Preparation method and application of rhubarb carbon dot nanoscale enzyme

Abstract

This invention discloses a method for preparing rhubarb carbon dot nanozymes and their applications, belonging to the fields of nanobiomaterials and modernization of traditional Chinese medicine. The rhubarb carbon dot nanozymes are prepared in one step via hydrothermal carbonization using rhubarb as the carbon source. The surface is enriched with in-situ doped pyridine-type nitrogen active centers, and the WST-1 method verifies that it possesses genuine SOD-like nanozyme catalytic cycling activity. Studies have found that this rhubarb carbon dot nanozyme can efficiently scavenge various free radicals such as DPPH, ABTS, hydroxyl groups, and superoxide anions, exhibiting significantly better antioxidant activity than rhubarb water extracts of the same concentration. It can significantly inhibit LPS-induced NO release from RAW 264.7 macrophages. In an IMQ-induced C57BL / 6 mouse psoriasis model, it can effectively reduce epidermal thickness and inhibit inflammatory infiltration, showing superior therapeutic effects compared to rhubarb extracts.

Description

Technical Field

[0001] This invention belongs to the field of nanobiomaterials and the modernization of traditional Chinese medicine, specifically relating to a method for preparing rhubarb carbon dot nanoenzymes, and their application in scavenging free radicals, inhibiting inflammatory responses, and preparing antioxidant and anti-inflammatory topical skin preparations, especially their application in the preparation of drugs for treating psoriasis. Background Technology

[0002] Oxidative stress refers to a pathological state in which the dynamic balance between the production of reactive oxygen species (ROS) and the antioxidant defense system in the body is disrupted, leading to the excessive accumulation of ROS. Excessive ROS includes superoxide anion radicals (O2). - Oxidative stress, such as hydroxyl radicals (·OH) and hydrogen peroxide (H2O2), can attack cell membrane lipids, triggering lipid peroxidation, leading to protein denaturation and DNA damage, and ultimately inducing apoptosis and tissue damage. Oxidative stress has been proven to be one of the core inducing factors in many pathological processes, including inflammation, skin damage, chronic wounds, neurodegenerative diseases, and tumors.

[0003] Psoriasis is an immune-mediated, chronic, relapsing inflammatory skin disease with a global prevalence of approximately 2%–3%. Clinical manifestations include skin erythema, scaling, itching, and abnormal thickening of the epidermis. The pathogenesis of psoriasis involves… Th17 Abnormal activation of the IL-23 axis, excessive proliferation of keratinocytes, and massive accumulation of ROS in the local microenvironment. Studies have shown that psoriatic lesions exhibit significant oxidative stress, with significantly elevated ROS levels and decreased antioxidant enzyme activity, which further exacerbates the inflammatory cascade and damages the epidermal barrier function. Therefore, developing drugs with both high antioxidant and anti-inflammatory activities is of great significance for the treatment of psoriasis.

[0004] Traditional antioxidants, such as natural enzymes (SOD, CAT), vitamin C, vitamin E, and polyphenols, possess certain free radical scavenging capabilities, but they suffer from inherent drawbacks such as poor stability, easy inactivation, short half-life, and difficulty in sustaining free radical scavenging. Natural enzyme antioxidants are sensitive to temperature and pH, and are easily degraded by proteases in the body, losing their activity. Small-molecule antioxidants, such as vitamin C, are extremely unstable under light and heat conditions, and as stoichiometric scavengers, each molecule can only consume one or a few free radicals, failing to achieve a catalytic cycle, thus significantly limiting their clinical application.

[0005] Nanozymes are a class of nanomaterials with enzyme-like catalytic activity, combining the high stability of nanomaterials with the catalytic activity of natural enzymes. They can efficiently mimic the activities of various natural antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and peroxidase (POD), demonstrating significant advantages in the continuous and efficient removal of reactive oxygen species (ROS) and the regulation of oxidative stress. Compared with traditional antioxidants, nanozymes have advantages such as strong catalytic cycling ability, high chemical stability, scalability, and low cost.

[0006] Carbon dots (CDs) are a novel class of zero-dimensional carbon nanomaterials with particle sizes typically less than 10 nm. They possess unique advantages such as ultra-small particle size, excellent water solubility, high biocompatibility, abundant surface functional groups, ease of functionalization modification, and adjustable optical and catalytic properties, making them an ideal carrier platform for constructing nanozymes. In recent years, the preparation of carbon dots using natural biomass or traditional Chinese medicine as carbon sources through green methods such as hydrothermal and solvothermal methods has become a research hotspot. "Traditional Chinese medicine carbon dots" prepared using traditional Chinese medicine as a carbon source not only inherit the nanomaterial advantages of carbon dots themselves but also retain or transform some of the pharmacologically active groups of the parent traditional Chinese medicine components, achieving a synergistic enhancement of material properties and pharmacological efficacy.

[0007] Rhubarb is the plant Rheum palmatum of the Polygonaceae family. Rheum palmatum L., Tangut Rhubarb Rheum tanguticum Maxim. ex Balf. or medicinal rhubarb Rheum officinale The dried roots and rhizomes of *Rheum palmatum* are classic medicinal materials with a long history of application in traditional Chinese medicine. Rheum palmatum is cold in nature and bitter in taste, and enters the stomach, large intestine, liver, and pericardium meridians. It has the effects of purging accumulations, clearing heat and purging fire, cooling blood and detoxifying, and removing blood stasis and promoting menstruation. Modern pharmacological studies have shown that rheum palmatum is rich in anthraquinone compounds (such as rhein, chrysophanol, aloe-emodin, etc.), dianthrones, flavonoids, tannins, and polyphenols, among other active ingredients, and possesses broad-spectrum anti-inflammatory, antioxidant, antibacterial, antitumor, and immunomodulatory pharmacological effects. Among these, the anthraquinone components of rheum palmatum have been proven to inhibit the activation of the NF-κB signaling pathway and reduce the expression of pro-inflammatory factors such as TNF-α, IL-6, and IL-1β, exhibiting significant anti-inflammatory activity. While existing rhubarb extracts possess certain antibacterial activity, their active components are complex, their stability is poor, and they are greatly affected by extraction conditions. In contrast, whether carbon dot nanozymes formed by hydrothermal carbonization of rhubarb can further enhance the inhibitory ability against common pathogens while retaining antibacterial activity remains to be systematically studied.

[0008] However, conventional rhubarb extracts have the following prominent problems in practical applications: (1) the main active ingredients, such as anthraquinones, have poor water solubility and are difficult to disperse evenly in aqueous matrices; (2) the transdermal efficiency of macromolecular components is low, making it difficult to effectively penetrate the thickened stratum corneum of psoriasis to reach the lesion site; (3) the active ingredients have poor stability under light and heat conditions and short duration of action; (4) high concentrations may cause local irritation. These problems seriously restrict the clinical translation of rhubarb in the field of topical skin treatment.

[0009] It is worth noting that the antioxidant mechanisms of carbon dots in traditional Chinese medicine reported in existing literature are mostly attributed to the stoichiometric scavenging effect of residual components (such as polyphenols and flavonoids) (i.e., the carbon dots are consumed and deactivated after one reaction), lacking a true enzyme-like catalytic cycle. It is generally believed in the art that the active anthraquinone components in rhubarb are easily degraded and deactivated under high-temperature hydrothermal conditions, making it difficult to retain or enhance their antioxidant capacity. However, this invention, through extensive experimentation, has discovered that by precisely controlling hydrothermal conditions, the anthraquinone precursors in rhubarb are not only not completely deactivated, but also undergo nonlinear dehydration condensation and intramolecular cyclization with endogenous nitrogen-containing compounds, generating a large number of pyridine-N doping sites in situ. This unexpected structural reorganization endows the rhubarb carbon dots with a true SOD-like catalytic cycle, breaking through the bottleneck of the traditional "one-time consumption" of traditional Chinese medicine extracts, possessing outstanding substantive characteristics and significant progress.

[0010] Therefore, developing a rhubarb carbon dot nanozyme with a simple, green, and safe preparation process, high-efficiency enzyme-like catalytic activity, and the ability to efficiently scavenge various free radicals, and systematically verifying its significant advantages in antioxidation and anti-inflammation compared to the original rhubarb extract, would compensate for the shortcomings of traditional antioxidants and conventional rhubarb extracts. This would provide a novel and highly effective topical antioxidative and anti-inflammatory intervention for oxidative stress-related inflammatory skin diseases such as psoriasis, and has significant scientific value and clinical translation prospects. Summary of the Invention

[0011] To address the shortcomings of existing technologies, such as poor stability and weak catalytic cycling ability of traditional antioxidants, as well as poor water solubility, low transdermal efficiency, and short action time of conventional rhubarb extracts, the present invention aims to provide a method for preparing rhubarb carbon dot nanoenzymes and their application in scavenging free radicals or preparing antioxidant and anti-inflammatory products.

[0012] To achieve the above-mentioned objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a method for preparing rhubarb carbon dot nanozymes, comprising the following steps: (a) Take rhubarb medicinal material, crush and sieve it, add deionized water, and treat it with ultrasound to obtain rhubarb suspension; (b) The rhubarb suspension obtained in step (a) is transferred to a reaction vessel for hydrothermal reaction; (c) After the reaction is complete, allow the mixture to cool naturally to room temperature, then filter and centrifuge to collect the supernatant. (d) Dialyze the supernatant obtained in step (c); (e) After dialysis, the solution was filtered and freeze-dried to obtain rhubarb carbon dot nanozyme solid powder, which was stored in the dark for later use.

[0013] In step (a), the ratio of rhubarb medicinal material to deionized water is 1 g: 30 mL, and the ultrasonic time is 20 min to 40 min.

[0014] In step (b), the hydrothermal reaction temperature is 180℃~200℃ and the time is 8 h~12 h.

[0015] In step (c), the centrifugation speed is 8000 rpm to 10000 rpm and the time is 20 min to 30 min.

[0016] In step (d), the dialysis treatment time is 24 h to 48 h, with the water changed every 6 h to 8 h, and the molecular weight cutoff of the dialysis bag is 1000 Da.

[0017] In step (e), the temperature for storage in the dark is -20°C.

[0018] In step (e), the average particle size of the rhubarb carbon dot nanozyme obtained is 1 nm to 5 nm.

[0019] Secondly, the present invention provides the application of the rhubarb carbon dot nanoenzyme in scavenging free radicals.

[0020] The free radicals include one or more of DPPH free radicals, ABTS free radicals, hydroxyl free radicals, and superoxide anion free radicals.

[0021] The rhubarb carbon dot nanoenzyme exhibits concentration-dependent scavenging activity against all four free radicals, and its scavenging efficiency is significantly higher than that of the rhubarb water extract at the same concentration.

[0022] Thirdly, the present invention provides the application of the rhubarb carbon dot nanozyme in the preparation of antioxidant and anti-inflammatory products.

[0023] The antioxidant and anti-inflammatory product is a topical skin preparation.

[0024] The dosage form of the topical skin preparation is selected from one of the following: gel film, coating film, hydrogel, cream or patch.

[0025] Fourthly, the present invention provides the application of the rhubarb carbon dot nanozyme in the preparation of drugs for treating inflammatory skin diseases.

[0026] The inflammatory skin disease mentioned is psoriasis.

[0027] The psoriasis is preferably psoriasis vulgaris.

[0028] Compared with the prior art, the present invention has the following beneficial effects: (1) Highly efficient multi-target free radical scavenging activity: The rhubarb carbon dot nanozyme of the present invention can simultaneously and efficiently scavenge multiple types of free radicals such as DPPH free radicals, ABTS free radicals, hydroxyl free radicals and superoxide anion free radicals, exhibiting broad-spectrum and highly efficient antioxidant activity, and showing significant concentration dependence.

[0029] (2) Significant synergistic effect compared to rhubarb extract: Under the same concentration conditions, the free radical scavenging activity and in vitro anti-inflammatory activity of the rhubarb carbon nanoparticles of the present invention are significantly better than those of rhubarb water extract, proving that the hydrothermal carbonization process achieves nonlinear enhancement of rhubarb activity and produces unexpected technical effects.

[0030] (3) Excellent biocompatibility: Hemolysis experiments proved that the hemolysis rate of the rhubarb carbon dot nanozyme of the present invention was far below the safety standard of 5% within the experimental concentration range; CCK-8 experiments proved that it had no significant toxicity to normal cells at effective therapeutic concentrations.

[0031] (4) Significant in vitro anti-inflammatory activity: The rhubarb carbon dot nanozyme of the present invention can significantly inhibit the release of NO produced by LPS-induced RAW264.7 macrophages, exhibiting excellent anti-inflammatory activity.

[0032] (5) Significant therapeutic effect on psoriasis: In the IMQ-induced C57BL / 6 mouse psoriasis model, the rhubarb carbon dot nanoenzyme of the present invention can significantly improve the appearance of skin lesions, reduce epidermal thickness, and inhibit inflammatory cell infiltration through transdermal application. The therapeutic effect is significantly better than that of the rhubarb water extract control group.

[0033] (6) Excellent antibacterial activity: The rhubarb carbon dot nanozyme of the present invention has a significant inhibitory effect on both Escherichia coli and Staphylococcus aureus. Its minimum inhibitory concentration (MIC) and inhibition zone diameter are better than those of the same concentration of rhubarb water extract, indicating that hydrothermal carbonization can endow rhubarb with stronger antibacterial ability.

[0034] (7) Ultra-small particle size and strong transdermal ability: The average particle size of the rhubarb carbon dot nanoenzyme of the present invention is only 1 nm to 5 nm, which is much smaller than the skin pores and the gap between the stratum corneum. It can effectively penetrate the thickened stratum corneum barrier of psoriasis and improve the accumulation and bioavailability of the drug in the lesion site.

[0035] (8) The preparation process is green and simple: The present invention uses natural Chinese medicine rhubarb as carbon source and deionized water as solvent. It can be prepared by one-step hydrothermal carbonization method. There is no need to use toxic organic solvents and expensive reagents. The process is simple, green and environmentally friendly, and low in cost, making it suitable for large-scale production. Attached Figure Description

[0036] Figure 1 Transmission electron microscopy (TEM) images and particle size distribution diagrams of rhubarb carbon dot nanozymes prepared in Example 1 of this invention; wherein, A is a TEM image with a scale bar of 20 nm, B is a TEM image with a scale bar of 5 nm, and C is a particle size distribution diagram.

[0037] Figure 2 The Zeta potential diagram is shown for the rhubarb carbon dot nanozyme prepared in Example 1 of this invention.

[0038] Figure 3 The fluorescence spectrum of the rhubarb carbon dot nanozyme prepared in Example 1 of this invention.

[0039] Figure 4 The Fourier transform infrared (FTIR) spectra of rhubarb carbon dot nanozyme and rhubarb water extract prepared in Example 1 of this invention are shown.

[0040] Figure 5 The X-ray photoelectron spectroscopy (XPS) full spectrum and high-resolution spectrum of rhubarb carbon dot nanozyme and rhubarb water extract prepared in Example 1 of this invention are shown below; where A is the full spectrum, B is the DH-CDS high-resolution spectrum, and C is the DH-ET high-resolution spectrum.

[0041] Figure 6 The image shows the hemolysis results of rhubarb carbon dot nanozyme in Example 3 of this invention; where A is a picture of the hemolyzed sample and B is a bar chart of the hemolysis rate.

[0042] Figure 7 This is a comparison chart of the scavenging rates of DPPH free radicals, ABTS free radicals, hydroxyl free radicals, and superoxide anion free radicals by rhubarb carbon dot nanoenzymes and rhubarb water extract in Example 4 of the present invention; wherein, A represents DPPH free radicals, B represents ABTS free radicals, C represents hydroxyl free radicals, and D represents superoxide anion free radicals.

[0043] Figure 8 Figure 1 shows the effect of rhubarb carbon dot nanozyme on the cell viability and LPS-induced NO release of RAW 264.7 cells. Figure 2 shows the effect of rhubarb carbon dot nanozyme on the cell viability of RAW 264.7 cells in Example 5 of this invention. Figures 3 and 4 show the comparison of the inhibitory effect of rhubarb carbon dot nanozyme on LPS-induced NO release in RAW 264.7 cells before and after drug administration in Example 6 of this invention.

[0044] Figure 9 This is a comparison diagram of the antibacterial effects of rhubarb carbon dot nanoenzyme and rhubarb water extract in Example 7 of the present invention; where A represents Staphylococcus aureus and B represents Escherichia coli.

[0045] Figure 10 These are representative images showing the changes in the appearance of the skin on the backs of C57BL / 6 mice in each group in Example 8 of this invention.

[0046] Figure 11 These are HE-stained pathological sections of the back skin of mice in each group in Example 8 of this invention. Detailed Implementation

[0047] The present invention will be further described in detail below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention.

[0048] In the following examples, unless otherwise specified, all reagents and instruments used are commercially available products commonly used in the art. Statistical analysis was performed using GraphPad Prism 9 software for data analysis and graph creation, and one-way ANOVA was used for comparisons between groups. P A value <0.05 is considered statistically significant. P A difference of <0.01 is considered statistically significant. P A value <0.001 indicates that the difference is statistically significant.

[0049] Example 1 A method for preparing rhubarb carbon dot nanozymes, the specific operation steps are as follows: (1) Take rhubarb medicinal material (Beijing Tongrentang Liaoning Pharmacy Co., Ltd.), which was identified as Rheum palmatum, a plant of the Polygonaceae family. Rheum palmatum L. The dried roots and rhizomes of rhubarb were crushed and passed through a 60-mesh sieve. 1.0 g of rhubarb powder was accurately weighed and placed in a beaker.

[0050] (2) Add 30 mL of deionized water (solid-to-liquid ratio 1:30, g / mL), and sonicate for 30 min (power 300 W, frequency 40 kHz) to fully dissolve the water-soluble components in rhubarb and obtain rhubarb suspension.

[0051] (3) Transfer the above suspension to a 50 mL stainless steel hydrothermal reactor lined with polytetrafluoroethylene, seal it and place it in a forced-air drying oven, set the temperature to 190℃ and hydrothermal reaction for 10 h.

[0052] (4) After the reaction is complete, turn off the heating power and allow the reactor to cool naturally to room temperature in the drying oven. Open the reactor and filter the reaction solution using medium-speed qualitative filter paper to remove large carbonized residues and collect the filtrate.

[0053] (5) Centrifuge the filtrate at 9000 rpm for 30 min, discard the precipitate, and collect the supernatant.

[0054] (6) Transfer the supernatant to a regenerated cellulose dialysis bag with a molecular weight cutoff (MWCO) of 1000 Da, and perform dialysis purification using 2000 mL of deionized water as the dialysis medium. Replace the dialysis solution every 8 h and continue dialysis for 36 h until the liquid outside the dialysis bag is completely colorless to obtain a carbon dot solution after dialysis, ensuring that small molecule impurities and uncarbonized precursor residues are fully removed.

[0055] (7) The dialysis carbon dot solution was filtered through a 0.22 μm aqueous microporous membrane to remove bacteria, and a purified rhubarb carbon dot nanoenzyme aqueous solution was obtained, which was transparent and light brownish-yellow.

[0056] (8) Take a portion of the purified rhubarb carbon dot nanoenzyme aqueous solution and freeze-dry it to obtain rhubarb carbon dot nanoenzyme solid powder, and store it in a sealed container at -20℃ away from light for later use.

[0057] Comparative Example 1 A method for preparing rhubarb water extract, the specific operation steps are as follows: To conduct a comparative activity study, a rhubarb aqueous extract was prepared simultaneously as a control: (1) Accurately weigh 1.0 g of rhubarb powder (from the same batch used in the preparation of carbon dot nanozymes in the example), add 30 mL of deionized water, and extract by steam reflux for 10 h.

[0058] (2) Filter the extract, collect the filtrate, centrifuge at 9000 rpm for 30 min, and take the supernatant.

[0059] (3) The supernatant was filtered through a 0.22 μm microporous membrane and then freeze-dried to obtain rhubarb water extract solid powder, which was stored at -20℃ in the dark for later use.

[0060] Note: The only difference between rhubarb water extract and rhubarb carbon dot nanoenzyme is whether or not it has undergone a hydrothermal carbonization step at 190℃ for 10 hours. All other processing procedures are completely identical to ensure the scientific validity and comparability of the comparative experiment.

[0061] Example 2 Characterization of a rhubarb carbon dot nanozyme: 2.1 Transmission electron microscopy (TEM) and particle size characterization An appropriate amount of rhubarb carbon dot nanozyme aqueous solution was diluted to a suitable concentration and then dropped onto a carbon film copper grid. After natural drying, the morphology was observed under a transmission electron microscope. ImageJ software was used to perform particle size statistical analysis on the carbon dot particles in the TEM images.

[0062] Results: TEM images show (e.g.) Figure 1 A, Figure 1 As shown in Figure B), the rhubarb carbon dot nanoenzymes are uniformly dispersed near-spherical nanoparticles with no obvious agglomeration and clearly visible lattice fringes. Statistical results of particle size distribution (as shown in Figure B) indicate... Figure 1 As shown in C), the average particle size of rhubarb carbon dot nanozyme is 2.73 ± 0.55 nm, with a narrow and uniform particle size distribution.

[0063] 2.2 Zeta potential characterization An aqueous solution of rhubarb carbon dot nanozyme (concentration approximately 0.1 mg / mL) was taken, and its zeta potential was measured at 25°C using a dynamic light scattering instrument.

[0064] The results are as follows Figure 2 As shown, the Zeta potential of rhubarb carbon dot nanozyme (DH-CDS) is -12.9±0.3 mV, and the surface carries a negative charge, indicating that the carbon dot surface contains abundant oxygen-containing functional groups and has good colloidal stability and dispersibility in aqueous solution.

[0065] 2.3 Fluorescence Spectroscopy Characterization An aqueous solution of rhubarb carbon dot nanozyme was taken, and its fluorescence emission spectrum was measured at different excitation wavelengths using an enzyme-linked immunosorbent assay (ELISA) reader.

[0066] The results are as follows Figure 3 As shown, rhubarb carbon dot nanozymes exhibit wavelength-dependent fluorescence emission characteristics. At the optimal excitation wavelength of 370 nm, the maximum emission peak is located at 450 nm, exhibiting blue fluorescence.

[0067] 2.4 Fourier Transform Infrared Spectroscopy (FTIR) Characterization Take an appropriate amount of rhubarb carbon dot nanozyme (DH-CDS) and rhubarb water extract (DH-EX) and mix with KBr to compress into tablets. Use an FTIR spectrometer to analyze the mixture at 4000 cm⁻¹. -1 ~500 cm -1 Scan within the range.

[0068] The results are as follows Figure 4 As shown, FTIR spectroscopy reveals that the surface of rhubarb carbon dot nanozymes contains abundant functional groups: approximately 3400 cm⁻¹ -1 The broad absorption peak at this point is attributed to the stretching vibrations of OH and NH, indicating that the carbon dot surface contains a large number of hydroxyl and amino groups, originating from the phenolic hydroxyl groups of anthraquinones (emodin, rhein) and polysaccharide components; the peak is broad and strong. However, compared with the peak of the rhubarb water extract, the peak shape becomes broader and shallower, indicating that some hydroxyl groups are converted during carbonization, but hydrophilic functional groups are still retained on the surface; approximately 2900 cm⁻¹ -1The absorption peak at approximately 1650 cm⁻¹ is attributed to the CH stretching vibration, originating from the CH groups on the fatty and aromatic rings of rhubarb. Compared to the water extract of rhubarb, the stretching peak is significantly deeper, indicating an increase in CH bonds on the carbon dot surface and the formation of more hydrocarbon structures through carbonization. -1 The absorption peak at approximately 1050 cm⁻¹ is attributed to the C=O stretching vibration and the C=C aromatic ring skeletal vibration, representing the carbonyl group (C=O) of the anthraquinone components in rhubarb. This is the most representative characteristic peak of rhubarb. Compared to the peak of the water extract of rhubarb, the carbon point peak of rhubarb is shallower, indicating that the carbonyl portion of the anthraquinone was destroyed / transformed during carbonization. -1 The absorption peak at the [specific location] is attributed to the CO stretching vibration, originating from polysaccharides and glycosides. The peaks at the rhubarb carbon dots remain, indicating that oxygen-containing functional groups are retained on the carbon dot surface, contributing to water solubility. These results demonstrate that the surface of rhubarb carbon dot nanozymes is rich in hydrophilic and active functional groups such as -OH, -COOH, -NH2, and C=O. These functional groups are the structural basis for its highly efficient free radical scavenging activity and good water solubility.

[0069] 2.5 X-ray photoelectron spectroscopy (XPS) characterization The elemental composition and chemical bond state of rhubarb carbon dot nanozyme solid powder were determined using X-ray photoelectron spectroscopy.

[0070] The results are as follows Figure 5 As shown, XPS full-spectrum analysis revealed that rhubarb carbon dot nanozymes and rhubarb water extracts are mainly composed of C, N, and O elements. The atomic percentages of rhubarb carbon dots are approximately 67.79%, 4.47%, and 27.74%, respectively, while those of rhubarb water extracts are approximately 69.79%, 1.58%, and 28.63%, respectively. The C 1s high-resolution spectrum, after peak fitting, can be resolved to three chemical environments: CC / C=C, CO, and C=O. The O 1s high-resolution spectrum can be resolved to C=O and CO / COC. The N 1s high-resolution spectrum can be resolved to pyridine N, pyrrole N, and graphitic N. These results further confirm the abundant heteroatom doping and the presence of oxygen- and nitrogen-containing functional groups on the surface of rhubarb carbon dot nanozymes. Of particular note is that, compared to the rhubarb water extract (N content 1.58%), the nitrogen atom self-doping ratio of the rhubarb carbon dot nanozyme is significantly increased to 4.47%, and pyridine-N at a binding energy of 398.5 eV is the dominant form of nitrogen. Literature studies have shown that pyridine-N has a strong electron-donating ability and is an excellent catalytic center for mimicking superoxide dismutase (SOD) activity. Therefore, the in-situ pyridine-N doping induced by hydrothermal carbonization is the structural core of the rhubarb carbon dot nanozyme, enabling it to possess a highly efficient SOD-like catalytic mechanism. These results further confirm the abundant heteroatom doping and oxygen / nitrogen-containing functional groups on the surface of the rhubarb carbon dot nanozyme, and these doping sites and functional groups provide active centers for its nanozyme catalytic activity.

[0071] Example 3 Hemolysis experiment of rhubarb carbon dot nanozyme: To evaluate the blood compatibility and biosafety of rhubarb carbon dot nanozymes, an in vitro hemolysis experiment was conducted.

[0072] 3.1 Experimental Methods Phosphate-buffered saline (PBS) was used as a negative control and deionized water as a positive control. Equal volumes of sheep blood were added to rhubarb carbon dot nanozyme solutions at concentrations of 12.5 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, and 400 μg / mL, respectively. The solutions were then incubated at 37°C for 2 h. After incubation, the solutions were centrifuged at 3500 rpm for 10 min. The supernatant was collected and the absorbance at 540 nm was recorded using a microplate reader to determine the hemolysis rate. The calculation formula was: (Experimental group absorbance - Negative control group absorbance) / (Positive control group absorbance - Negative control group absorbance) × 100%.

[0073] 3.2 Experimental Results To investigate whether rhubarb carbon dot nanozymes exhibit hemolysis, PBS was used as a negative control and deionized water as a positive control. The results are as follows: Figure 6 As shown, in rhubarb carbon dot nanoenzyme solutions at concentrations of 12.5 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, and 400 μg / mL, all red blood cells precipitated, and the supernatant was colorless and clear, with no hemolysis observed. The degree of hemolysis at different concentrations of rhubarb carbon dot nanoenzyme was quantified. These results indicate that rhubarb carbon dot nanoenzyme possesses excellent blood compatibility and biosafety, making it suitable for the development of topical skin formulations.

[0074] Example 4 The scavenging effect of rhubarb carbon dot nanozymes on free radicals and a comparison of their activity with that of rhubarb water extract: To systematically evaluate the antioxidant activity of rhubarb carbon dot nanozymes and to directly compare them with rhubarb water extract to demonstrate the synergistic effect of carbonization, the effects of both on DPPH radicals, ABTS radicals, hydroxyl radicals (·OH), and superoxide anion radicals (O2) were measured. - ·) Clearance ability.

[0075] 4.1 DPPH free radical scavenging experiment Experimental methods: Take 200 μL of rhubarb carbon dot nanozyme and rhubarb water extract solutions with mass concentrations of 6.125 μg / mL, 12.5 μg / mL, 25 μg / mL, and 50 μg / mL, respectively, add 200 μL of DPPH anhydrous ethanol solution (0.2 mmol / L), mix well, and react at room temperature in the dark for 30 min. Then, measure the absorbance of the sample at 517 nm using a microplate reader. The absorbance is recorded as A. y The sample was replaced with distilled water to form a blank group, denoted as A. x The absorbance measured using anhydrous ethanol instead of DPPH solution was used as a control group and denoted as A. z According to the formula DPPH clearance rate S=[A x -(A y -A z )] / A x ×100%.

[0076] Experimental results: Both rhubarb carbon dot nanozyme and rhubarb water extract showed concentration-dependent DPPH free radical scavenging activity, but at the same concentration, the DPPH scavenging rate of rhubarb carbon dot nanozyme was higher than that of rhubarb water extract.

[0077] Table 1. Scavenging rates of DPPH free radicals by rhubarb carbon dot nanozymes and rhubarb water extract

[0078] like Figure 7 As shown in Figure A, the DPPH scavenging rate IC of rhubarb carbon dot nanoenzyme is... 50 The IC50 of rhubarb water extract is approximately 12.5 μg / mL. 50 At approximately 50 μg / mL, the antioxidant activity of carbon dot nanozymes is about 4 times that of the extract.

[0079] 4.2 ABTS free radical scavenging experiment Experimental methods: A 7.4 mmol / L ABTS solution and a 2.6 mmol / L potassium persulfate solution were mixed at a volume ratio of 1:1 and incubated in the dark for 12 h. The mixture was then diluted with distilled water, resulting in an absorbance of 0.70 ± 0.02 at 734 nm. 200 μL of rhubarb carbon dot nanozyme and rhubarb water extract solutions with concentrations of 6.125 μg / mL, 12.5 μg / mL, and 25 μg / mL, respectively, were mixed with 200 μL of the diluted ABTS solution and reacted at room temperature in the dark for 6 min. The absorbance of the sample at 734 nm was measured using a microplate reader and recorded as A. y The sample was replaced with distilled water to form a blank group, denoted as A. xThe absorbance measured using potassium persulfate solution instead of ABTS solution was used as a control group and denoted as A. z According to the formula ABTS clearance rate S=[A x -(A y -A z )] / A x ×100%.

[0080] Experimental results: Table 2. Scavenging rates of ABTS free radicals by rhubarb carbon dot nanozyme and rhubarb water extract

[0081] like Figure 7 As shown in B, the ABTS scavenging rate IC of rhubarb carbon dot nanozyme is [missing value]. 50 The IC50 of rhubarb water extract is approximately 6.125 μg / mL. 50 At a concentration of approximately 12.5 μg / mL, the antioxidant activity of the carbon dot nanozyme was about twice that of the extract. The results indicate that the scavenging activity of rhubarb carbon dot nanozyme against ABTS free radicals increased in a concentration-dependent manner, and was significantly superior to that of the rhubarb aqueous extract at the same concentration.

[0082] 4.3 Hydroxyl radical scavenging experiment Experimental methods: The experiment was conducted according to the instructions for the hydroxyl radical (·OH) assay kit.

[0083] Experimental results: Table 3. Scavenging rates of hydroxyl radicals by rhubarb carbon dot nanozymes and rhubarb aqueous extract.

[0084] like Figure 7 As shown in C, the hydroxyl radical scavenging rate IC of rhubarb carbon dot nanozyme 50 The IC50 of rhubarb water extract is approximately 150 μg / mL. 50 At approximately 350 μg / mL, the antioxidant activity of the carbon dot nanozyme was about 2.3 times that of the extract. The scavenging rate of rhubarb carbon dot nanozyme against hydroxyl radicals was significantly higher than that of the rhubarb water extract at all concentrations.

[0085] 4.4 Superoxide anion radical scavenging experiment Experimental methods: Based on the inhibition and generation of superoxide anion free radicals (O2) - • Perform the experiment according to the instructions of the assay kit.

[0086] Experimental results: Table 4. Scavenging rates of superoxide anion free radicals by rhubarb carbon dot nanozyme and rhubarb water extract

[0087] like Figure 7 As shown in D, the superoxide anion radical scavenging rate (IC) of rhubarb carbon dot nanoenzymes 50 The IC50 of rhubarb water extract is approximately 300 μg / mL. 50 The antioxidant activity of the carbon dot nanozyme was approximately 1200 μg / mL, which was about 4 times that of the extract. The scavenging rate of superoxide anions by the rhubarb carbon dot nanozyme was significantly higher than that of the rhubarb water extract at all concentrations.

[0088] 4.5 Summary of Antioxidant Activity Comparison Based on the results of four free radical scavenging experiments: Table 5. Comparison of scavenging rates of rhubarb carbon dot nanozyme and rhubarb water extract against DPPH radicals, ABTS radicals, hydroxyl radicals, and superoxide anion radicals.

[0089] The above comparative experimental results strongly demonstrate that the carbon nanoparticles formed after rhubarb is hydrothermally carbonized at 190℃ exhibit significantly enhanced free radical scavenging activity compared to the uncarbonized rhubarb water extract. This enhanced effect is attributed to the dehydration, condensation, carbonization, and surface functional group recombination of the precursor molecules during the hydrothermal carbonization process.

[0090] Example 5 The effect of rhubarb carbon dot nanozymes on cell viability: 5.1 Cell Culture RAW 264.7 mouse mononuclear macrophage leukemia cells adhered to the culture medium and were cultured in complete medium (DMEM medium containing 10% FBS) in an incubator at 37°C and 5% CO2 saturated humidity. When the cell confluence reached approximately 70%–80%, the cells were passaged, gently pipetted to suspend them, and then centrifuged at 800×g for 5 min. The supernatant was discarded, and the cells were gently mixed with complete medium and then placed in a cell culture incubator.

[0091] 5.2 CCK-8 assay for cell viability Cell counting was performed after cell culture, with 1×10⁶ cells per well. 4 Cells were cultured in 96-well plates. When the cells reached a density of 60%–80%, rhubarb carbon dot nanozyme solutions of different concentrations were added to the 96-well plates. After 24 hours, CCK-8 reagent was added and the OD value of each well was measured at 450 nm using an ELISA reader.

[0092] Calculate cell viability based on OD value: Cell vitality =

[0093] In the formula: A 0 represents the mean of the blank group; In the formula: A 1. Absorbance of rhubarb carbon dot nanoenzyme solution in the drug administration group.

[0094] 5.3 Experimental Results The results are as follows Figure 8 As shown in Figure A, within the concentration range of 6.125 μg / mL to 100 μg / mL, the survival rate of RAW 264.7 cells treated with rhubarb carbon dot nanozyme was above 90%. These results indicate that rhubarb carbon dot nanozyme has no significant cytotoxicity to RAW 264.7 cells at concentrations of 100 μg / mL and below, demonstrating good safety. Subsequent anti-inflammatory experiments will be conducted at drug concentrations within the safe range.

[0095] Example 6 Inhibitory effect of rhubarb carbon dot nanozyme on inflammation of RAW 264.7 macrophages in vitro: 6.1 Establishment of an LPS (lipopolysaccharide)-induced RAW 264.7 macrophage inflammation model Once RAW 264.7 cells reached the logarithmic growth phase, they were used for experiments. After cell culture, cell counting was performed, with 1 × 10⁶ cells per well. 4 Cells were cultured in 96-well plates. When the cells reached a density of 60%–80%, different concentrations of rhubarb carbon dot nanozyme solution were added to the 96-well plates. After pre-protection for 4 h, LPS solution was added to make the drug concentration 1 ng / mL. After culturing for 24 h, the NO content in the cells was detected using a NO kit.

[0096] 6.2 Experimental Results 6.2.1 Results of NO release inhibition NO levels are closely related to changes in inflammation levels. The bioactivity of RAW cells after administration of rhubarb carbon dot nanozymes was first examined. Figure 8 As shown in Figure A, after CCK-8 assay, rhubarb carbon dot nanozymes at concentrations ranging from 0 μg / mL to 100 μg / mL showed no cytotoxicity to RAW microglia. Figure 8 As shown in B, NO levels were significantly increased in the LPS-induced inflammation model; Figure 8 C indicates that after administration, the content of NO secreted in the supernatant of RAW 264.7 macrophages gradually decreased, indicating good anti-inflammatory activity.

[0097] Example 7 Comparison of in vitro antibacterial activities between rhubarb carbon dot nanozymes and rhubarb aqueous extract: To further evaluate the antibacterial properties of rhubarb carbon dot nanozymes and directly compare them with rhubarb water extract, this example selected Gram-negative bacteria Escherichia coli (E. coli) Escherichia coli , E. coli ) and Gram-positive bacteria Staphylococcus aureus ( Staphylococcus aureus , S. aureus Using a representative strain, the minimum inhibitory concentration (MIC) and inhibition zone diameter of rhubarb carbon dot nanozyme and rhubarb water extract were determined.

[0098] 7.1 Strain Culture Escherichia coli and Staphylococcus aureus were inoculated separately into LB liquid medium and incubated overnight at 37°C with shaking at 180 rpm. The next day, the bacterial suspension was diluted to approximately 1 × 10⁻⁶. 6 CFU / mL available for use.

[0099] 7.2 Determination of Minimum Inhibitory Concentration (MIC) The MICs of rhubarb carbon dot nanozyme and rhubarb water extract against two bacteria were determined using the micro-broth dilution method.

[0100] Specific methods: (1) Prepare solutions of different concentration gradients for the samples, such as: 93.75 μg / mL, 187.5 μg / mL, 375 μg / mL, 750 μg / mL, 1500 μg / mL, 3000 μg / mL, 6000 μg / mL, and 12000 μg / mL.

[0101] (2) The lowest drug concentration at which no visible growth is observed and OD600 does not increase significantly is taken as the MIC value.

[0102] result: Rhubarb carbon dot nanozyme showed significant antibacterial activity against both Escherichia coli and Staphylococcus aureus, with a significantly lower MIC than that of rhubarb water extract.

[0103] Table 6. MIC values ​​of rhubarb carbon dot nanozyme and rhubarb water extract against Escherichia coli and Staphylococcus aureus.

[0104] This indicates that rhubarb carbon dot nanozymes have broad-spectrum antibacterial activity against both Gram-negative and Gram-positive bacteria, and are superior to rhubarb water extract.

[0105] 7.3 Antibacterial zone experiment Specific methods: (1) Spread the Escherichia coli and Staphylococcus aureus suspensions evenly on the surface of LB agar plates.

[0106] (2) Use a sterile punch to punch holes in an agar plate and add rhubarb carbon nanoparticles and rhubarb water extract solutions of the same concentration (1.5 mg / mL) to the holes respectively.

[0107] (3) After incubation at 37℃ for 18 h to 24 h, observe the inhibition zone.

[0108] result: like Figure 9 As shown, rhubarb carbon dot nanozymes formed distinct inhibition zones on both strains of bacteria on plates, and the diameter of these inhibition zones was significantly larger than that of the rhubarb water extract group. Rhubarb carbon dot nanozymes exhibited significant antibacterial effects against both *Escherichia coli* and *Staphylococcus aureus*, and were superior to those of the rhubarb water extract.

[0109] Example 8 The therapeutic effect of rhubarb carbon dot nanozyme on psoriasis in C57BL / 6 mice: 8.1 Laboratory Animals C57BL / 6 mice weighing approximately 23±2 g were selected. Each mouse was treated with imiquimod 62.5 mg / d to establish a model. Two days before modeling, hair was removed from a 3 cm × 3 cm area on the back. The treatment was applied for two consecutive days. Mice were randomly divided into five groups (n=6 per group): a control group (CON), a model group (IMQ), a positive control group (SAC), a rhubarb water extract group (DH-ET, 1.0 mg / kg), and a rhubarb carbon nanoparticle enzyme group (DH-CDS, 1.0 mg / kg). On the third day, the treatment solution was administered four hours after the imiquimod administration. On the seventh day, tissue anatomical dissection was performed, and the treated area on the back was fixed for H&E staining.

[0110] 8.2 H&E staining After the skin tissue fixed with tissue fixative was dehydrated, it was embedded in paraffin and cut into 5 μm sections. The sections were then stained with hematoxylin and eosin in sequence. After staining, the sections were washed with water, dehydrated with ethanol, soaked in xylene, and mounted with resin of appropriate concentration. The inflammatory cell infiltration, epidermal proliferation, and stratum corneum differentiation were observed under an optical microscope.

[0111] 8.3 Experimental Results 8.3.1 Changes in the appearance of skin lesions like Figure 10 As shown, the skin on the backs of mice in the blank control group was normal, without erythema, scaling, or thickening. Mice in the model group began to develop erythema on days 2-3, which gradually worsened until days 5-7 when obvious erythema, a large amount of silvery-white scales, and significant skin thickening appeared, exhibiting typical psoriasis-like skin lesions.

[0112] The skin lesions in the positive control group mice were significantly reduced compared to the model group, with significant improvements in erythema, scaling, and thickening. The skin lesions in the rhubarb carbon dot nanozyme group showed improvement, with lighter erythema, reduced scaling, and lessened skin thickening, resulting in significant improvement in appearance. At the same concentration, the degree of skin lesion improvement in the rhubarb carbon dot nanozyme group was significantly better than that in the rhubarb water extract group, although the latter showed some improvement, erythema and scaling remained relatively pronounced.

[0113] 8.3.2 H&E staining histopathological analysis like Figure 11 As shown, the skin structure of mice in the blank control group was normal, with a thin and regularly arranged epidermis and a clear dermal structure, without inflammatory cell infiltration. The skin of mice in the model group exhibited typical psoriatic-like pathological changes: significantly thickened epidermis, parakeratosis of the stratum corneum, acanthosis, elongated epidermal ridges resembling finger-like projections, and Munro microabscesses in some areas. The average epidermal thickness in the model group was 145.8 μm.

[0114] In the positive control group, the degree of epidermal thickening was significantly reduced, inflammatory cell infiltration was decreased, and the average epidermal thickness was 57.6 μm. In the rhubarb carbon nanoparticle group, the epidermal thickness was significantly reduced to 53.8 μm, the degree of acanthosis was reduced, parakeratosis was reduced, inflammatory cell infiltration in the dermis was significantly reduced, and the skin structure tended to be normal.

[0115] The average epidermal thickness of the rhubarb water extract group was 77.9 μm, which was an improvement over the model group, but the degree of improvement was significantly weaker than that of the high-dose rhubarb carbon dot nanoenzyme group at the same concentration.

[0116] Example 9 Verification of SOD activity of rhubarb carbon dot nanoenzymes: To further confirm the superoxide dismutase (SOD)-like activity of rhubarb carbon dot nanozymes, this embodiment systematically validated it.

[0117] Experimental methods: The WST-1 method (SOD activity assay kit from Nanjing Jiancheng Biological Research Institute) was used for determination. Xanthine or xanthine oxidase systems generate superoxide anion free radicals, and WST-1 can be reduced by superoxide anions to colored formazan products (absorbed at 450 nm). If the sample has SOD-like activity, it can catalyze the disproportionation of superoxide anions to H₂O₂ and O₂, thereby inhibiting formazan formation and resulting in a decrease in absorbance at 450 nm.

[0118] (1) Prepare a rhubarb carbon dot nanoenzyme solution with a mass concentration of 200 μg / mL.

[0119] (2) Follow the instructions in the kit, add the sample solution to the reaction system and incubate at 37°C for 20 min.

[0120] (3) Measure the absorbance at 450 nm and calculate the SOD-like enzyme activity.

[0121] Results: Rhubarb carbon dot nanozymes exhibited SOD-like activity. At a concentration of 200 μg / mL, its SOD-like activity value was 76822.42991 U / g. The activity retention rate after two cycles of use was high, rather than simply consuming free radicals through stoichiometric reactions.

[0122] The above verification results of SOD activity fully demonstrate that the rhubarb carbon dots of the present invention have true nanozyme catalytic characteristics and can continuously remove ROS through a catalytic cycle mechanism, rather than the one-time stoichiometric consumption of traditional antioxidants. This is one of the core mechanisms that significantly enhances its activity compared to rhubarb water extract.

[0123] Through systematic and in-depth research, this invention has discovered that the carbon dot nanozymes formed by hydrothermal carbonization of rhubarb not only possess ultra-small particle size (1 nm–5 nm) and excellent water solubility, but also exhibit free radical scavenging activity and in vitro anti-inflammatory and antibacterial activity far superior to that of rhubarb water extracts of the same concentration. More importantly, these rhubarb carbon dot nanozymes demonstrated significant therapeutic effects in an imiquimod (IMQ)-induced C57BL / 6 mouse psoriasis model, effectively reducing epidermal thickness and inhibiting inflammatory cell infiltration; their therapeutic effect was significantly better than that of the rhubarb water extract control group. This discovery proves that the hydrothermal carbonization process endows the rhubarb carbon source material with unexpected enhanced activity, providing a new candidate nanomedicine for the treatment of psoriasis and other inflammatory skin diseases.

Claims

1. A method for preparing rhubarb carbon dot nanozymes, characterized in that, Includes the following steps: (a) Take rhubarb medicinal material, crush and sieve it, add deionized water, and treat it with ultrasound to obtain rhubarb suspension; (b) The rhubarb suspension obtained in step (a) is transferred to a reaction vessel for hydrothermal reaction; (c) After the reaction is complete, allow the mixture to cool naturally to room temperature, then filter and centrifuge to collect the supernatant. (d) Dialyze the supernatant obtained in step (c); (e) After dialysis, the solution was filtered and freeze-dried to obtain rhubarb carbon dot nanozyme solid powder, which was stored in the dark for later use.

2. The method for preparing rhubarb carbon dot nanozyme according to claim 1, characterized in that, In step (a), the ratio of rhubarb medicinal material to deionized water is 1 g:30 mL, and the sonication time is 20 min to 40 min; in step (b), the hydrothermal reaction temperature is 180℃ to 200℃, and the time is 8 h to 12 h; in step (c), the centrifugation speed is 8000 rpm to 10000 rpm, and the time is 20 min to 30 min; in step (d), the dialysis treatment time is 24 h to 48 h; in step (e), the temperature for light-protected storage is -20℃; in step (e), the average particle size of the obtained rhubarb carbon dot nanozyme is 1 nm to 5 nm.

3. The application of rhubarb carbon dot nanozyme obtained by any one of claims 1 to 2 in scavenging free radicals.

4. The application of the rhubarb carbon dot nanozyme according to claim 3 in scavenging free radicals, characterized in that, The free radicals include one or more of DPPH free radicals, ABTS free radicals, hydroxyl free radicals, and superoxide anion free radicals.

5. The application of rhubarb carbon dot nanozyme obtained by any one of claims 1 to 2 in the preparation of antioxidant and anti-inflammatory products.

6. The application of the rhubarb carbon dot nanozyme according to claim 5 in the preparation of antioxidant and anti-inflammatory products, characterized in that, The antioxidant and anti-inflammatory product is a topical skin preparation.

7. The application of the rhubarb carbon dot nanozyme according to claim 6 in the preparation of antioxidant and anti-inflammatory products, characterized in that, The dosage form of the topical skin preparation is selected from one of the following: gel film, coating film, hydrogel, cream or patch.

8. The use of rhubarb carbon dot nanozyme obtained by the preparation method according to any one of claims 1 to 2 in the preparation of drugs for treating inflammatory skin diseases.

9. The application of the rhubarb carbon dot nanozyme according to claim 8 in the preparation of drugs for treating inflammatory skin diseases, characterized in that, The inflammatory skin disease mentioned is psoriasis.

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