derived from theobromine reduce the staining area on the back of the zebrafish, indicating that the content of β-galactosidase is reduced, indicating that the carbon quantum dots derived from theobromine have the property of delaying aging. Preferably, preferably, 0.025 mg / ml of the carbon quantum dot solution is prepared, and its therapeutic effect is determined using a zebrafish aging model.

[0021] The synthesis method and application of the theobromine-derived carbon quantum dots have the following beneficial effects: (1) The carbon quantum dots derived from theobromine in the present invention have fluorescent properties and can be used for biological imaging.

[0022] (2) The carbon quantum dots derived from theobromine in the present invention have the function of delaying aging and can be used as medicines and cosmetics for the development and treatment of anti-aging related drugs.

[0023] (3) The theobromine-derived carbon quantum dots of the present invention are not limited to delaying aging. Based on their anti-inflammatory function, they can also be used in the development of anti-inflammatory related drugs, cosmetics and the treatment of diseases.

[0024] (4) The theobromine-derived carbon quantum dots of the present invention are not limited to delaying aging. Based on their antioxidant function, they can be used as food additives, drugs and cosmetics in the anti-inflammatory field.

[0025] The above content only lists some of the benefits of the present invention. In addition to medicines, foods, health products, cosmetics, medical devices, etc., the benefits are not limited to the fields of construction, decoration, electrical appliances, kitchen utensils and other daily necessities, ceramic products, metal products, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The fluorescence emission spectrum of theobromine-derived carbon quantum dots (2LL008 CDs) in the system of the present invention; Figure 2 It is the ultraviolet absorption spectrum of theobromine-derived carbon quantum dots (2LL008 CDs) in the system of the present invention; Figure 3 This is a fluorescence imaging image of zebrafish larvae of the theobromine-derived carbon quantum dots (2LL008 CDs) of the present invention; Figure 4 The biosafety assessment of the theobromine-derived carbon quantum dots (2LL008 CDs) of the present invention; Figure 5 The β-galactosidase staining diagram and statistical diagram of the theobromine-derived carbon quantum dots (2LL008 CDs) of the present invention on the zebrafish aging model; Figure 6 A schematic diagram of the effect of theobromine-derived carbon quantum dots (2LL008 CDs) of the present invention on reactive oxygen species (ROS) and malondialdehyde (MDA) in a zebrafish aging model; Figure 7 The theobromine-derived carbon quantum dots (2LL008 CDs) of the present invention have an effect on the zebrafish aging model nrf2 / nqo1 Schematic diagram of the effects on mRNA levels; Figure 8 The theobromine-derived carbon quantum dots (2LL008 CDs) of the present invention have an effect on inflammatory factors in zebrafish aging models il1b, il6, tnfa mRNA Schematic diagram of the impact of levels. DETAILED DESCRIPTION

[0027] Combine the following Figures 1 to 8 , the present invention is further described: Embodiment 1: This Example 1 provides a method for preparing theobromine-derived carbon quantum dots (2LL008 CDs), which specifically comprises the following steps: a. The molar ratio of theobromine, citric acid and ethylenediamine is 2:4:15, deionized water is 20 ml, and the mixture is stirred evenly with a glass rod, then transferred to a polytetrafluoroethylene liner and placed in a reactor.

[0028] b. Place the reactor in a heating box and set the reaction conditions to 120-240°C for 2-10 hours, preferably 200°C for 10 hours.

[0029] c. After the reaction is completed, centrifuge and precipitate at 8000-15000 rpm for 5-30 min. Preferably, the centrifuge and precipitate are at 12000 rpm for 20 min.

[0030] d. Take the centrifugal supernatant and put it into a dialysis bag. The dialysis condition is a dialysis bag with a molecular weight of 300-2000 Da and a dialysis time of 1-3 days. The lyophilization is performed in a freeze dryer to obtain theobromine-derived carbon quantum dot powder. The preferred dialysis condition is a dialysis bag with a molecular weight of 500 Da and a dialysis time of 2 days.

[0031] e. Prepare a carbon quantum dot solution, and test its fluorescence and ultraviolet absorption curves by fluorescence spectrophotometer and ultraviolet absorption spectrometer respectively. Figures 1 to 2 shown.

[0032] Figure 1 The fluorescence emission spectrum of 2LL008 CDs shows that 2LL008 CDs has no excitation dependence and has an obvious peak. When excited at 350 nm, 2LL008 CDs has the strongest blue fluorescence emission at around 440 nm.

[0033] Figure 2 This is the UV absorption spectrum of 2LL008 CDs, which has an obvious absorption peak at 260 nm. 260 nm corresponds to the π−π* absorption of the aromatic structure in the structure.

[0034] f. Place 3 dpf zebrafish in 2LL008 CDs solution for 4 h and take fluorescent photos. Figure 3 shown.

[0035] Figure 3 This is a fluorescent imaging picture of zebrafish larvae. The zebrafish added to the 2LL008 CDs solution clearly emit blue light.

[0036] Conclusion: Through the analysis of fluorescence emission spectrum and ultraviolet absorption diagram, the results show that the present invention synthesized fluorescent carbon quantum dots, and its fluorescence property can be used for biological imaging.

[0037] Embodiment 2: This Example 2 provides an assessment of the biosafety concentration of theobromine-derived carbon quantum dots (2LL008 CDs) in zebrafish embryos, specifically comprising the following steps: a. Prepare a 0-0.2 mg / ml carbon quantum dot solution, and use zebrafish embryos to determine its biosafety concentration. Preferably, prepare 0.01, 0.02, 0.05, 0.1, and 0.2 mg / ml carbon quantum dot solutions.

[0038] b. 3 hpf zebrafish embryos were placed in carbon quantum dot solutions of different concentrations. The spontaneous movement frequency was counted at 24 h, the heart rate was counted at 48 h, the hatching rate was counted at 54 h, and the survival rate was counted at 96 h. The results are shown in the figure. Figure 4 shown.

[0039] c. Conclusion: If Figure 4 As shown, theobromine-derived carbon quantum dots have no significant difference in spontaneous movement frequency, heart rate, hatching rate, and survival rate compared with the control group within a concentration of 0.1 mg / ml, and do not affect the normal development of zebrafish embryos. According to the linear regression method, the half-lethal concentration of theobromine-derived carbon quantum dots is 0.16 mg / ml, while the therapeutic concentration used in the aging model is 0.025 mg / ml. Its effective concentration is far below the critical value of the safe concentration, indicating that theobromine-derived carbon quantum dots have excellent biosafety. Its excellent biosafety can be applied to the research and development of biological imaging, drugs, cosmetics, and health products.

[0040] Embodiment 3: This Example 3 provides a theobromine-derived carbon quantum dots (2LL008 CDs) for delaying zebrafish aging model, specifically comprising the following steps: a. Prepare a 0.01-0.1 mg / ml carbon quantum dot solution and use a zebrafish aging model to determine its therapeutic effect. Preferably, prepare a 0.025 mg / ml carbon quantum dot solution and use a zebrafish aging model to determine its therapeutic effect.

[0041] b. Azobisisobutyramidine hydrochloride (AAPH) was used as an aging inducer to treat zebrafish to establish an aging model. c. The β-galactosidase staining method can be used to find out whether the drug has the function of delaying aging, such as Figure 5 shown.

[0042] d. Use biochemical kits to detect antioxidant-related indicators ROS and MDA, and use qPCR technology to detect mRNA levels NRF2 / NQO1 Changes, such as Figures 6 to 7 shown.

[0043] e. Detection of inflammatory factors at the mRNA level using qPCR technology il1b, il6, tnfa mRNA Level changes, such as Figure 8 shown.

[0044] The conclusion is: Figure 5 As shown in the figure, the β-galactosidase staining results showed that the back staining of zebrafish in the AAPH-induced aging model group deepened, while theobromine-derived carbon quantum dots reduced the degree of staining on the back of zebrafish, indicating that the content of β-galactosidase in zebrafish decreased, indicating that theobromine-derived carbon quantum dots have the characteristics of delaying aging. It can be applied to the development and treatment of anti-aging related drugs and cosmetics. Figure 6-7 As shown in Figure 2, theobromine-derived carbon quantum dots reduced the levels of ROS and MDA, lowered the level of oxidative stress in vivo, and regulated the oxidative stress-related signaling pathway Nrf2 / Nqo1. Figure 8 As shown, theobromine-derived carbon quantum dots regulate inflammation-related factors IL1B, IL6, and TNFA, reducing the level of inflammation in the body. Its anti-inflammatory and antioxidant functions can be used in the fields of medicine and cosmetics to solve skin inflammation problems such as anti-inflammatory acne and can be used in the development of anti-aging skin care products.

[0045] 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 protection scope of the present invention.

Claims

1. A method for synthesizing theobromine-derived carbon quantum dots, comprising the following steps: Step 1, theobromine, citric acid and ethylenediamine are dissolved in water; Step 2, generating a carbon quantum dot stock solution through a hydrothermal reaction; Step 3, centrifuging the carbon quantum dot stock solution obtained in step 2 to obtain a supernatant; Step 4: dialyze the supernatant in step 3, and freeze-dry to finally obtain theobromine-derived carbon quantum dot powder.

2. The method for synthesizing theobromine-derived carbon quantum dots according to claim 1, characterized in that: In step 1, the molar ratio of theobromine, citric acid and ethylenediamine is 2:4:

15.

3. The method for synthesizing theobromine-derived carbon quantum dots according to claim 1, characterized in that: The hydrothermal reaction conditions in step 2 are 120-240° C. for 2-10 hours.

4. The method for synthesizing theobromine-derived carbon quantum dots according to claim 1, characterized in that: In step 3, the centrifugal precipitation conditions are 8000-15000 rpm and the centrifugation time is 5-30 min; or / and, the dialysis conditions in step 4 are a dialysis bag with a molecular weight of 300-2000 Da and a dialysis time of 1-3 days, and the theobromine-derived carbon quantum dot powder is freeze-dried in a freeze dryer.

5. A theobromine-derived carbon quantum dot, characterized in that: It is prepared by the synthesis method described in any one of claims 1 to 4.

6. A drug for treating and / or preventing aging, characterized in that: Including the theobromine-derived carbon quantum dots as described in claim 5.

7. A skin care product with anti-inflammatory and antioxidant functions, characterized in that: Including the theobromine-derived carbon quantum dots as described in claim 5.

8. An application of theobromine-derived carbon quantum dots for biological safety, characterized in that: The biosafety concentration of theobromine-derived carbon quantum dots described in claim 5 was determined using zebrafish embryos. The half-lethal concentration of theobromine-derived carbon quantum dots and the therapeutic concentration used in the aging model were lower than the critical value of the safety concentration, indicating that theobromine-derived carbon quantum dots have excellent biosafety.

9. An application of theobromine-derived carbon quantum dots for delaying aging, characterized in that: The therapeutic effect of the theobromine-derived carbon quantum dots described in claim 5 was determined using a zebrafish aging model. The theobromine-derived carbon quantum dots reduced the staining area on the back of the zebrafish, indicating that the content of β-galactosidase was reduced, indicating that theobromine-derived carbon quantum dots have the property of delaying aging.

10. A biological imaging material, characterized in that: The carbon quantum dots derived from theobromine as claimed in claim 5 are tested for fluorescence emission and ultraviolet absorption characteristics; through analysis of the fluorescence emission spectrum and the ultraviolet absorption diagram, it is found that the carbon quantum dots have fluorescence emission characteristics that are dependent on the excitation wavelength and can be used for biological imaging.

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