Salvia miltiorrhiza and panax notoginseng residue carbon dots with broad-spectrum free radical scavenging ability, and preparation method and application thereof

Abstract

The application provides a salvia miltiorrhiza and panax notoginseng residue carbon dot with broad-spectrum free radical scavenging ability and a preparation method and application thereof, the carbon dot takes the salvia miltiorrhiza and panax notoginseng residue with a mass ratio of 1:1 as raw materials, adds a natural low eutectic solvent, is then transferred to a reaction kettle, and after reaction, is filtered, dialyzed and freeze-dried to obtain the salvia miltiorrhiza and panax notoginseng residue carbon dot; wherein the volume ratio of the added natural low eutectic solvent to the sum of the mass of the panax notoginseng residue and the salvia miltiorrhiza residue is 1:10 muL / mg, and the natural low eutectic solvent is a system formed by betaine and lactic acid at a molar ratio of 1:1. The carbon dot has good structural uniformity and surface functionalization characteristics, the particle size is in the nanometer range, and the surface is rich in oxygen-containing and nitrogen-containing functional groups, and these structural characteristics are beneficial to the scavenging of free radicals.

Description

Technical Field

[0001] This invention belongs to the field of bio-nanomaterials, and in particular relates to a carbon dot of Danshen-Sanqi medicinal residue with broad-spectrum free radical scavenging ability, its preparation method and application. Background Technology

[0002] Currently, free radical scavengers mostly utilize small-molecule antioxidants such as vitamin C, polyphenols, and plant extracts. However, these substances typically suffer from poor stability, limited scope of action, and easy degradation, making it difficult to maintain sustained antioxidant activity in complex environments. Meanwhile, carbon dots, due to their good water dispersibility, surface modifiability, and good biocompatibility, are gradually becoming an important research direction for novel antioxidant nanomaterials. Existing studies have shown that biomass such as fruit peels, straw, and algae can be used as carbon sources to prepare carbon dots with certain free radical scavenging capabilities.

[0003] However, existing technologies still have the following shortcomings: First, the residue after extraction of traditional Chinese medicine is usually treated as waste, and its residual active ingredients are not effectively utilized. Second, existing methods for preparing biomass carbon dots do not adequately utilize this high-value biomass resource of traditional Chinese medicine residue, especially lacking a technical solution that combines green solvent-assisted extraction with in-situ carbon dot preparation. Third, existing research on natural eutectic solvents mainly focuses on the extraction of plant active ingredients, with limited research on their ability to simultaneously promote the enrichment of active precursors in the residue and further promote the formation of antioxidant carbon dots. Therefore, developing a new method for preparing carbon dots with broad-spectrum free radical scavenging capabilities using medicinal residue as raw material and natural eutectic solvents as an auxiliary system has significant research and application value. Summary of the Invention

[0004] In view of this, the present invention aims to propose a carbon dot of Danshen-Sanqi medicinal residue with broad-spectrum free radical scavenging ability, its preparation method and application, so as to solve the problems of low utilization rate of medicinal residue resources, insufficient preparation methods of green antioxidant nanomaterials and limited free radical scavenging range of existing antioxidants in the prior art.

[0005] To achieve the above objectives, the technical solution of the present invention is implemented as follows: A method for preparing carbon dots from *Salvia miltiorrhiza* and *Panax notoginseng* residue with broad-spectrum free radical scavenging ability involves dispersing *Panax notoginseng* residue and *Salvia miltiorrhiza* residue in deionized water at a mass ratio of 1:1, then adding a natural eutectic solvent, transferring the mixture to a reaction vessel, reacting at 180℃ for 12 h, and finally filtering, dialyzing, and freeze-drying to obtain carbon dots from *Salvia miltiorrhiza* and *Panax notoginseng* residue. The volume ratio of the added natural eutectic solvent to the sum of the masses of *Panax notoginseng* and *Salvia miltiorrhiza* residue is 1:10 μL / mg, and the natural eutectic solvent is a system formed by betaine and lactic acid in a molar ratio of 1:1.

[0006] Furthermore, the filtration method is as follows: centrifuge at 6000-8000 rpm for 10-20 min, and then filter the supernatant through a 0.22 μm filter membrane.

[0007] Furthermore, the dialysis method is as follows: use a dialysis bag with a molecular weight cutoff of 1000-1200 Da to dialyze in deionized water for 20-24 hours, and change the external solution every 6-8 hours during this period.

[0008] Furthermore, the freeze-drying conditions are: -50℃ to -45℃, for a time of 28-30 hours.

[0009] Furthermore, the carbon dots of the prepared Danshen-Sanqi residue are spherical or nearly spherical particles with a particle size of 2.32-3.96 nm, and the surface contains hydroxyl, carboxyl and nitrogen-containing functional groups.

[0010] The present invention also provides carbon dots of Danshen-Sanqi residue prepared by the preparation method described above.

[0011] The present invention also provides an application of carbon dots from tanshinone-Panax notoginseng residue as described above, wherein the carbon dots from tanshinone-Panax notoginseng residue are used for the scavenging of free radicals, including the scavenging of DPPH free radicals, superoxide anions, NO free radicals and hydroxyl free radicals.

[0012] Compared with existing technologies, the carbon dots of Danshen-Sanqi residue with broad-spectrum free radical scavenging ability, their preparation method, and applications described in this invention have the following advantages: (1) This invention uses Panax notoginseng and Salvia miltiorrhiza residue as carbon sources to realize the high-value utilization of waste Chinese medicine residue and improve the resource utilization level of residue.

[0013] (2) This invention introduces a natural eutectic solvent-assisted system, which not only promotes the enrichment of antioxidant precursors in the residue, but also promotes their conversion into functional carbon dots during the hydrothermal process. Screening results show that the carbon dots obtained from the system with a 1:1 molar ratio of betaine to lactic acid have the best DPPH radical scavenging effect, while the natural eutectic solvent itself shows almost no DPPH radical scavenging ability, indicating that its main role is to promote extraction and conversion.

[0014] (3) The carbon dots obtained by the present invention have good structural uniformity and surface functionalization characteristics. The particle size is in the nanoscale range and the surface is rich in oxygen-containing and nitrogen-containing functional groups. These structural characteristics are beneficial to their scavenging of free radicals.

[0015] (4) The carbon dots obtained in this invention have a broad-spectrum free radical scavenging ability, and can simultaneously scavenge DPPH free radicals, superoxide anions, NO free radicals and hydroxyl free radicals. Among them, the DPPH free radical scavenging experiment showed that its scavenging ability was better than that of ascorbic acid and traditional decoction control; the results of the other three free radical experiments also showed that the carbon dots had significant scavenging effects on different reactive oxygen species and reactive nitrogen species, indicating that the material has a wide free radical response range and good antioxidant application potential.

[0016] (5) The process of this invention uses water as the main reaction medium and the amount of natural eutectic solvent added is small. The preparation process is green and simple, and it is suitable for further expansion into a general preparation strategy for functional nanomaterials derived from drug residues. Attached Figure Description

[0017] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings: Figure 1 The following are state diagrams of 30 groups of natural eutectic solvents prepared in Example 2 at room temperature; Figure 2 The image shows the DPPH radical scavenging results of the carbon dots prepared in Example 3; Figure 3 The image shows the DPPH radical scavenging results of the carbon dots prepared in Example 4; Figure 4 The image shows the DPPH radical scavenging results of the carbon dots prepared in Example 5; Figure 5 The image shows the superoxide anion scavenging results of the carbon dots prepared in Example 5; Figure 6 The image shows the NO radical scavenging results of the carbon dots prepared in Example 5; Figure 7 This is a graph showing the hydroxyl radical scavenging results of the carbon dots prepared in Example 5; Figure 8 A comparison chart of the DPPH radical scavenging capabilities of the carbon dots prepared in Example 6; Figure 9 Fourier transform infrared spectrum of the betaine-lactic acid natural eutectic solvent prepared in Example 7; Figure 10 The hydrogen nuclear magnetic resonance image of the betaine-lactic acid natural eutectic solvent prepared in Example 7; Figure 11 Characterization of the carbon dots prepared in Example 7; a) Scanning electron microscope image of the carbon dots; b) Magnified image of the carbon dots; c) Particle size distribution map of the carbon dots; d) Ultraviolet-visible spectrum scan. Figure 12Fluorescence emission characteristics of carbon dots prepared in Example 7; a) Increased carbon dot concentration under sunlight, b) Increased carbon dot concentration under ultraviolet light; Figure 13 The fluorescence properties of the carbon dots prepared in Example 7 are shown below: a) Two-dimensional fluorescence spectrum; b) Fluorescence excitation and emission spectrum; c) Fluorescence lifetime curve; d) Fourier transform-infrared spectrum. Figure 14 The structural characteristics of the carbon dots prepared in Example 7 are shown in Figure a; X-ray photoelectron spectroscopy (XPS) spectrum is shown in Figure b; C1s spectrum is shown in Figure c; and O1s spectrum is shown in Figure d. Figure 15 The graph shows the scavenging results of the carbon dots prepared in Example 7 on DPPH radicals, superoxide anions, NO radicals, and hydroxyl radicals; a represents DPPH radicals; b represents superoxide anions; c represents NO radicals; and d represents hydroxyl radicals. Figure 16 Results of DPPH radical scavenging experiments on different materials; Figure 17 Curves showing the change in DPPH free radical scavenging over time for different groups; Figure 18 Comparison of DPPH free radical scavenging results among different groups; Figure 19 Stability evaluation of the carbon dots prepared in Example 7; a) Temperature conditions; b) pH conditions; c) Storage conditions. Detailed Implementation

[0018] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0019] In this document, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0020] In this document, when values ​​are described as ranges, it should be understood that such disclosure includes disclosure of all possible subranges within that range, as well as the specific numerical values ​​falling within that range, regardless of whether the specific numerical value or specific subrange is explicitly specified.

[0021] In this article, the terms "multiple" or "more than" are used unless otherwise specified, referring to a quantity greater than or equal to 2. For example, "one or more" means one or more types.

[0022] In this document, the terms "preferred" and "more preferred" are used only to describe implementation methods or embodiments with better effects, and should be understood as not constituting a limitation on the scope of protection of this invention.

[0023] In this document, terms such as "further" are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of this invention.

[0024] In this article, the term "and / or" describes an association between objects, indicating that three relationships can exist. For example, A and / or B means: A or B, or A and B.

[0025] In this document, the term "about" means + / - 10% of a specified value, preferably + / - 5%, and more preferably + / - 1%.

[0026] In this article, the terms “include,” “including,” “have,” “contain,” etc., are all open-ended terms, meaning that they include but are not limited to.

[0027] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar to or equivalent to those described herein may be used in the implementation or testing of this invention.

[0028] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0029] Example 1: Preparation of Panax notoginseng and Salvia miltiorrhiza residue Weigh 5g each of dried Panax notoginseng powder and Salvia miltiorrhiza powder, and add 100mL of 70% ethanol solution to each. Reflux extraction is performed at 80℃ for 2 hours. Each herb is extracted twice to remove as many extractable components as possible. After extraction, filter and collect the insoluble residue, washing thoroughly with ethanol. Dry the residue at 60℃ to constant weight, pulverize, and pass through a 30-mesh sieve to obtain Panax notoginseng residue and Salvia miltiorrhiza residue, respectively, for later use.

[0030] Example 2: Preparation of natural eutectic solvent Using betaine and L-proline as candidate hydrogen bond acceptors (HBAs) and glucose (D-Glucose), lactic acid, citric acid, malonate acid, and succinic acid as candidate hydrogen bond donors (HBDs), 30 groups of natural eutectic solvents were prepared by combining them in molar ratios of 1:1, 1:2, and 2:1, respectively. During preparation, the corresponding hydrogen bond acceptors and hydrogen bond donors are added to a container and continuously heated and stirred at 80°C until a homogeneous and transparent liquid is formed, thus obtaining the corresponding natural eutectic solvent. The resulting system includes betaine-glucose (Bet-Glu), betaine-lactic acid (Bet-Lac), betaine-citric acid (Bet-Ca), betaine-malonic acid (Bet-Mal), betaine-succinic acid (Bet-Suc), L-proline-glucose (L-Pro-Glu), L-proline-lactic acid (L-Pro-Lac), L-proline-citric acid (L-Pro-Ca), L-proline-malonic acid (L-Pro-Mal), and L-proline-succinic acid (L-Pro-Suc).

[0031] Table 1. Composition of natural eutectic solvents

[0032] The obtained natural eutectic solvent, such as Figure 1 As shown in Table 1, nine systems, numbered 4, 5, 7, 11, 18, 19, 20, 23, and 25, were transparent liquids at room temperature, while the other systems were viscous liquids. Clarity, homogeneity, phase separation, and apparent viscosity were used as initial screening criteria. Since low viscosity is generally beneficial for mass transfer, precursor release, and hydrothermal conversion, the relatively high-viscosity combinations L-Pro-Lac (1:1) (numbered 19) and Bet-Lac (1:2) (numbered 5) were excluded. Therefore, seven other groups of naturally occurring eutectic solvents with good physical stability were selected for subsequent experiments.

[0033] Example 3: Screening of natural eutectic solvents The seven natural eutectic solvents with good physical stability obtained from the initial screening in Example 2 were used to prepare carbon dots from Panax notoginseng and Salvia miltiorrhiza residues, respectively. The preparation methods are as follows: Weigh out 150 mg of Panax notoginseng residue (PN) and 150 mg of Salvia miltiorrhiza residue (SM) obtained in Example 1, respectively, and disperse them in 30 mL of deionized water. Then add 30 μL of each of the seven natural eutectic solvents obtained in Example 2. After thorough mixing, transfer the mixture to a 50 mL stainless steel reactor lined with polytetrafluoroethylene and carry out a hydrothermal reaction at 180 °C for 12 h.

[0034] After the reaction was completed, the mixture was allowed to cool naturally to room temperature. Larger precipitate particles were removed by centrifugation at 8000 rpm for 20 min. The supernatant was then filtered through a 0.22 μm polyethersulfone membrane and dialyzed in deionized water for 24 h using a dialysis bag with a molecular weight cutoff of 1000 Da. The external solution was changed every 8 h during dialysis. After dialysis, the mixture was freeze-dried at -45℃ for 30 h to obtain Danshen-Sanqi residue carbon dots (SM-PN-CDs) powder.

[0035] The antioxidant properties of the obtained carbon dots (SM-PN-CDs) from different groups of Tanshinone-Panax notoginseng residue were evaluated using a DPPH free radical scavenging experiment. Figure 2 As shown in the figure, the DPPH group is a blank control, without any carbon dots of drug residue. It can be seen from the figure that when the natural eutectic solvent formed by betaine and lactic acid (Bet-Lac) in a molar ratio of 1:1 is used, the DPPH free radical scavenging ability of the carbon dots is the best. Therefore, this system is determined to be the preferred natural eutectic solvent.

[0036] Example 4: Optimization of hydrothermal reaction temperature The natural eutectic solvent Bet-Lac, screened in Example 3, was used in the experiment. The mass ratio of Panax notoginseng residue to Salvia miltiorrhiza residue was maintained at 1:1. Carbon dots were prepared at hydrothermal reaction temperatures of 160℃, 180℃, and 200℃, with other conditions the same as in Example 3. The antioxidant capacity of the carbon dots obtained under different temperature conditions was evaluated using a DPPH free radical scavenging experiment.

[0037] The results are as follows Figure 3 As shown, the carbon dots prepared at 180℃ exhibited the highest DPPH radical scavenging ability. At 160℃, the resulting carbon dots showed lower antioxidant capacity, presumably related to insufficient carbonization. When the temperature increased to 200℃, the scavenging ability of the resulting carbon dots decreased, possibly due to damage to some key surface functional groups at high temperatures. Therefore, a reaction temperature of 180℃ is preferred.

[0038] Example 5: Optimization of the ratio of Panax notoginseng and Salvia miltiorrhiza residue The natural eutectic solvent Bet-Lac, screened in Example 3, was used in the experiment. The hydrothermal reaction temperature was 180℃. Five combinations of Panax notoginseng and Salvia miltiorrhiza were used: pure Salvia miltiorrhiza (SM), Salvia miltiorrhiza: Panax notoginseng = 2:1, Salvia miltiorrhiza: Panax notoginseng = 1:1, Salvia miltiorrhiza: Panax notoginseng = 1:2, and pure Panax notoginseng (PN). Different carbon dots were prepared under the same conditions as in Example 3. The scavenging effects on DPPH radicals, superoxide anions, NO radicals, and hydroxyl radicals were investigated. A blank control group was set up without any added medicinal residue carbon dots.

[0039] The results are as follows Figures 4-7 As shown, the system with a higher proportion of Panax notoginseng residue exhibits stronger scavenging ability for DPPH free radicals and superoxide anions, while the system with a higher proportion of Salvia miltiorrhiza residue demonstrates superior scavenging ability for hydroxyl free radicals. A comprehensive comparison of the scavenging abilities of the four types of free radicals reveals that carbon dots obtained when the mass ratio of Panax notoginseng residue to Salvia miltiorrhiza residue is 1:1 exhibit a more balanced and broader-spectrum free radical scavenging ability. Therefore, a mass ratio of Panax notoginseng residue to Salvia miltiorrhiza residue of 1:1 is the preferred method.

[0040] Example 6: Comparison of co-hydrothermal preparation and post-mixing methods To verify the effect of the co-hydrothermal reaction on the properties of carbon dots, samples were prepared using the following two methods: The first method is co-hydrothermal preparation, which involves mixing Panax notoginseng residue and Salvia miltiorrhiza residue in a set ratio and then carrying out a hydrothermal reaction in the same reaction system. The second method is post-mixing preparation, which involves preparing carbon dots from Panax notoginseng residue and carbon dots from Salvia miltiorrhiza residue separately, and then physically mixing them in the appropriate proportions.

[0041] The DPPH free radical scavenging capacity of samples obtained by the two methods was compared under the ratios of Danshen:Sanqi=2:1, Danshen:Sanqi=1:1, and Danshen:Sanqi=1:2.

[0042] The results are as follows Figure 8 As shown, the DPPH radical scavenging effect of carbon dots prepared by co-hydrothermal reaction was significantly better than that of the post-mixed sample. This indicates that during the hydrothermal reaction, the precursor components of Panax notoginseng residue and Salvia miltiorrhiza residue underwent more complete synergistic carbonization and surface functional group integration, which is beneficial for forming carbon dots with higher antioxidant activity. Furthermore, for the 1:1 ratio with better radical scavenging ability, the preparation method had a greater impact.

[0043] In summary, the optimal parameters for preparing carbon dots from Danshen-Sanqi residue were selected, namely, using a molar ratio of 1:1 of betaine and lactic acid (Bet-Lac) as a natural eutectic solvent, a mass ratio of 1:1 between Sanqi residue and Danshen residue, a reaction temperature of 180℃, and a co-hydrothermal method for preparing carbon dots.

[0044] Example 7: Preparation of carbon dots from Danshen-Sanqi residue Carbon dots from Danshen-Sanqi residue were prepared according to the screening conditions described above: 150 mg of Panax notoginseng residue and 150 mg of Salvia miltiorrhiza residue prepared in Example 1 were weighed and dispersed in 30 mL of deionized water. Then, 30 μL of betaine-lactic acid natural eutectic solvent was added and mixed thoroughly. The mixture was then transferred to a 50 mL stainless steel reactor lined with polytetrafluoroethylene and subjected to hydrothermal reaction at 180 °C for 12 h to obtain a Salvia miltiorrhiza-Panax notoginseng residue carbon dot solution.

[0045] After the reaction was completed, the carbon dot solution of Danshen-Sanqi residue was allowed to cool naturally to room temperature. It was then centrifuged at 8000 rpm for 20 min to remove larger precipitate particles. The supernatant was filtered through a 0.22 μm polyethersulfone filter membrane and then dialyzed in deionized water for 24 h using a dialysis bag with a molecular weight cutoff of 1000 Da. The external solution was changed every 8 h during the dialysis process. After the dialysis was completed, the solution was freeze-dried at -45℃ for 30 h to obtain carbon dot powder derived from Danshen-Sanqi residue.

[0046] The carbon dot powder prepared under the above conditions has a mass of 23.743 mg, and the yield is approximately 8% relative to the total mass of the added drug residue.

[0047] 1. The preferred betaine-lactic acid natural eutectic solvent was characterized by Fourier transform infrared spectroscopy and hydrogen nuclear magnetic resonance.

[0048] Fourier transform infrared results as follows Figure 9 As shown, this indicates that the stretching vibration peak of OH in lactic acid changes from 3380 cm⁻¹. -1 Move to 3360cm -1 This indicates that significant hydrogen bonding has formed in the system; the characteristic C=O peak of lactic acid is located at 1702 cm⁻¹. -1 The C=O characteristic peak of betaine is located at 1625 cm⁻¹. -1 After forming a natural eutectic solvent, the relevant C=O absorption peak shifts to 1720 cm⁻¹. -1 The CN stretching vibration peak in betaine is approximately 1480 cm⁻¹. -1 This can still be observed in natural eutectic solvents. These results indicate that betaine and lactic acid form a stable eutectic system primarily through non-covalent hydrogen bonding.

[0049] The results of hydrogen nuclear magnetic resonance are as follows Figure 10 The results show that characteristic proton signals of both betaine and lactic acid are present in the natural eutectic solvent, and the proton chemical shifts related to hydroxyl and carboxyl groups in lactic acid change, but no new covalent bond characteristic signals appear, further proving that the formation of the preferred natural eutectic solvent mainly depends on hydrogen bonding.

[0050] 2. Characterize the morphology and structure of carbon dots.

[0051] from Figure 11 a- Figure 11 As can be seen from c, the obtained carbon dots are uniformly dispersed spherical or near-spherical particles, with a particle size distribution ranging from approximately 2.32 to 3.96 nm and an average particle size of approximately 3.2 nm. The local lattice fringe spacing is approximately 0.163 nm, corresponding to the crystal plane structure of graphitized carbon.

[0052] Figure 11 As can be seen from d, a significant absorption peak appears in the UV-Vis absorption spectrum near 282 nm, indicating the presence of aromatic sp in the carbon dots. 2 Structural domain.

[0053] 3. Fluorescence emission is an inherent characteristic of carbon dots. The carbon dot solution obtained after dialysis was taken and diluted 10-fold, 100-fold, and 1000-fold, respectively. The fluorescence emission characteristics of the carbon dot solution at the original concentration, after 10-fold, 100-fold, and 1000-fold dilutions were then detected. Figure 12 "a" represents the state of the carbon point solution under sunlight. From left to right, the values ​​are: original concentration, 10-fold dilution, 100-fold dilution, and 1000-fold dilution. The original concentration solution is yellow. Figure 12 b shows the state of the carbon dot solution under ultraviolet light. From left to right, the values ​​are the original concentration, 10-fold dilution, 100-fold dilution, and 1000-fold dilution. It can be seen that the fluorescence darkens as the carbon dot concentration decreases. The obtained carbon dots exhibit stable blue fluorescence under ultraviolet light irradiation.

[0054] 4. Figure 13 and Figure 14 The fluorescence and structural properties of the carbon dots were tested.

[0055] Figure 13 It can be seen that the maximum emission value is distributed in the 420-510nm region, with a narrow excitation range of 360-410nm. According to... Figure 13 As shown in b, as the photoluminescence excitation wavelength increases from 300 nm to 400 nm, the photoluminescence (PL) wavelength redshifts from 453 nm to 481 nm. Multiple emission energy levels together cause a significant excitation wavelength-dependent emission phenomenon. The PL decay curves of SM-PN-CDs were measured under EPLED excitation at 378 nm using an Edinburgh spectrometer, as shown in [the figure]. Figure 13 c.

[0056] Figure 13 d indicates that the carbon dot surface contains functional groups such as OH or NH, C=C, COC, etc.

[0057] Figure 14 The data shows that SM-PN-CDs contain C (64.33%), O (29.06%), and N (6.61%). Figure 14In the C1s spectrum of b, CC / C=C (284.81 eV), CO (286.26 eV) and C=N / C=O (288.25 eV). Figure 14 The O1s spectrum of d contains C=O (531.46 eV) and CO (532.75 eV). Furthermore, according to... Figure 14 The N1s spectrum of c shows three components: graphite-N, pyrrole-N, and pyridine-N. This indicates that the surface of SM-PN-CDs has abundant functional groups, which is beneficial to its water dispersibility and free radical scavenging performance.

[0058] Example 8: Free Radical Scavenging Experiment The following experiments were conducted on the carbon dots obtained in Example 7.

[0059] 1. DPPH free radical scavenging experiment Different volumes of carbon dot solutions obtained after dialysis in Example 7, namely 0, 50, 100, 200, 250, and 500 μL, were mixed with 2 mL of 0.1 mM DPPH ethanol solution, with a system pH of 7.0. A 1 mg / mL ascorbic acid (AA) solution was used as a positive control group, and carbon dots prepared from the residue of a traditional decoction obtained by boiling equal masses of Panax notoginseng and Salvia miltiorrhiza in water at 100°C for 2 hours were used as the residue group. Carbon dot solutions were prepared in the same way, and the absorbance at 517 nm was measured using a UV-Vis spectrophotometer.

[0060] The results are as follows Figure 15 As shown in Figure a, the curve from top to bottom indicates that the absorbance gradually decreases as the amount of carbon dots added gradually increases, demonstrating that carbon dots can effectively scavenge DPPH free radicals. Further quantitative analysis shows that the DPPH free radical scavenging ability of the obtained carbon dots is significantly dose-dependent.

[0061] Figure 16 The carbon dots prepared by the present invention are superior to those prepared by the carbon dots prepared from the residue of Panax notoginseng and Salvia miltiorrhiza after decoction of natural substance ascorbic acid (AA) (residue group).

[0062] In addition, 500 μL of carbon dot solution prepared according to the present invention was used as the carbon dot-solvent group; carbon dot solution prepared by grinding equal masses of Panax notoginseng and Salvia miltiorrhiza into powder was used as the raw material powder group; carbon dot solution prepared by decocting equal masses of Panax notoginseng and Salvia miltiorrhiza with water at 100°C for 2 hours was used as the residue group; and a natural eutectic solvent formed by equal amounts of betaine and lactic acid (Bet-Lac) in a molar ratio of 1:1 was used as the solvent group. The blank control group did not contain carbon dot solution or natural eutectic solvent. The UV-Vis absorbance at 517 nm was measured every 30 seconds over a 10-minute period to obtain... Figure 17 and Figure 18 .

[0063] As can be seen from the figure, the natural eutectic solvent itself has almost no DPPH free radical scavenging activity. However, the carbon dots (SM-PN-CDs) prepared from the medicinal residue with the assistance of the natural eutectic solvent have a significantly better scavenging ability than the carbon dots from the medicinal residue without the addition of the natural eutectic solvent, and are comparable to the carbon dots prepared from the original medicinal powder. This indicates that the natural eutectic solvent mainly plays a role in promoting the extraction and transformation of active precursors in the medicinal residue in this invention.

[0064] 2. Superoxide anion scavenging experiment 400 μL of riboflavin solution, 400 μL of L-methionine solution, 400 μL of nitroblue tetrazolium solution, and different volumes (0, 50, 100, 200, 250, and 500 μL) of carbon dot solution obtained after dialyzing in Example 7 were mixed. Each solution was prepared in 0.01 M PBS buffer (pH 7.4). The concentrations of riboflavin, L-methionine, and nitroblue tetrazolium were 66.7 μM, 43.3 mM, and 25 μM, respectively. The mixtures were irradiated under a 30 W fluorescent lamp for 10 min, and the absorbance was measured at 560 nm.

[0065] The results are as follows Figure 15 b. The curve from top to bottom shows that as the amount of carbon dots added gradually increases, the absorbance at 560 nm is relatively high when no carbon dots are added; as the amount of carbon dots added increases, the absorbance at 560 nm decreases significantly, indicating that the carbon dots prepared by this invention can effectively remove superoxide anions and have a free radical scavenging ability similar to superoxide dismutase.

[0066] 3. NO free radical scavenging experiment Take 200 μL of a 200 mM sodium nitroprusside solution and incubate at 37 °C for 30 min to generate NO radicals. Then add different volumes (0, 50, 100, 200, 250, and 500 μL) of the carbon dot solution obtained after dialyzing in Example 7, and bring the volume to 2 mL with 0.1 M, pH 7.4 PBS buffer. Next, add 50 μL of a 10 mg / mL p-aminobenzenesulfonic acid solution and 50 μL of a 10 mg / mL naphthylethylenediamine hydrochloride solution to the system. After reacting for 10 min, measure the absorbance at 540 nm.

[0067] The results are as follows Figure 15 c. As the amount of carbon dots added increases, the absorbance at 540 nm continues to decrease, indicating that the carbon dots prepared by this invention can effectively inhibit NO free radicals from participating in the formation of colored azo products, and exhibit good NO free radical scavenging ability.

[0068] 4. Hydroxyl radical scavenging experiment Take 300 μL of 8.8 mM hydrogen peroxide solution, 100 μL of 100 mM TMB solution, and different volumes of carbon dot solutions obtained after dialyzing in Example 7 (0, 50, 100, 200, 250, and 500 μL), add them to 0.01 M PBS buffer (pH 7.4), and pre-incubate for 5 min. Then add 20 μL of 100 mM ferrous sulfate solution to start the Fenton reaction, and measure the absorbance at 652 nm every 30 s for 5 min.

[0069] The results are as follows Figure 15 d. Without the addition of carbon dots, the system rapidly generates oxidation products, exhibiting high absorbance at 652 nm. As the amount of carbon dots added increases, the absorbance gradually decreases, indicating that the carbon dots obtained in this invention have a good scavenging effect on hydroxyl radicals. Since hydroxyl radicals have a strong oxidizing ability, this experimental result further proves that the carbon dots obtained in this invention have strong broad-spectrum antioxidant potential.

[0070] Example 9: Stability Evaluation Using DPPH free radical scavenging ability as an evaluation index, the carbon dot solution obtained after dialysis in Example 7 was used to investigate the stability of the obtained carbon dots under different temperatures, different pH values ​​and different storage conditions.

[0071] In the thermal stability experiment, the carbon dot solution and the DPPH reaction system were reacted at 10-90℃ for 10 min respectively, and their free radical scavenging ability was measured. The results are as follows: Figure 19 a. The carbon dots obtained by this invention maintain strong free radical scavenging activity in the range of 10-90℃, and still have high scavenging efficiency at 70-90℃.

[0072] In the pH stability experiment, the DPPH reaction system was adjusted to pH 2-12 using acid-base solutions, and the DPPH free radical scavenging capacity of the carbon dot solution was measured under each pH condition. The results are as follows: Figure 19 b. Carbon dots maintain strong free radical scavenging ability over a wide pH range, with better performance under near-neutral conditions.

[0073] In the storage stability experiment, the carbon dot solution was stored for 5 days under light-protected room temperature, light-protected room temperature nitrogen atmosphere, and light-protected refrigeration at 4°C. Its DPPH free radical scavenging ability was measured daily. The results are as follows: Figure 19 c. 4℃ light-proof refrigeration and nitrogen protection conditions can maintain the free radical scavenging activity of carbon dots well, while the activity decay is more obvious under normal room temperature conditions. This indicates that the carbon dots obtained by this invention have good storage stability, but using light-proof, low temperature or inert gas protection is more conducive to maintaining their performance.

[0074] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing carbon dots from *Salvia miltiorrhiza*-*Panax notoginseng* residue with broad-spectrum free radical scavenging ability, characterized in that: Panax notoginseng residue and Salvia miltiorrhiza residue in a mass ratio of 1:1 were dispersed in deionized water, then a natural eutectic solvent was added, and the mixture was transferred to a reaction vessel. After reacting at 180°C for 12 hours, the mixture was filtered, dialyzed, and freeze-dried to obtain carbon dots from the Salvia miltiorrhiza residue. The volume ratio of the added natural eutectic solvent to the sum of the masses of the Panax notoginseng residue and Salvia miltiorrhiza residue was 1:10 μL / mg. The natural eutectic solvent was a system formed by betaine and lactic acid in a molar ratio of 1:

1. The prepared carbon dots from the Salvia miltiorrhiza residue were spherical or nearly spherical particles with a particle size of 2.32-3.96 nm and contained hydroxyl, carboxyl, and nitrogen-containing functional groups on their surface.

2. The method for preparing carbon dots from Danshen-Sanqi residue with broad-spectrum free radical scavenging ability according to claim 1, characterized in that: The filtration method is as follows: centrifuge at 6000-8000 rpm for 10-20 min, and filter the supernatant through a 0.22 μm filter membrane.

3. The method for preparing carbon dots from Danshen-Sanqi residue with broad-spectrum free radical scavenging ability according to claim 1, characterized in that: The dialysis method is as follows: use a dialysis bag with a molecular weight cutoff of 1000-1200 Da and dialyze in deionized water for 20-24 hours, changing the external solution every 6-8 hours during the period.

4. The method for preparing carbon dots from Danshen-Sanqi residue with broad-spectrum free radical scavenging ability according to claim 1, characterized in that: The freeze-drying conditions are: -50℃ to -45℃, for 28-30 hours.

5. A carbon dot of Danshen-Sanqi residue prepared by the preparation method according to any one of claims 1-4.

6. An application of the carbon dots of Danshen-Sanqi residue as described in claim 5, characterized in that: The carbon dots from the Danshen-Sanqi medicinal residue are used for the scavenging of free radicals for non-disease diagnosis and treatment purposes, including the scavenging of DPPH free radicals, superoxide anions, NO free radicals, and hydroxyl free radicals.

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