Ligusticum wallichii carbon dots, medicine and application

Abstract

The invention relates to the technical field of traditional Chinese medicine carbon dots, in particular to ligusticum wallichii carbon dots, a medicine and application. The ligusticum wallichii carbon dots disclosed by the invention are obtained by carbonizing ligusticum wallichii by a hydrothermal method, wherein the hydrothermal method carbonization is a hydrothermal reaction at 175-195 DEG C for 8-12 hours; research finds that the ligusticum wallichii carbon dots have the characteristic of treating myocardial apoptosis caused by myocardial ischemia reperfusion injury, and compared with ligusticum wallichii carbon dots synthesized by ligustrazine and a pyrolysis method, the ligusticum wallichii carbon dots have the advantage that the heart function can be better recovered. The invention provides a new candidate drug for treating myocardial ischemia reperfusion injury.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine carbon dots, and relates to a chuanxiong carbon dot, a drug and an application thereof. Background Art

[0002] Traditional Chinese medicine carbon dots are a class of nano-scale carbon-based materials extracted or synthesized from traditional Chinese medicine, which have the characteristics of small size, good biocompatibility, excellent fluorescence performance, etc. In recent years, traditional Chinese medicine carbon dots have shown potential application value in the fields of bioimaging, drug delivery and disease treatment, and have attracted much attention especially in the nano-transformation of active ingredients of natural drugs.

[0003] The research on traditional Chinese medicine carbon dots began in the early 21st century. With the development of nanotechnology, researchers found that the carbon dots extracted from traditional Chinese medicine not only retained some active ingredients of the parent drug, but also had unique physical and chemical properties. For example, ginseng carbon dots have been proven to have antioxidant and anti-inflammatory effects, and astragalus carbon dots have shown significant effects in immunomodulation. In addition, traditional Chinese medicine carbon dots have also been used in the fields of cancer treatment, neuroprotection and antibacterial. For example, coptis carbon dots significantly improved the anti-cancer effect by targeted drug delivery, while salvia carbon dots showed potential advantages in the treatment of cardiovascular diseases. However, at present, the research on traditional Chinese medicine carbon dots mostly focuses on a single disease model, lacking systematic exploration of its biological mechanism and in-depth research on cross-field applications.

[0004] Traditional Chinese medicine carbon dots have the advantages of natural source, low toxicity, diverse functions, etc., and show broad prospects especially in the field of disease treatment. However, its research is still in its infancy and there are obvious deficiencies: one is that the biological mechanism of traditional Chinese medicine carbon dots has not been deeply explored, lacking systematic research on its action targets and signal pathways; the other is that the application research in the field of disease treatment is relatively limited, mostly focusing on a single disease model, lacking interdisciplinary and multi-field collaborative exploration. Therefore, deeply studying the biological mechanism of traditional Chinese medicine carbon dots and expanding its application scope are the key problems that need to be solved urgently at present. Summary of the Invention

[0005] In order to solve the deficiencies of the prior art, the purpose of the present invention is to provide a chuanxiong carbon dot, a drug and an application thereof.

[0006] To achieve the above purpose, the present invention adopts the following technical solutions: In the first aspect of the present invention, a chuanxiong carbon dot is provided, which is obtained by hydrothermal carbonization of chuanxiong, and the hydrothermal carbonization is carried out by hydrothermal reaction at 175°C to 195°C for 8h to 12h.

[0007] The present invention discovers through research that the chuanxiong carbon dot obtained by hydrothermal carbonization not only has the characteristic of efficiently down-regulating cardiomyocyte apoptosis proteins, but also can better restore heart function than tetramethylpyrazine and the chuanxiong carbon dot synthesized by pyrolysis method.

[0008] Further, after the hydrothermal reaction is completed, impurities are removed by centrifugation, and then the Chuanxiong carbon dots are obtained through dialysis purification.

[0009] Further, the cut-off molecular weight of the dialysis is 400 Da to 600 Da.

[0010] Further, the average particle size of the Chuanxiong carbon dots is between 2 nm and 4 nm.

[0011] In the second aspect of the present invention, a drug is provided, which comprises the Chuanxiong carbon dots, and a pharmaceutically acceptable excipient or carrier. The drug is used for:

[0012] 1) inhibiting cardiomyocyte apoptosis; 2) treating myocardial ischemia-reperfusion injury.

[0013] After adding appropriate excipients, the drug can be prepared into acceptable pharmaceutical preparations, such as granules, capsules, tablets, injections, mixtures, oral liquids, syrups or other liquid preparations. The excipients that can be added vary according to the dosage form prepared. Exemplarily, optional excipients are suspending agents, thickening agents, flavoring agents, coloring agents, preservatives, penetrants, pH regulators, wetting agents, etc. Those skilled in the art can select the specific types of excipients according to the properties of the Chuanxiong carbon dots, the properties of the excipients and the requirements of the preparation.

[0014] In the third aspect of the present invention, an application of the Chuanxiong carbon dots in the preparation of a drug for inhibiting cardiomyocyte apoptosis is provided.

[0015] In the fourth aspect of the present invention, an application of the Chuanxiong carbon dots in the preparation of a drug for treating ischemia-reperfusion injury is provided.

[0016] Further, the ischemia-reperfusion injury is myocardial ischemia-reperfusion injury.

[0017] The beneficial effects of the present invention are as follows: (1) The present invention uses the hydrothermal method to prepare Chuanxiong carbon dots with water as the medium under high temperature and high pressure conditions. The raw materials used are inexpensive, the synthesis process is convenient and fast, and it has a wide range of applications, with great commercial potential.

[0018] (2) The Chuanxiong carbon dots prepared by the one-step hydrothermal method in the present invention show a strong down-regulating ability for a variety of apoptotic proteins including Caspase3, Caspase9, and Bax.

[0019] (3) The Chuanxiong carbon dots prepared in the present invention use Chinese herbal medicine as the precursor material, have a strong effect of down-regulating cardiomyocyte apoptosis, and can achieve the treatment of in vivo myocardial ischemia-reperfusion injury while ensuring good biosafety. Description of the Drawings

[0020] Figure 1 Particle size detection results of the ligusticum wallichii carbon dots prepared in Example 1 of the present invention. A is the observation image obtained by transmission electron microscopy, and B is the statistical result of particle size distribution.

[0021] Figure 2 Raman spectrum of the ligusticum wallichii carbon dots prepared in Example 1 of the present invention.

[0022] Figure 3 Expression of apoptotic proteins in rat cardiomyocytes after treatment of rats with myocardial ischemia-reperfusion injury with the ligusticum wallichii carbon dots prepared in Example 1 of the present invention. 1 to 4 are Caspase3, Caspase9, Bax, and β-actin in sequence.

[0023] Figure 4 Echocardiogram of the hearts of rats after treatment of rats with myocardial ischemia-reperfusion injury with the ligusticum wallichii carbon dots prepared in Example 1 of the present invention, ligustrazine, and the ligusticum wallichii carbon dots synthesized by the pyrolysis method.

[0024] Figure 5 Left ventricular ejection fraction and left ventricular fractional shortening of rats after treatment of rats with myocardial ischemia-reperfusion injury with the ligusticum wallichii carbon dots prepared in Example 1 of the present invention, ligustrazine, and the ligusticum wallichii carbon dots synthesized by the pyrolysis method. A is the left ventricular ejection fraction of rats, and B is the left ventricular fractional shortening; a to e are the Control group, MIRI group, ligustrazine group, LC-CDs1 group, and LC-CDs2 group in sequence. Detailed implementation manners

[0025] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments, but it should not be construed as a limitation of the present invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well-known to those skilled in the art. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0026] Currently, the artificially developed traditional Chinese medicine carbon dots have the disadvantages of unclear biological mechanisms and poor treatment effects. The present invention proposes to synthesize ligusticum wallichii carbon dots by the hydrothermal method. The synthesis process is convenient and fast, and has a wide range of applications, with great commercial potential.

[0027] In one or more embodiments, during the process of preparing ligusticum wallichii carbon dots by the hydrothermal method, the temperature is 175°C to 195°C, and the time is 8h to 12h.

[0028] In one or more embodiments, the ligusticum wallichii carbon dots synthesized by the hydrothermal method are centrifuged to remove impurities, and then dialysis is carried out. The cut-off molecular weight of dialysis is 400Da to 600Da.

[0029] In some embodiments, the average particle size of the ligusticum wallichii carbon dots prepared by the hydrothermal method is 2nm to 4nm.

[0030] In some embodiments, the I of the Raman spectrum of the ligusticum wallichii carbon dots prepared by the hydrothermal method D / I G is between 0.8 and 1.0.

[0031] A typical embodiment of the present invention provides an application of ligusticum wallichii carbon dots in the preparation of products for inhibiting cardiomyocyte apoptosis.

[0032] In some embodiments, the ligusticum wallichii carbon dots prepared by the hydrothermal method have a significant effect of down-regulating the expression of cardiomyocyte apoptosis proteins, and have a significant down-regulating effect on the apoptosis proteins Caspase3, Caspase9, and Bax.

[0033] Another embodiment of the present invention provides an application of ligusticum wallichii carbon dots in the preparation of drugs for treating myocardial ischemia-reperfusion injury.

[0034] In some embodiments, the administration method of the drug for treating myocardial ischemia-reperfusion injury is in-situ injection. Specifically, the dosage of ligusticum wallichii carbon dots, ligustrazine, and ligusticum wallichii carbon dots synthesized by the pyrolysis method for in-situ injection is 1 mg / kg to 3 mg / kg.

[0035] In order to enable those skilled in the art to more clearly understand the technical solutions of the present invention, the technical solutions of the present invention will be described in detail below with specific embodiments.

[0036] The ligusticum wallichii in the following examples is fresh Bozhou ligusticum wallichii purchased from Anhui Risheng Biotechnology Co., Ltd.

[0037] Example 1: Preparation of a kind of ligusticum wallichii carbon dots.

[0038] The fresh Bozhou ligusticum wallichii purchased on Taobao is dried and ground into powder. Weigh 100 mg of ligusticum wallichii powder and disperse it in 10 mL of water. After mixing, transfer it to a 30 mL polytetrafluoroethylene high-pressure reaction kettle and carry out hydrothermal reaction at 180 °C for 12 h. After the reaction is completed, transfer the high-pressure reaction kettle to room temperature for cooling. After the high-pressure reaction kettle is completely cooled, open the reaction kettle, transfer the reaction product to a 10 mL centrifuge tube, centrifuge at 8000 rpm / min for 30 min, take the supernatant to remove impurities, and repeat this step three times. Transfer the final supernatant into a 500 Da dialysis bag and dialyze for 48 h for purification, changing the water every 5 h.

[0039] Example 2: Preparation of a kind of ligusticum wallichii carbon dots.

[0040] Fresh Ligusticum chuanxiong Hort. is dried in the sun and ground into powder. Weigh 100 mg of Ligusticum chuanxiong Hort. powder and disperse it in 10 mL of water. After mixing, transfer it to a 30 mL polytetrafluoroethylene high-pressure reaction kettle, and carry out hydrothermal reaction at 195 °C for 12 h. After the reaction is completed, transfer the high-pressure reaction kettle to room temperature for cooling. After the high-pressure reaction kettle is completely cooled, open the reaction kettle, transfer the reaction product to a 10 mL centrifuge tube, centrifuge at a speed of 8000 rpm / min for 30 min, take the supernatant to remove impurities, and repeat this step three times. Transfer the final supernatant into a dialysis bag with a molecular weight cut-off of 500 Da and dialyze for 48 h for purification, changing the water every 5 h.

[0041] Example 3: Preparation of Ligusticum chuanxiong Hort. carbon dots.

[0042] Fresh Ligusticum chuanxiong Hort. is dried in the sun and ground into powder. Weigh 100 mg of Ligusticum chuanxiong Hort. powder and disperse it in 10 mL of water. After mixing, transfer it to a 30 mL polytetrafluoroethylene high-pressure reaction kettle, and carry out hydrothermal reaction at 180 °C for 8 h. After the reaction is completed, transfer the high-pressure reaction kettle to room temperature for cooling. After the high-pressure reaction kettle is completely cooled, open the reaction kettle, transfer the reaction product to a 10 mL centrifuge tube, centrifuge at a speed of 8000 rpm / min for 30 min, take the supernatant to remove impurities, and repeat this step three times. Transfer the final supernatant into a dialysis bag with a molecular weight cut-off of 500 Da and dialyze for 48 h for purification, changing the water every 5 h.

[0043] Example 4: Preparation of Ligusticum chuanxiong Hort. carbon dots.

[0044] Fresh Ligusticum chuanxiong Hort. is dried in the sun and ground into powder. Weigh 100 mg of Ligusticum chuanxiong Hort. powder and disperse it in 10 mL of water. After mixing, transfer it to a 30 mL polytetrafluoroethylene high-pressure reaction kettle, and carry out hydrothermal reaction at 180 °C for 12 h. After the reaction is completed, transfer the high-pressure reaction kettle to room temperature for cooling. After the high-pressure reaction kettle is completely cooled, open the reaction kettle, transfer the reaction product to a 10 mL centrifuge tube, centrifuge at a speed of 8000 rpm / min for 30 min, take the supernatant to remove impurities, and repeat this step three times. Transfer the final supernatant into a dialysis bag with a molecular weight cut-off of 400 Da and dialyze for 48 h for purification, changing the water every 5 h.

[0045] Example 5: Preparation of Ligusticum chuanxiong Hort. carbon dots.

[0046] Fresh Ligusticum chuanxiong Hort. was dried in the sun and ground into powder. 100 mg of Ligusticum chuanxiong Hort. powder was weighed and dispersed in 10 mL of water. After mixing, it was transferred to a 30 mL polytetrafluoroethylene high-pressure reaction kettle and hydrothermally reacted at 180 °C for 12 h. After the reaction was completed, the high-pressure reaction kettle was transferred to room temperature for cooling. After the high-pressure reaction kettle was completely cooled, it was opened, and the reaction product was transferred to a 10 mL centrifuge tube. It was centrifuged at 8000 rpm / min for 30 min to take the supernatant to remove impurities, and this step was repeated three times. The final supernatant was transferred into a dialysis bag with a molecular weight cut-off of 600 Da and dialyzed for 48 h for purification, and the water was changed every 5 h.

[0047] In Examples 1 to 5, carbon dots of Ligusticum chuanxiong Hort. with the expected effects were prepared, and the properties of the carbon dots of Ligusticum chuanxiong Hort. prepared in each example were basically the same. Therefore, only the carbon dots of Ligusticum chuanxiong Hort. prepared in Example 1 will be taken as an example for description below.

[0048] Figure 1 Figure 1 is a transmission electron microscope of carbon dots of Ligusticum chuanxiong Hort., which is used to observe the morphological characteristics of the carbon dots of Ligusticum chuanxiong Hort. The results show that the carbon dots of Ligusticum chuanxiong Hort. are spherical structures with a size of 2 nm to 4 nm under the transmission electron microscope. The small size indicates that the carbon dots of Ligusticum chuanxiong Hort. have a higher specific surface area and can play a more powerful role.

[0049] Figure 2 It shows that the carbon dots of Ligusticum chuanxiong Hort. have a graphite morphology and surface defects, and I D / I G is 0.91, and the I D / I G of the carbon dots of Ligusticum chuanxiong Hort. prepared in other examples is between 0.8 and 1.0.

[0050] Experimental Example 1 Down-regulation effect of carbon dots of Ligusticum chuanxiong Hort. on apoptotic proteins The carbon dots of Ligusticum chuanxiong Hort. were used to test their down-regulation of apoptotic proteins. The specific operation steps are as follows: Healthy adult SD rats were selected and randomly divided into: Blank control group: labeled as Control group.

[0051] Model group: labeled as MIRI group.

[0052] Drug group: labeled as MIRI+LC-CDs group.

[0053] Before modeling, in the MIRI+LC-CDs group, ligustrazine carbon dots solution was injected in situ at the cardiac site, with the administration concentration of 3 mg / kg. The Control group and the MIRI group were injected with an equal amount of normal saline. The myocardial ischemia-reperfusion injury model was established by ligating the left anterior descending branch of the coronary artery. Specifically, after anesthetizing the rats, the chest cavity was incised to expose the heart. After ligating the left anterior descending branch for 30 minutes, the ligature was released to restore blood flow for 120 minutes. The rats were sacrificed 24 hours after the modeling was completed. The hearts were quickly removed, and the left ventricular myocardial tissues were separated. One part was used for protein extraction, and the other part was stored in liquid nitrogen for later use. The myocardial tissues were minced and then homogenized with RIPA lysis buffer. After lysing on ice for 30 minutes, centrifugation was performed at 12,000 rpm and 4°C for 15 minutes. The supernatant was taken, and the protein concentration was measured by the BCA method and adjusted to a uniform concentration for later use. Equal amounts of protein samples were subjected to SDS-PAGE electrophoresis and transferred to a PVDF membrane. After blocking with 5% skim milk for 1 hour, primary antibodies against Caspase3, Caspase9, and Bax at a dilution ratio of 1:1000 were added and incubated overnight at 4°C. After washing the membrane with TBST, secondary antibody labeled with HRP at a dilution ratio of 1:5000 was added and incubated at room temperature for 1 hour. ECL chemiluminescence reagent was used for development. As Figure 3 shown, compared with the Control group, the expressions of Caspase3, Caspase9, and Bax in the MIRI group were increased, while the expressions of Caspase3, Caspase9, and Bax in the MIRI+LC-CDs group were lower than those in the MIRI group, indicating that ligustrazine carbon dots can effectively down-regulate the expression of apoptotic proteins and inhibit cardiomyocyte apoptosis.

[0054] Experimental Example 2 Functional improvement effects of ligustrazine carbon dots, ligustrazine, and ligustrazine carbon dots synthesized by the pyrolysis method on myocardial ischemia-reperfusion injury Ligustrazine carbon dots synthesized by the pyrolysis method: 20 grams of honeysuckle were ground into powder in a crucible, and then heated to 350°C in a muffle furnace and maintained for 1 hour. After calcination, the powder was ground, and then boiled in 1 L of ultrapure water for 1.5 h. After cooling to room temperature, impurities were removed by centrifugation, and then purified by dialysis. The cut-off molecular weight of the dialysis was 400 Da - 600 Da.

[0055] Healthy adult SD rats were selected and randomly divided into: Blank control group: labeled as the Control group.

[0056] Model group: labeled as the MIRI group.

[0057] Ligustrazine drug group: labeled as the ligustrazine group.

[0058] Ligustrazine carbon dots group synthesized by the pyrolysis method: labeled as the LC-CDs1 group.

[0059] Ligusticum chuanxiong carbon dot drug group: labeled as LC-CDs2 group.

[0060] Before modeling, the ligustrazine drug group, the Ligusticum chuanxiong carbon dot drug group, and the Ligusticum chuanxiong carbon dots synthesized by the pyrolysis method were injected in situ through the heart, and the Control group and the MIRI group were injected with an equal amount of normal saline. The myocardial ischemia-reperfusion injury model was established by ligating the left anterior descending branch of the coronary artery. Specifically, after anesthetizing the rats, the chest was opened to expose the heart. After ligating the left anterior descending branch for 30 minutes, the ligature was released to restore blood flow for 120 minutes. The rat hearts were examined by ultrasound 24 hours after the modeling was completed.

[0061] As Figure 4 shown, it was observed that the Ligusticum chuanxiong carbon dot drug group could more effectively restore the impaired cardiac function of the rat heart caused by reperfusion than the ligustrazine drug group and the Ligusticum chuanxiong carbon dots synthesized by the pyrolysis method. The systolic and diastolic functions of the heart were restored, indicating that the Ligusticum chuanxiong carbon dots provided by the present invention could effectively improve cardiac ischemia-reperfusion injury.

[0062] As Figure 5 shown, it was observed that the Ligusticum chuanxiong carbon dot drug group could more effectively restore the left ventricular ejection fraction and the left ventricular fractional shortening rate of the rat heart than the ligustrazine drug group and the Ligusticum chuanxiong carbon dots synthesized by the pyrolysis method, indicating that the Ligusticum chuanxiong carbon dots provided by the present invention could effectively improve the cardiac function injury caused by cardiac ischemia-reperfusion.

[0063] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications once they learn the basic creative concept. Therefore, the appended claims are intended to be construed to include the preferred embodiments as well as all changes and modifications falling within the scope of the present invention.

Claims

1. A chuanxiong carbon dot, characterized in that It is obtained by hydrothermal carbonization of Ligusticum chuanxiong, and the hydrothermal carbonization is carried out by hydrothermal reaction at 175°C to 195°C for 8h to 12h.

2. The chuanxiong carbon dots according to claim 1, characterized in that, After the hydrothermal reaction is completed, impurities are removed by centrifugation, and the Ligusticum chuanxiong carbon dots are obtained by dialysis purification.

3. The chuanxiong carbon dots according to claim 2, characterized in that, The cut-off molecular weight of the dialysis is 400Da to 600Da.

4. The chuanxiong carbon dots according to claim 1, characterized in that, The average particle size of the Ligusticum chuanxiong carbon dots is 2nm to 4nm.

5. A drug, characterized in that, It comprises the Ligusticum chuanxiong carbon dots described in any one of claims 1 to 4, and pharmaceutically acceptable excipients; the drug is used for: 1) inhibiting cardiomyocyte apoptosis; 2) treating myocardial ischemia-reperfusion injury.

6. Use of the Ligusticum chuanxiong carbon dots described in any one of claims 1 to 4 in the preparation of a drug for inhibiting cardiomyocyte apoptosis.

7. Use of the Ligusticum chuanxiong carbon dots described in any one of claims 1 to 4 in the preparation of a drug for treating ischemia-reperfusion injury.

8. The application according to claim 6, wherein The ischemia-reperfusion injury is myocardial ischemia-reperfusion injury.

Images