Use of peony root derived carbon dots in preparation of drugs for treating myocarditis

Abstract

The application discloses application of cortex moutan derived carbon dots in preparation of a drug for treating myocarditis. Cortex moutan derived carbon dots (MCDs) obtained by one-step hydrothermal method and high-temperature heating have good biocompatibility, exhibit excellent ROS resistance in vitro and in vivo, and can significantly remove •OH, •O2, 1 O2. The MCDs can effectively inhibit the oxidative damage and pro-inflammatory factor level of myocarditis cells and acute myocarditis mice. The MCDs are easy to prepare, simple to synthesize, can improve the heart function of acute myocarditis mice and effectively relieve myocardial damage, and provide a new technology and new preparation for treatment of myocarditis.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine application technology, specifically relating to the use of peony bark-derived carbon dots in the preparation of drugs for treating myocarditis. Background Technology

[0002] Myocarditis is an inflammatory disease of the myocardium, where inflammation causes swelling of the myocardial layer and impairs the contractile function of myocardial cells. If myocarditis is particularly severe and cardiac function cannot meet the body's needs, it can further develop into heart failure, and some cases of acute fulminant myocarditis can even lead to death. Myocarditis can be caused by a variety of infectious or non-infectious factors, such as viruses, immune system activation, or exposure to toxins and drugs. Recently, myocarditis has been considered a rare complication of the Covid-19 mRNA vaccine. Depending on the extent of organ involvement, myocarditis presents with a variety of clinical manifestations, such as chest pain with other uncomplicated clinical symptoms, new-onset or worsening heart failure, chronic heart failure, life-threatening hemodynamic impairment (acute fulminant myocarditis), and life-threatening arrhythmias or conduction disturbances (such as persistent ventricular arrhythmias, atrioventricular block, and sudden death).

[0003] Treatment options for myocarditis include addressing arrhythmias and heart failure according to standard guidelines, as well as treating the underlying cause. While various treatment options exist for myocarditis, the efficacy of medications remains limited. For example, existing drugs such as vitamin C, coenzyme Q10, and astragalus, while offering some protection to the myocardium, have limited effectiveness, and treatment outcomes vary significantly among patients. Therefore, new treatment strategies and agents are urgently needed for the treatment of myocarditis, especially acute myocarditis.

[0004] In recent years, carbon dots, a novel class of zero-dimensional nanomaterials with a particle size of less than 10 nm, have attracted increasing attention due to their high biocompatibility, low cost, and antioxidant enzyme-like activity. Using traditional Chinese medicines rich in various active ingredients as carbon sources to prepare herbal-derived carbon dots with special functions such as anti-inflammatory and antioxidant effects represents an important direction for the interdisciplinary development of traditional Chinese medicine in disease treatment and nanomedicine.

[0005] Moutan bark is a classic Chinese herbal medicine that enters the heart, liver, and kidney meridians, possessing the effects of clearing heat and cooling blood, promoting blood circulation and removing blood stasis. Modern pharmacological studies have found that paeonol, the main active ingredient in moutan bark, has significant therapeutic effects in treating atherosclerosis, thrombosis, reperfusion injury, arrhythmia, and hypertension, and shows broad prospects in cardiovascular drug screening and development. However, there are currently few reports on the treatment of myocarditis with moutan bark. Therefore, further research on moutan bark and in-depth development of its application in the treatment of cardiovascular diseases, especially exploring its therapeutic potential in myocarditis, are problems that need to be solved. Summary of the Invention

[0006] Purpose of the invention: The technical problem to be solved by the present invention is to provide the application of peony bark-derived carbon dots in the preparation of drugs for treating myocarditis.

[0007] Technical solution: In order to solve the above-mentioned technical problems, the present invention provides the application of peony bark-derived carbon dots in the preparation of drugs for treating myocarditis.

[0008] The average particle size of the carbon dots derived from peony bark is 4.8 ± 0.5 nm, and the lattice size of the carbon dots derived from peony bark is 0.22 nm ± 0.07 nm.

[0009] The MCDs are uniform in size, rich in active groups such as amino and carboxyl groups on their surface, and have an average particle size of 4.8 nm and a zeta potential of -15.4 mV.

[0010] The concentration of the carbon dots derived from peony bark is 10–100 μg / mL.

[0011] The peony bark-derived carbon dots are prepared by a one-step hydrothermal method using peony bark as a raw material.

[0012] The hydrothermal method specifically includes: grinding peony bark into peony bark powder, dissolving it in ultrapure water, and placing it in a hydrothermal reactor for heating and reaction; cooling after the reaction, opening the reactor, taking out the product, and sequentially filtering, vacuum filtering, dialysis, and vacuum freeze-drying it to obtain the final product, which is peony bark-derived carbon dots.

[0013] The mass-to-volume ratio of the peony bark powder to ultrapure water is 1:5g / mL to 1:50g / mL.

[0014] The hydrothermal reaction time for the heating reaction is 3-12 hours, and the temperature range is 160-300℃. Preferably, the temperature is 180℃ and the reaction time is 6 hours.

[0015] The myocarditis mentioned includes acute myocarditis. The drug mentioned is a single-ingredient preparation or a compound preparation.

[0016] Among these applications are the anti-inflammatory and antioxidant effects of peony bark-derived carbon dots on myocarditis cells.

[0017] The applications include downregulating oxidative stress and inflammatory factor levels in myocarditis cells and / or inhibiting inflammatory cell infiltration, inflammatory factor and oxidative damage levels in mouse hearts by using peony bark-derived carbon dots, thereby maintaining normal cardiac function and improving myocardial injury in mice.

[0018] Beneficial effects: Compared with the prior art, the present invention has the following advantages: The peony bark-derived carbon dots (MCDs) obtained by the one-step hydrothermal high-temperature heating method of the present invention have good biocompatibility and exhibit excellent anti-ROS ability in vivo and in vitro, and can significantly scavenge ·OH, ·O2-, 1 O2. MCDs can effectively inhibit oxidative damage and pro-inflammatory factor levels in myocarditis cells and mice with acute myocarditis. The MCDs proposed in this invention are easy to prepare and simple to synthesize, and can improve cardiac function and effectively alleviate myocardial damage in mice with acute myocarditis, providing a new technology and formulation for the treatment of myocarditis. Attached Figure Description

[0019] Figure 1 (a) is a method for preparing carbon dots MCDs from peony bark; Figure 1 (b) TEM images of MCDs; Figure 1 (c) HRTEM images of MCDs, with white arrows indicating the crystal lattice; Figure 1 (d) AFM plot of MCDs; Figure 1 (e) FT-IR plots of MCDs;

[0020] Figure 2 (a) Raman characteristic peaks of MCDs; Figure 2 (b) XRD patterns of MCDs; XPS measurements of MCDs: Figure 2 (c) XPS plot; Figure 2 (d) C1s spectrum, Figure 2 (e) N1s spectrum, Figure 2 (f) O1s spectrum;

[0021] Figure 3 (a) Measurement of zeta potential in MCDs; Figure 3 (b) Hydrated particle size of MCDs; Figure 3 (c) ABTS assay (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) assay) to determine the in vitro anti-total ROS ability of MCDs; Figure 3 (d) Salicylic acid assay to determine the resistance of MCDs to ·OH; Figure 3 (e) Pyrogallol assay to determine the MCDs' resistance to ··O2 - Ability; Figure 3 (f) SOSG (single oxygen sensor green) method for determining MCDs resistance 1 O2's ability;

[0022] Figure 4 (a) Cell viability assay after incubation of different concentrations of MCDs with RAW264.7 or HL-1 for 48 h; Figure 4 (b) Cell viability assay of HL-1 cells co-incubated with MCDs and LPS or H2O2 at different time points; Figure 4 (c) The DCFH probe was used to determine the anti-total ROS ability of MCDs in vivo under LPS or H2O2 induction for 48 h; Figure 4 (d) qPCR detection of the effects of MCDs on HL-1 cells after 48 h of induction with or without LPS or H2O2. Figure 4 (d)IL-6, Figure 4 (e)TNF-α and Figure 4 (f) Regulatory level of IL-1α;

[0023] Figure 5 (a) Schematic diagram of animal experiments; Figure 5 (b) Anatomical diagram of the mouse heart; Figure 5 (c) Mouse cardiac index (heart weight / body weight); Figure 5 (d) H&E pathological section of mouse heart; Figure 5 (e) Left image, ejection fraction (EF); Right image, left ventricular fractional shortening (FS); Figure 5 (f) Serum creatine kinase (CK) and lactate dehydrogenase (LDH) levels; Figure 5 (g) Serum levels of inflammatory factors IL-6 and TNF-α; Figure 5 (h) The ratio of NAD+ / NADH in myocardial tissue; Figure 5 (i) The content of MDA in myocardial tissue. Detailed Implementation

[0024] Example 1: Preparation, characterization, and in vitro antioxidant capacity determination of carbon dots MCDs from peony bark.

[0025] 1. Preparation and characterization of carbon dot MCDs from peony bark

[0026] Take 5g of peony bark (purchased from Anhui Huchuntang Chinese Herbal Pieces Co., Ltd., batch number: 240301, origin: Jiangsu) and grind it into powder. Dissolve it in 50mL of ultrapure water and place it in a 150mL PTFE-lined hydrothermal reactor. After assembly, heat it in a high-temperature oven at 180℃ for 6 hours. After cooling overnight, open the reactor, take out the heated product, and filter it sequentially, then perform vacuum filtration (0.22μm), dialyze (72h), and vacuum freeze-dry. The resulting final product is MCDs, which should be stored at -20℃ for long-term storage.

[0027] The particle size and surface morphology of MCDs were measured using high-resolution transmission electron microscopy (JEM-2100F, Japan) and atomic force microscopy (Bruker Dimension ICON, Germany) in tapping mode, respectively. Figure 1 This image visually demonstrates the process of heating 5g of Cortex Moutan in a hydrothermal reactor at 180℃ for 6 hours, followed by dialysis for 72 hours and vacuum freeze-drying to obtain Cortex Moutan-derived carbon dots (MCDs). The final product of MCDs obtained was 0.65g, with a yield of 13%. Transmission electron microscopy (TEM) showed that the MCDs were uniform in size, conformed to a Gaussian distribution, and had an average particle size of 4.8±0.5nm. Figure 1 b). High-resolution transmission electron microscopy (HRTEM) further revealed that the lattice structure of the MCDs was within 0.22 nm ± 0.07 nm. Figure 1 c) indicates that it possesses graphene structural characteristics. Atomic force microscopy (AFM) provides higher resolution for characterizing nanoparticle size, and its results show that the height of the MCDs is around 4.85 nm, which is consistent with the results of TEM. Figure 1 d). To further investigate the functional groups on the surface of MCDs, we used Fourier transform infrared spectroscopy (FT-IR) to detect the MCDs. The results showed that the functional groups of MCDs at 3421 cm⁻¹... -1 2921cm -1 1625cm -1 1043cm -1 The presence of broad peaks nearby is attributed to OH / NH, CH, CN, and CO groups on the surface of the MCDs. Furthermore, FT-IR analysis of the peony bark powder revealed that the functional groups on the MCD surface are consistent with the active groups of peony bark, suggesting that the MCDs may retain the biological efficacy of peony bark.

[0028] To further determine the structure and composition of MCDs, we first scanned and detected them using Raman spectroscopy. The Raman characteristic peaks and diffraction peaks of the MCDs were measured using a Raman spectrometer (Horiba LabRAM HREvlution, Japan) and an X-ray diffractometer (Rigaku SmartLab SE), respectively. The functional groups and composition of the MCDs were determined using a Fourier transform infrared spectroscopy (FT-IR 650S, Tianjin Gangdong) and an X-ray photoelectron spectroscopy (Thermo Kalpha, USA). Figure 2 As shown in a, MCDs are at 1380cm -1 and 1576cm -1 It exhibits typical Raman characteristic peaks, the D peak and the G peak. The D peak mainly originates from the stretching vibrations of C-C bonds and C=C double bonds, while the G peak originates from sp... 2The stretching vibrations of hybrid carbon atoms and π electrons are both Raman characteristic peaks of graphene, especially the high-intensity G peak, reflecting that MCDs possess a single-layer graphene structure. Simultaneously, X-ray diffraction (XRD) results show that MCDs have a diffraction peak at 22.31°. Figure 2 b), which corresponds to the graphene plane (002), matches Raman's data, strongly suggesting that the MCDs are graphene. We also analyzed the composition of the MCDs using X-ray photoelectron spectroscopy (XPS). For example... Figure 2 As shown in Figure c, MCDs are mainly composed of C, O, and N, accounting for 52.6%, 41.03%, and 6.17% respectively. XPS full spectrum analysis shows that MCDs contain C1s, N1s, and O1s peaks, located at 286.1 eV, 400.1 eV, and 531.1 eV respectively. XPS analysis of C1s further shows characteristic peaks at 284.4 eV, 285.8 eV, 286.2 eV, and 287.5 eV, indicating the presence of CO / CN, C=N, C=C / CC, and C=O bonds, respectively. XPS analysis of N1s shows characteristic peaks at 399.5 eV and 400.1 eV corresponding to NH and CN, while XPS analysis of O1s shows characteristic peaks at 531.1 eV and 532.4 eV corresponding to C=O and C-OH / COC. These results indicate that MCDs are monolayer graphene carbon dots with surfaces rich in active groups such as amino and carboxyl groups.

[0029] 2. Determination of the in vitro antioxidant capacity of MCDs

[0030] MCDs were dissolved in water, and their surface potential (zeta potential) and hydrated particle size were measured. Data showed that the MCDs carried a negative charge on their surface, with an average zeta potential of -15.4 mV. Figure 3 a). The average hydrated particle size of MCDs is 16.2 nm, which is due to the slight aggregation of MCDs in aqueous solution. Figure 3 b). The presence of unpaired electrons and surface defects on the surface of carbon dots often endows them with the ability to scavenge free radicals (ROS). Therefore, the ABTS free radical scavenging ability test kit (Boxbio, AKAO012C) was first used to test the in vitro total antioxidant capacity of different concentrations of MCDs (0, 10, 25, 50, 100 μg / mL) at different time points (10 min, 30 min, 1 h, 2 h, 4 h, 6 h, 12 h, 24 h). Figure 3As shown in c and Table 1, the higher the concentration of MCDs, the stronger their ability to scavenge total ROS, i.e., the higher their antioxidant capacity. 100 μg / mL of MCDs can scavenge nearly 80% of total ROS after 24 hours. The scavenging efficiency of total reactive oxygen species (ROS) was determined using the ABTS free radical scavenging ability test kit method. The specific calculation formula is: ABTS total ROS scavenging rate = (OD of blank group) / (OD of blank group). 405 nm -OD of the test group 405 nm +Control group OD 405 nm )÷ Blank group OD 405 nm ×100%.

[0031] Table 1. Scavenging efficiency (%) of MCDs at different concentrations for total reactive oxygen species (ROS)

[0032] 10min 0 0 5 8 17 30min 0 2 9 16 18 1h 0 5 11 16 21 2h 0 8 8 17 25 4h 0 6 14 24 32 6h 0 5 15 25 44 12h 0 8 20 31 50 24h 0 12 28 42 81

[0033] To further investigate the ROS scavenging ability of MCDs, we examined their scavenging efficiency against three different ROS using the salicylic acid method, the pyrogallol autoxidation method, and the singlet oxygen green fluorescent probe (SOSG). Experimental data showed that MCDs can significantly scavenge hydroxyl radicals ·OH (…). Figure 3 d), Superoxide anion·O2 - ( Figure 3 e) and singlet oxygen 1 O2( Figure 3 f).

[0034] Example 2: Cellular Experiment of Carbon Dot MCDs from Moutan Cortex

[0035] MCDs powder was dissolved in ultrapure water to prepare a 10 mg / mL aqueous solution, which was then added to 96-well plates containing RAW264.7 (mouse mononuclear macrophage leukemia cells) and HL-1 (mouse cardiomyocytes) cells that had been seeded and adhered overnight. The seeding density was 5 × 10⁶ cells per well. 4 The final concentrations of MCDs were 0, 10, 25, 50, and 100 μg / mL. After incubation for 48 h, CCK-8 reagent was added, and the absorbance (OD) at 450 nm was measured using a microplate reader. The effect of MCDs on cell viability was determined by the CCK-8 assay. Figure 4 As shown in Figure a, aqueous solutions of MCDs at different concentrations were not toxic to RAW264.7 and HL-1 cells and did not affect cell viability, indicating that MCDs have high biocompatibility.

[0036] To further investigate the intrinsic regulatory mechanism of MCDs on myocarditis cells, we first explored the effect of MCDs on the oxidative level of myocarditis cells using the DCFH probe (intracellular total ROS probe). Both LPS and H2O2 can induce cellular oxidative stress, leading to the secretion of inflammatory factors. Therefore, we stimulated HL-1 cells with LPS at a final concentration of 5 μg / mL and H2O2 at 100 μM to construct two parallel myocarditis cell models. After HL-1 cells were co-incubated with MCDs and LPS or H2O2 at a final concentration of 50 μg / mL for 48 h, the total ROS fluorescent probe DCFH (Beyotime Biotechnology, S0033S) was added. Images were taken using an inverted fluorescence microscope, and the fluorescence was quantitatively analyzed using ImageJ. Figure 4 As shown in b, MCDs significantly rescued the cell viability of HL-1 myocarditis cells induced by LPS / H2O2, and the longer the incubation time, the better the rescue effect. The results indicate that MCDs can effectively inhibit ROS production in HL-1 cells induced by LPS / H2O2 and regulate the oxidative level of myocarditis cells. Figure 4 c). Simultaneously, we also examined the effect of MCDs on the inflammatory level of myocarditis cells. The results showed that MCDs significantly downregulated the expression of pro-inflammatory factors in myocarditis cells, including IL-6 (c). Figure 4 d) TNF-α Figure 4 e) and IL-1α ( Figure 4 f). Similarly, after 48 hours of incubation, RNA was extracted from each group of cells, and the mRNA levels of IL-6, TNF-α, and IL-1α were detected. These results indicate that MCDs have good biocompatibility and can salvage the cell viability of myocarditis cells by inhibiting oxidative stress and inflammatory factor levels.

[0037] Example 3: Animal Experiments with Carbon Dot MCDs from Moutan Bark

[0038] To evaluate the therapeutic effect of MCDs on myocarditis in vivo, we established an acute myocarditis model in male C57BL / 6j mice by intraperitoneal injection of LPS (10 mg / kg, 0.2 mL). Eighteen 8-week-old C57BL / 6j mice were randomly divided into three groups: PBS (healthy group), LPS (model group), and MCDs (treatment group). One hour before the LPS intraperitoneal injection, 100 μL of PBS / MCDs was injected into the tail vein of the mice. One hour later, the mice were injected intraperitoneally with LPS (10 mg / kg, 0.2 mL) to establish the acute myocarditis model. The healthy group was controlled by intraperitoneal injection of 0.2 mL of PBS. Six hours later, echocardiography was performed on all mice. Eight hours later, the mice were euthanized and all mice were dissected. Figure 5 a).

[0039] like Figure 5b and Figure 5 As shown in Figure c, the cardiac index (heart weight / body weight) of mice in the LPS group was significantly higher than that in the PBS and MCDs groups. However, after early intervention with MCDs, there was no significant difference between the MCDs group and the PBS group. This result indicates that MCDs effectively alleviated the inflammation-induced cardiac enlargement in mice caused by LPS. Further pathological sections from the H&E section showed that early intervention with MCDs resulted in significantly less inflammatory cell infiltration in the heart of the mice compared to the LPS group. Figure 5 d). To further investigate cardiac function in mice, we used echocardiography to assess cardiac function in each group of mice 6 hours after intraperitoneal injection of LPS. Ejection fraction (EF) and left ventricular fractional shortening (FS) are two important indicators for evaluating cardiac function. Data showed that LPS significantly inhibited EF and FS in mice, causing cardiac dysfunction, which was significantly improved after intervention with MCDs. Figure 5 e). In addition, we also examined the activities of serum markers of myocardial injury, such as creatine kinase (CK) and lactate dehydrogenase (LDH). Figure 5 As shown in f, the results indicate that MCDs can significantly reverse LPS-induced upregulation of CK and LDH, demonstrating a good therapeutic effect on myocardial injury in mice. Pro-inflammatory factors IL-6 and TNF-α were also detected. Data showed that LPS significantly increased serum IL-6 and TNF-α levels, but MCD intervention improved this situation and downregulated inflammation levels in mice. Figure 5 g). Oxidative damage is a significant cause of myocarditis in mice, therefore we measured NAD+ in mice. + / NADH and MDA levels. The results showed that LPS induced NAD in mice. + Disorders of NADH and elevated MDA levels exacerbate oxidative stress in mouse cardiomyocytes, while intervention with MCDs can help the heart resist oxidative damage. Figure 5 h and Figure 5 i). In summary, drug administration of MCDs can significantly inhibit the levels of pro-inflammatory factors and oxidative stress in mice with myocarditis, maintain normal cardiac function and volume, and improve and alleviate the progression of acute myocarditis in mice.

Claims

1. The application of peony bark-derived carbon dots as the sole active ingredient in the preparation of drugs for treating myocarditis, characterized in that, The average particle size of the peony bark-derived carbon dots is 4.8 ± 0.5 nm, and the lattice size is 0.22 nm ± 0.07 nm. The peony bark-derived carbon dots are prepared from the traditional Chinese medicine peony bark using a one-step hydrothermal method. The hydrothermal method specifically includes: grinding peony bark into powder, dissolving it in ultrapure water, and placing it in a hydrothermal reactor for heating and reaction; cooling after the reaction, opening the reactor, removing the product, and sequentially filtering, vacuum filtering, dialysis, and vacuum freeze-drying to obtain the final product, which is the peony bark-derived carbon dots. The mass-to-volume ratio of peony bark powder to ultrapure water is 1:5 g / mL to 1:50 g / mL. The hydrothermal reaction time is 3-12 h, and the temperature range is 160-300℃.

2. The application according to claim 1, characterized in that, The concentration of the carbon dots derived from peony bark is 10~100 μg / mL.

3. The application according to claim 1, characterized in that, The applications include the anti-inflammatory and antioxidant effects of peony bark-derived carbon dots on myocarditis cells.

4. The application according to claim 1, characterized in that, The applications include downregulating oxidative stress and inflammatory factor levels in myocarditis cells and / or inhibiting inflammatory cell infiltration, inflammatory factor and oxidative damage levels in mouse hearts by using peony bark-derived carbon dots, maintaining normal cardiac function and improving myocardial injury in mice.

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