Huang-qi carbon nanoparticles, a preparation method and application thereof

Abstract

The present application relates to the technical field of medicine, in particular to a carbon nanoparticle of rhizoma polygonati and a preparation method and application thereof. The present application successfully constructs a preparation method of the carbon nanoparticle of rhizoma polygonati. The carbon nanoparticle of rhizoma polygonati prepared by the method of the present application has excellent in-vitro antioxidant capacity, biological safety and definite liver protection activity, and can realize the liver protection effect through the multi-pathway synergy of "metabolic regulation-antioxidant defense-mitochondrial protection", and significantly prevent alcohol-induced hepatocyte damage. The present application provides experimental and theoretical support for analyzing the generation rule of nano active substances in the processing of rhizoma polygonati, developing the nano mechanism research of traditional Chinese medicine processing, and provides a new research and development idea for developing new low-toxicity and high-efficiency liver protection traditional Chinese medicine nano preparations.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a Polygonatum odoratum carbon nanoparticle, its preparation method, and its application. Background Technology

[0002] With changing lifestyles in modern society, the incidence of alcoholic liver injury induced by factors such as excessive alcohol consumption and oxidative stress disorders continues to rise. This disease can gradually progress to hepatitis, liver fibrosis, and even cirrhosis, posing a serious threat to human health. Existing chemical hepatoprotective drugs often have drawbacks such as significant side effects and insufficient safety with long-term use. Therefore, developing natural, low-toxicity drugs with diverse target areas for the prevention and treatment of alcoholic liver injury has significant clinical value.

[0003] Polygonatum sibiricum Red, the dried rhizome of a plant in the genus Polygonatum of the Liliaceae family, is a traditional Chinese medicine with a long history of medicinal use, often considered both food and medicine. Traditional Chinese medicine believes that Polygonatum is sweet and neutral in nature, entering the spleen, lung, and kidney meridians. It has the functions of tonifying qi and nourishing yin, strengthening the spleen, moistening the lungs, and benefiting the kidneys, and is often used to improve symptoms such as spleen and stomach qi deficiency, fatigue, dry cough due to lung deficiency, insufficient essence and blood, and soreness of the lower back and knees. Modern pharmacology shows that Polygonatum contains various active substances such as polysaccharides, saponins, and flavonoids. Its extracts can reduce alcoholic liver damage and inhibit oxidative stress and liver inflammation. However, existing Polygonatum liver-protecting preparations mostly use crude extracts of the rhizome or single purified components, which generally suffer from problems such as large molecular size, poor cell membrane permeability, and low bioavailability in vivo. Furthermore, the extraction process is complex and costly, making it difficult to fully realize its liver-protective effects.

[0004] The interdisciplinary integration of nanotechnology and traditional Chinese medicine offers a new approach to addressing the shortcomings of traditional Chinese medicine efficacy. Carbon nanoparticles, as low-toxicity and highly biocompatible nanomaterials, possess excellent cell penetration and antioxidant capabilities. The formation of carbon nanoparticles derived from traditional Chinese medicine typically relies on the dehydration, condensation, polymerization, and carbonization reactions of organic matter such as polysaccharides, proteins, and amino acids under heating conditions. The high-temperature and high-humidity environment of the nine-steaming and nine-drying process of Polygonatum sibiricum can promote the Maillard reaction and carbonization nucleation of polysaccharides and amino acids, ultimately forming carbon nanoparticles, which have the potential to develop novel hepatoprotective nanoparticle formulations. Currently, there is no systematic characterization of carbon nanoparticles generated from the processing of Polygonatum sibiricum and no research on their protective activity against alcoholic liver injury.

[0005] Based on this, the present invention uses nine-steamed and nine-dried Polygonatum as raw material to prepare Polygonatum carbon nanoparticles, analyzes its physicochemical characteristics through various characterization methods, and verifies its hepatoprotective activity, aiming to fill the existing research gap and provide experimental basis and theoretical support for the development of natural nano-hepatoprotective drugs. Summary of the Invention

[0006] The purpose of this invention is to provide a Polygonatum carbon nanoparticle and its preparation method.

[0007] Another object of the present invention is to provide the use of the above-mentioned Polygonatum carbon nanoparticles in the preparation of medicaments for the prevention or treatment of alcoholic liver injury.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The specific steps of the preparation method of Polygonatum carbon nanoparticles of the present invention are as follows: S1. Weigh out the nine-steamed and nine-dried Polygonatum rhizome, add 5-15 times the weight of the medicinal material to decoct 1-5 times, each time for 30-90 minutes; filter, combine the decoctions, and concentrate to one-third of the original liquid to obtain concentrated liquid. S2. Add 1-5 times the volume of anhydrous ethanol to the concentrate, let stand for 12-36 hours at a low temperature of 2-8℃, and then filter. S3. Take the filtrate and put it into a dialysis bag with a molecular weight cutoff of 300 Da. Dialyze with deionized water for 5-10 days, changing the water several times during the process. After dialysis, filter the solution using a microporous membrane and freeze-dry the filtrate to obtain the final product.

[0009] Preferably, in the preparation method of Polygonatum carbon nanoparticles of the present invention, step S1 specifically involves: weighing Polygonatum that has been steamed and dried nine times, adding 8-12 times the weight of the medicinal material to deionized water and decocting 2-4 times, each time for 40-80 minutes; filtering, combining the decoctions, and concentrating to one-third of the original liquid to obtain a concentrated liquid.

[0010] In a further preferred embodiment, in the preparation method of Polygonatum carbon nanoparticles of the present invention, step S1 specifically involves: weighing Polygonatum that has been steamed and dried nine times, adding 10 times the weight of the medicinal material to deionized water and decocting it three times, each time for 60 minutes; filtering, combining the decoctions, and concentrating it to one-third of the original liquid to obtain a concentrated liquid.

[0011] Preferably, in the preparation method of Polygonatum carbon nanoparticles of the present invention, step S2 specifically involves: adding anhydrous ethanol at a volume of 2-4 times that of the concentrated liquid, allowing it to stand for 20-28 hours at a low temperature of 3-5°C, and then filtering.

[0012] In a further preferred embodiment, in the preparation method of Polygonatum carbon nanoparticles of the present invention, step S2 specifically involves: adding anhydrous ethanol at a volume of 3 times that of the concentrated liquid, allowing it to stand for 24 hours at a low temperature of 4°C, and then filtering.

[0013] Preferably, in the preparation method of Polygonatum carbon nanoparticles of the present invention, the dialysis time in step S3 is 6-8 days.

[0014] In a further preferred embodiment, in the preparation method of Polygonatum carbon nanoparticles of the present invention, the dialysis time in step S3 is 7 days.

[0015] Preferably, in the preparation method of Polygonatum carbon nanoparticles of the present invention, the pore size of the microporous filter membrane in step S3 is 0.22 μm.

[0016] The Polygonatum carbon nanoparticles of this invention are prepared using the above-described preparation method.

[0017] The application of the Polygonatum carbon nanoparticles described in this invention in the preparation of drugs for the prevention or treatment of alcoholic liver injury.

[0018] The beneficial effects of this invention are: 1. The Polygonatum carbon nanoparticles prepared in this invention possess excellent biological application potential. Characterization using TEM, XRD, XPS, and FT-IR confirmed that the carbon nanoparticles are uniform, well-dispersed, and have a particle size of 10±2.4 nm. Their amorphous crystal structure exhibits enhanced reactivity. The material is primarily composed of carbon, oxygen, and nitrogen, with a surface rich in hydrophilic functional groups such as hydroxyl, amino, carbonyl, and ether bonds, exhibiting excellent water solubility and cell permeability while retaining the structural characteristics of Polygonatum polysaccharide derivatives. These characterization data corroborate each other, clearly elucidating the structure-activity relationship between raw material components, nanostructure, and biological activity. This provides experimental and theoretical support for analyzing the generation rules of nano-active substances during the processing of Polygonatum and for conducting research on the nano-mechanism of traditional Chinese medicine processing.

[0019] 2. The Polygonatum carbon nanoparticles prepared in this invention possess excellent in vitro antioxidant activity and biocompatibility. DPPH free radical scavenging experiments showed that low concentrations of Polygonatum carbon nanoparticles could rapidly achieve high free radical scavenging efficiency (69.34% scavenging rate at 1 mg / mL). CCK-8 cytotoxicity experiments showed that low concentrations (0.5-1 mg / mL) promoted the proliferation of HepG2 hepatocytes, and cell viability at 2 mg / mL was not significantly different from the normal control group. Only high concentrations above 4 mg / mL showed cell inhibition, indicating that the Polygonatum carbon nanoparticles have low overall toxicity and good biocompatibility.

[0020] 3. The Polygonatum sibiricum carbon nanoparticles prepared in this invention can significantly prevent alcohol-induced hepatocellular damage and possess clear hepatoprotective activity. In an ethanol-induced HepG2 hepatocellular damage model, pretreatment within the full concentration range of 0.5-8 mg / mL can stably maintain hepatocellular viability, and the cell survival rate is significantly better than that of the damage model control group. Mechanism of action studies show that the hepatoprotective effect of these Polygonatum sibiricum carbon nanoparticles relies on the synergistic action of multiple pathways: metabolic regulation, antioxidant defense, and mitochondrial protection; increasing ADH activity to accelerate alcohol metabolism and reduce the accumulation of toxic acetaldehyde; upregulating SOD activity and reducing MDA content to alleviate cell membrane oxidative damage and reduce LDH leakage; and activating skin CI activity to stabilize mitochondrial energy metabolism function, forming a complete liver protection chain of "source detoxification, blocking oxidative damage, and repairing organelles."

[0021] 4. The Polygonatum carbon nanoparticles provided by this invention combine the advantages of both nanomaterials and the traditional Chinese medicine Polygonatum, exhibiting outstanding industrialization and medicinal value. On one hand, the nanoparticle size facilitates their entry into cells to exert their effects, resulting in good water solubility and high absorption efficiency, while fully preserving the original pharmacological activity of Polygonatum. On the other hand, the research conclusions of this invention corroborate existing research results on the protection of liver mitochondria and regulation of respiratory chain complexes by Polygonatum preparations, confirming that the use of Polygonatum carbon nanoparticles for the prevention and treatment of alcoholic liver injury has reliable theoretical support, providing a novel research and development approach for developing new, low-toxicity, and highly effective hepatoprotective traditional Chinese medicine nanoparticle preparations. Attached Figure Description

[0022] Figure 1 TEM image of Polygonatum odoratum carbon nanoparticles; Figure 2 The XRD pattern of Polygonatum odoratum carbon nanoparticles; Figure 3 The X-ray photoelectron spectra of Polygonatum odoratum carbon nanoparticles are shown in the figure (A is the XPS full spectrum of PS-CNs, B is the C1s spectrum of PS-CNs, C is the N1s spectrum of PS-CNs, and D is the O1s spectrum of PS-CNs). Figure 4 The infrared spectrum of Polygonatum odoratum carbon nanoparticles; Figure 5 The DPPH free radical scavenging rate of Polygonatum carbon nanoparticles; Figure 6 The survival rate of HepG2 cells after treatment with different concentrations of Polygonatum odoratum carbon nanoparticles; Figure 7 The cell survival rate of HepG2 cells after alcohol damage following pretreatment with different concentrations of Polygonatum odoratum carbon nanoparticles; Figure 8 The effect of different concentrations of Polygonatum odoratum carbon nanoparticles on the activity of various enzymes in cells of an alcohol-induced injury model (in the figure: A is ADH, B is LDH, C is MDA, D is SOD, and E is CI). Detailed Implementation

[0023] The technical solution of the present invention will be described in detail below with reference to specific embodiments. These embodiments are for explanation and illustration only and do not constitute a limitation on the technical solution of the present invention.

[0024] Example 1

[0025] The preparation method of Polygonatum odoratum carbon nanoparticles is as follows: Weigh out the nine-steamed and nine-dried Polygonatum rhizome, add 10 times its weight of deionized water and decoct three times, 60 min each time; filter, combine the decoctions, and concentrate to one-third of the original liquid to obtain a concentrated liquid; add 3 times the volume of anhydrous ethanol to the concentrated liquid, and let it stand at 4℃ for 24 h; filter, put the filtrate into a dialysis bag with a molecular weight cutoff of 300 Da and dialyze with deionized water for 7 days, changing the water multiple times during the period; after dialysis, filter using a 0.22 μm microporous membrane, and freeze-dry the filtrate to obtain the final product.

[0026] Example 2

[0027] The preparation method of Polygonatum odoratum carbon nanoparticles is as follows: Weigh out the nine-steamed and nine-dried Polygonatum rhizome, add 15 times its weight of deionized water and decoct for 90 min; filter, concentrate the decoction to one-third of the original liquid to obtain a concentrated liquid; add 1 volume of anhydrous ethanol to the concentrated liquid, and let stand at 2℃ for 36 h; filter, put the filtrate into a dialysis bag with a molecular weight cutoff of 300 Da and dialyze with deionized water for 5 days, changing the water several times during the period; after dialysis, filter with a 0.22 μm microporous membrane, and freeze-dry the filtrate to obtain the final product.

[0028] Example 3

[0029] The preparation method of Polygonatum odoratum carbon nanoparticles is as follows: Weigh out the nine-steamed and nine-dried Polygonatum rhizome, add five times the weight of the medicinal material in deionized water and decoct five times, 30 min each time; filter, combine the decoctions, and concentrate to one-third of the original liquid to obtain a concentrated liquid; add five times the volume of the concentrated liquid in anhydrous ethanol, and let it stand at 8℃ for 12 h; filter, put the filtrate into a dialysis bag with a molecular weight cutoff of 300 Da and dialyze with deionized water for 10 days, changing the water multiple times during the period; after dialysis, filter using a 0.22 μm microporous membrane, and freeze-dry the filtrate to obtain the final product.

[0030] Example 4

[0031] The preparation method of Polygonatum odoratum carbon nanoparticles is as follows: Weigh out the nine-steamed and nine-dried Polygonatum rhizome, add 8 times the weight of the medicinal material to deionized water and decoct 4 times, 40 min each time; filter, combine the decoctions, and concentrate to one-third of the original liquid to obtain a concentrated liquid; add 2 times the volume of the concentrated liquid to anhydrous ethanol, and let stand at 3℃ for 28 h; filter, put the filtrate into a dialysis bag with a molecular weight cutoff of 300 Da and dialyze with deionized water for 6 days, changing the water multiple times during the period; after dialysis, filter using a 0.22 μm microporous membrane, take the filtrate and freeze-dry to obtain the final product.

[0032] Example 5

[0033] The preparation method of Polygonatum odoratum carbon nanoparticles is as follows: Weigh out the nine-steamed and nine-dried Polygonatum rhizome, add 12 times its weight of deionized water and decoct twice, 80 min each time; filter, combine the decoctions, and concentrate to one-third of the original liquid to obtain a concentrated liquid; add 4 times the volume of anhydrous ethanol to the concentrated liquid, and let it stand at 5℃ for 20 h; filter, put the filtrate into a dialysis bag with a molecular weight cutoff of 300 Da and dialyze with deionized water for 8 days, changing the water multiple times during the period; after dialysis, filter using a 0.22 μm microporous membrane, and freeze-dry the filtrate to obtain the final product.

[0034] Example 6

[0035] Drugs for preventing alcoholic liver damage were prepared using Polygonatum sibiricum carbon nanoparticles from Examples 1-5.

[0036] Example 7

[0037] Drugs for treating alcoholic liver injury were prepared using Polygonatum odoratum carbon nanoparticles from Examples 1-5.

[0038] To further verify the reliability of the present invention, the inventors conducted a series of experiments, as follows: 1. Experimental Methods 1.1 Extraction and preparation of Polygonatum odoratum carbon nanoparticles A certain amount of nine-steamed and nine-dried Polygonatum was weighed and decocted three times with 10 times the weight of the medicinal material in deionized water, each time for 60 minutes. The decoctions from the three decoctions were combined, filtered, and one-third of the original liquid was concentrated to obtain a concentrated liquid. Then, three times the volume of the concentrated liquid in anhydrous ethanol was added, and the mixture was allowed to stand at 4°C for 24 hours. The polysaccharides and other substances that were precipitated were filtered out and then placed in a dialysis bag with an Mw 300 Da and dialyzed with deionized water for 7 days, with frequent water changes during the period. After dialysis, the precipitates were removed using a 0.22 μm microporous membrane, and the solution was freeze-dried to obtain Polygonatum carbon nanoparticles (PS-CNs). The collected nanoparticles were stored at 4°C in the dark.

[0039] 1.2 Characterization of Polygonatum odoratum carbon nanoparticles 1.2.1 TEM The morphology, particle size distribution, and microstructure of Polygonatum carbon nanoparticles were observed using a transmission electron microscope (TEM, Talos F200SG2S / TEM, Thermo Scientific FEI, USA). A copper mesh was used for observation at 200 kV. Before use, the sample was dissolved in pure water and sonicated for 2 h.

[0040] 1.2.2 XRD The scan was performed using a Smartlab X-ray diffractometer (XRD, Smartlab, Rigaku Corporation, Japan) at a speed of 10° / min, with a scan range of 10-90°.

[0041] 1.2.3 XPS The elemental composition of the sample was detected using a Nexsa X-ray electron spectrometer at a power of 150 W, and the chemical bonds contained in each element were determined by peak fitting.

[0042] 1.2.4 FT-IR The Polygonatum odoratum carbon nanoparticle sample was scanned 32 times using an IRTracer-100 Fourier transform infrared spectrometer, with a scanning range of 500-4000 cm⁻¹. -1 .

[0043] 1.3 Antioxidant capacity of Polygonatum odoratum carbon nanoparticles The antioxidant capacity of Polygonatum odoratum carbon nanoparticles was tested using a DPPH oxygen free radical scavenging experiment. 1 mL of different concentrations of Polygonatum odoratum carbon nanoparticle solutions were placed in 1.5 mL centrifuge tubes for later use. A blank control group was also prepared by adding 1 mL of anhydrous ethanol. 1 mL of 0.2 mmol / L DPPH solution was added to each of the centrifuge tubes containing the different concentrations of Polygonatum odoratum carbon nanoparticle solutions and the blank control, and the mixture was rapidly vortexed to ensure complete reaction. The centrifuge tubes were incubated at room temperature in the dark for 30 min. After the reaction, the absorbance of the supernatant in each tube was measured at 517 nm using a UV-Vis spectrophotometer. The data were analyzed to evaluate the antioxidant capacity of the Polygonatum odoratum carbon nanoparticles.

[0044] 1.4 In vitro cytotoxicity of Polygonatum odoratum carbon nanoparticles The in vitro cytotoxicity of different concentrations of Polygonatum odoratum carbon nanoparticles co-cultured with HepG2 cells for 24 h was evaluated using the Cell Counting Kit-8 (CCK8) assay. After resuscitation, HepG2 cells were seeded into culture flasks containing complete culture medium (DMEM medium containing 10% fetal bovine serum and 1% penicillin antibiotics) and cultured in a 37°C, 5% CO2 incubator. When the cells reached the logarithmic growth phase, they were digested with trypsin to adjust the cell concentration to 1×10⁶ cells / year. 4 Cells were seeded at a density of 100 μL / mL into 96-well cell culture plates and incubated for 24 h to allow cell adhesion. Different concentrations (0.5 mg / mL, 1 mg / mL, 2 mg / mL, 4 mg / mL, 8 mg / mL) of Polygonatum sibiricum carbon nanoparticles were added to the experimental groups and incubated for 24 h. The solution was then aspirated, and the cells were washed three times with PBS, followed by incubation with 10% CCK-8 solution for another 2 h. The absorbance at 450 nm was measured using a microplate reader to calculate cytotoxicity.

[0045] 1.5 Investigation into the performance and mechanism of preventing alcoholic liver injury 1.5.1 Performance evaluation in preventing alcoholic liver injury After resuscitation, HepG2 cells were seeded into culture flasks containing complete culture medium (DMEM medium containing 10% fetal bovine serum and 1% penicillin-dextrose antibody) and cultured in a 37°C, 5% CO2 incubator. When the cells reached the logarithmic growth phase, they were digested with trypsin to adjust the cell concentration to 1×10⁶ cells / year. 4 Cells were seeded at a density of 100 μL / mL into 96-well cell culture plates and incubated for 24 h to allow cell adhesion. Different concentrations of Polygonatum sibiricum carbon nanoparticle solutions were added to the experimental groups and incubated for 24 h. The solution was then aspirated, and the cells were washed three times with PBS, followed by incubation with 10% ethanol for 1 h. After washing three times with PBS, 10% CCK-8 solution was added and incubated for 2 h. The absorbance at 450 nm was measured using a microplate reader to calculate cell viability.

[0046] 1.5.2 Research on the mechanism of preventing alcoholic liver injury Based on the results of in vitro cytotoxicity and anti-alcoholic liver injury performance studies, Polygonatum odoratum carbon nanoparticles at concentrations of 0.5 mg / mL, 1 mg / mL, and 2 mg / mL were selected for experiments. Blank, model, and experimental groups were established. After stimulation with ethanol solution for 1 h, cells were washed three times with PBS, and then the cells were collected. The activities of various enzymes within the cells (alcohol dehydrogenase (ADH); lactate dehydrogenase (LDH); malondialdehyde (MDA); superoxide dismutase (SOD); and mitochondrial respiratory chain complex I (CI)) were measured according to the kit instructions.

[0047] 2. Experimental Results 2.1 Characterization of Polygonatum odoratum carbon nanoparticles 2.1.1 TEM TEM morphology images of Polygonatum carbon nanoparticles are shown below. Figure 1 As shown. By Figure 1 It can be seen that PS-CNs have a uniform overall morphology, are irregular particles, have good dispersibility, and have a particle size of 10±2.4 nm.

[0048] 2.1.2 XRD XRD pattern of Polygonatum carbon nanoparticles as follows Figure 2 As shown. By Figure 2 It can be seen that PS-CNs have a broad and weak diffuse peak near 2θ=22°, without obvious sharp peaks, and exhibit an overall amorphous characteristic.

[0049] 2.1.3 XPS X-ray photoelectron spectroscopy of Polygonatum carbon nanoparticles is as follows: Figure 3As shown in the figure. Analysis of the experimental results indicates that PS-CNs are mainly composed of carbon, nitrogen, and oxygen, with elemental proportions of 60.02% (carbon), 4.07% (nitrogen), and 35.91% (oxygen), respectively. In the C1s spectrum, three different energy peaks are observed at 283.3, 284.8, and 286.4 eV. Based on the different binding energies and chemical states, these three peaks are considered to be inorganic carbon (or graphite-like carbon), CO bond, and C / C bond, respectively. The N 1s spectrum shows three different bond energy peaks: 398.6 (-C=NC-pyridine nitrogen), 400.5 (C3-N quaternary nitrogen), and 405 eV (ON). The O 1s spectrum shows three different bond energy peaks at 532.2, 531.3, and 529.9 eV, corresponding to CO bond (ether bond COC), C=O carbon-oxygen double bond (carbonyl, carboxyl, amide carbonyl), and -OH hydroxyl group, respectively. The XRD and XPS results corroborated each other, revealing the crystal structure, elemental composition, and surface chemical characteristics of Polygonatum carbon nanoparticles, providing experimental evidence for elucidating the structure-activity relationship of precursor composition, structural characteristics, and biological activity.

[0050] 2.1.4 FT-IR The infrared spectrum of Polygonatum carbon nanoparticles is as follows: Figure 4 As shown. The results indicate that 3400-3300 cm... -1 A broad and strong absorption peak is observed at this point, attributed to the stretching vibrations of OH and NH, indicating that the surface is rich in hydrophilic groups such as hydroxyl and amino groups. 2930 cm⁻¹ -1 The weak absorption peak corresponds to the stretching vibration of CH saturated alkyl groups. (1710 cm⁻¹) -1 The C=O carbonyl stretching vibration at this point indicates the presence of carboxyl and ester groups. (1620 cm⁻¹) -1 The strong peak at 1100-1050 cm⁻¹ is a characteristic of C=C and amide bond conjugated framework vibrations, typical of carbon nanoparticles. -1 The COC and CO stretching vibrations at the site reflect the polysaccharide-derived structure.

[0051] 2.2 Antioxidant capacity of Polygonatum odoratum carbon nanoparticles Based on the excellent antioxidant capacity of Polygonatum sibiricum, we designed an experimental system to evaluate the DPPH free radical scavenging ability of PS-CNs and the vitamin C (Vc) positive control group. The DPPH free radical scavenging rate of Polygonatum sibiricum carbon nanoparticles is as follows: Figure 5 As shown in the figure. The results showed that the clearance rate of PS-CNs rapidly increased to about 69.34% at low concentrations (≤1 mg / mL), and then slowly decreased with increasing concentration, dropping to about 49.98% at 10 mg / mL.

[0052] 2.3 In vitro cytotoxicity of Polygonatum odoratum carbon nanoparticles This study evaluated the changes in cell viability of HepG2 cells after co-culturing with PS-CNs at concentrations ranging from 0.5 to 8 mg / mL for 24 hours using the CCK-8 assay. The results are as follows: Figure 6 As shown in the figure. The results showed that at low concentrations (0.5-1 mg / mL), the viability of HepG2 cells was significantly higher than that of the control group (Control); at high concentrations (2-8 mg / mL), the viability of HepG2 cells began to decrease significantly; at a concentration of 2 mg / mL, there was no difference in viability between HepG2 cells and the control group (p>0.05); at concentrations of 4-8 mg / mL, the viability of HepG2 cells decreased significantly (0.0001<p≤0.001), and the inhibitory effect increased with increasing concentration.

[0053] 2.4 Investigation on the performance and mechanism of Polygonatum odoratum carbon nanoparticles in preventing alcoholic liver injury 2.4.1 Study on the performance of Polygonatum odoratum carbon nanoparticles in preventing alcoholic liver injury This study used an alcohol-induced HepG2 cell model to systematically evaluate the hepatoprotective activity of PS-CNs at concentration gradients of 0.5–8 mg / mL using the CCK-8 assay. Ethanol treatment was used to simulate hepatic oxidative stress injury, while pre-incubation with carbon nanoparticles was used to examine their intervention effect on the injury. The results are as follows: Figure 7 As shown in the figure. The results showed that PS-CNs maintained stable cell viability between 56% and 66% within the range of 0.5-8 mg / mL, which was significantly higher than that of the positive control group.

[0054] 2.4.2 Study on the mechanism of Polygonatum odoratum carbon nanoparticles in preventing alcoholic liver injury To investigate the mechanism by which PS-CNs prevent alcohol-induced injury, this study used various reagent kits to examine changes in intracellular ADH, LDH, MDA, and SOD levels. Additionally, changes in mitochondrial complex (CⅠ) levels were measured. The results are as follows: Figure 8 As shown. The results showed that after HepG2 cells were pretreated with PS-CNs and then subjected to alcohol injury, the intracellular ADH content increased with increasing concentration. Figure 8 A), at a concentration of 2 mg / mL, reached 27.242 U / mL, which was significantly higher than that of the model group (16.777 U / mL) and the blank control group (10.155 U / mL); intracellular LDH also gradually increased with the increase of pretreated carbon nanoparticle concentration. Figure 8B), at a concentration of 2 mg / mL, the concentration reached 0.432 U / mL, significantly higher than the model group (0.163 U / mL) and close to the blank control group (0.847 U / mL); after PS-CNs pretreatment, at a Polygonatum carbon nanoparticle concentration of 2 mg / mL, the MDA concentration was 1.304 nmol / mL, significantly lower than the model group without any drug treatment (1.583 nmol / mL). Figure 8 C); HepG2 cells pretreated with PS-CNs followed by alcohol injury showed an intracellular SOD content of 418.489 U / mgprot at a concentration of 2 mg / mL of Polygonatum sibiricum carbon nanoparticles. Figure 8 D), compared with the ethanol model group (259.345 U / mgprot), the intracellular SOD activity in the PS-CNs treatment group was significantly increased; after PS-CNs pretreatment, the cell CⅠ activity increased in a concentration-dependent manner with increasing drug concentration (D). Figure 8 E), at high concentration (2 mg / mL), it can recover to 61.46%, which is significantly higher than that of the model group (p<0.001).

[0055] Mechanistic studies have confirmed that the hepatoprotective effect of PS-CNs is not achieved through a single pathway, but rather through a synergistic multi-pathway approach involving metabolic regulation, antioxidant defense, and mitochondrial protection. PS-CNs upregulate alcohol dehydrogenase (ADH) activity, accelerating alcohol metabolism in hepatocytes and reducing the accumulation of toxic byproducts such as acetaldehyde, thus mitigating alcohol damage at its source. They also increase superoxide dismutase (SOD) activity, reduce malondialdehyde (MDA) levels, inhibit lipid peroxidation, reduce free radical damage to cell membranes, and decrease lactate dehydrogenase (LDH) leakage. Furthermore, they enhance mitochondrial respiratory chain complex I activity, protecting mitochondrial structure and function and maintaining cellular energy metabolism stability. As a core target of alcohol damage, mitochondrial function recovery is crucial for hepatocyte survival, which is the core mechanism by which PS-CNs exert their potent hepatoprotective effect. These three mechanisms work synergistically to form a complete protective chain of "reducing the source of damage, blocking the damage process, and repairing damaged structures." Polygonatum carbon nanoparticles significantly reduced MDA levels and increased SOD activity in a liver injury model, suggesting that traditional Chinese medicine-derived carbon nanoparticles may enter cells due to their small particle size and exert a hepatoprotective effect by enhancing the activity of antioxidant enzyme systems. Similarly, in studies on alleviating metabolic-related fatty liver disease, Polygonatum pills significantly protected against hepatic mitochondrial damage, inhibited the increase of MDA in hepatic mitochondria, and reduced SOD, GSH, ATP synthase, Complex I, and Complex II. Network pharmacology predicted that this effect was related to the PI3K / AKT signaling pathway—reporting the protective effect of Polygonatum components on respiratory chain complex I, which is highly consistent with the finding of increased CI activity in this study, further confirming the general theory that "these types of carbon nanoparticles have advantages such as good water solubility, high biocompatibility, low toxicity, and easy absorption, while retaining the original pharmacological activity of traditional Chinese medicine."

[0056] 3. Conclusion This study successfully constructed carbon nanoparticles of Polygonatum odoratum, systematically characterized their structure and activity, and revealed that the synergistic carbonization of natural carbon nanoparticles and polysaccharides is the key to the formation of highly active products. This provides experimental support for understanding the generation law of nano-components during the nine-steaming and nine-drying process of Polygonatum odoratum, and also provides a reference model and theoretical support for the nano-scale research on the processing mechanism of traditional Chinese medicine.

[0057] Although the present invention has been described in detail above with general descriptions, specific embodiments, and experiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for preparing Polygonatum odoratum carbon nanoparticles, characterized in that, The specific steps are as follows: S1. Weigh out the nine-steamed and nine-dried Polygonatum rhizome, add 5-15 times the weight of the medicinal material to decoct 1-5 times, each time for 30-90 minutes; filter, combine the decoctions, and concentrate to one-third of the original liquid to obtain concentrated liquid. S2. Add 1-5 times the volume of anhydrous ethanol to the concentrate, let stand for 12-36 hours at a low temperature of 2-8℃, and then filter. S3. Take the filtrate and put it into a dialysis bag with a molecular weight cutoff of 300 Da. Dialyze with deionized water for 5-10 days, changing the water several times during the period. After dialysis, filter it with a microporous membrane and freeze-dry the filtrate to obtain the final product.

2. The method for preparing Polygonatum carbon nanoparticles according to claim 1, characterized in that, Step S1 is as follows: Weigh out the nine-steamed and nine-dried Polygonatum, add 8-12 times the weight of the medicinal material to decoct 2-4 times, each time for 40-80 minutes; filter, combine the decoctions, and concentrate to one-third of the original liquid to obtain concentrated liquid.

3. The method for preparing Polygonatum carbon nanoparticles according to claim 2, characterized in that, Step S1 is as follows: Weigh out the nine-steamed and nine-dried Polygonatum, add 10 times the weight of the medicinal material to deionized water and decoct 3 times, 60 minutes each time; filter, combine the decoctions, and concentrate to one-third of the original liquid to obtain concentrated liquid.

4. The method for preparing Polygonatum carbon nanoparticles according to claim 1, characterized in that, Step S2 specifically involves adding 2-4 times the volume of the concentrated liquid in anhydrous ethanol, allowing it to stand at a low temperature of 3-5℃ for 20-28 hours, and then filtering.

5. The method for preparing Polygonatum carbon nanoparticles according to claim 4, characterized in that, Step S2 specifically involves adding anhydrous ethanol at a volume of 3 times that of the concentrated liquid, allowing it to stand at 4°C for 24 hours, and then filtering.

6. The method for preparing Polygonatum carbon nanoparticles according to claim 1, characterized in that, The dialysis time in step S3 is 6-8 days.

7. The method for preparing Polygonatum carbon nanoparticles according to claim 6, characterized in that, The dialysis time in step S3 is 7 days.

8. The method for preparing Polygonatum carbon nanoparticles according to claim 1, characterized in that, The pore size of the microporous filter membrane mentioned in step S3 is 0.22 μm.

9. Polygonatum carbon nanoparticles prepared by the preparation method described in claim 1.

10. The use of Polygonatum carbon nanoparticles as described in claim 9 in the preparation of medicaments for the prevention or treatment of alcoholic liver injury.

Images