A method for preparing selenized Ophiopogon japonicus polysaccharide and its uses

Abstract

This invention discloses a method for preparing selenized Ophiopogon japonicus polysaccharide, its structural characterization, and its applications. The preparation method includes: dissolving Ophiopogon japonicus polysaccharide in dilute nitric acid, adding sodium selenite, reacting at 50–90°C for 4–12 hours, adjusting the pH to neutral, and obtaining selenized Ophiopogon japonicus polysaccharide after dialysis, concentration, and freeze-drying. Structural characterization was performed using ultraviolet spectroscopy, infrared spectroscopy, and scanning electron microscopy, showing that selenium was successfully linked to the polysaccharide molecule in the form of selenite ester. Multiple cell experiments showed that: CCK-8 assay results indicated that the polysaccharide had no significant toxicity to RAW264.7 cells in the concentration range of 25–100 μg / mL; Griess assay and ELISA results showed that the polysaccharide significantly inhibited LPS-induced release of NO, TNF-α, and IL-6 from macrophages in a concentration-dependent manner; GC-MS analysis of the metabolite lactate revealed its anti-inflammatory mechanism at the metabolic level. Animal experiments showed that the selenized Ophiopogon japonicus polysaccharide significantly reduced the inflammatory response in a mouse skin burn model and promoted wound healing. The process of this invention is simple and the conditions are mild. The selenized Ophiopogon japonicus polysaccharide prepared has good anti-inflammatory activity and can be used to prepare drugs for treating skin burns and inflammation.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a method for preparing selenized Ophiopogon japonicus polysaccharide, its structural characterization, and its application in the preparation of anti-inflammatory drugs, particularly drugs for treating skin burns and inflammation. Background Technology

[0002] Burns are a common type of surgical trauma in clinical practice. Patients often experience a series of problems after injury, including local microcirculatory disturbances, increased oxidative stress, inflammation and edema, and secondary infections; in severe cases, it can even lead to shock. While commonly used anti-inflammatory drugs have some effect, long-term use may cause adverse reactions such as liver and kidney toxicity or contact dermatitis. Therefore, finding a naturally derived drug that can exert its effects through multiple pathways, including anti-inflammatory and antioxidant effects, with fewer side effects is of significant clinical importance.

[0003] Ophiopogon japonicus, the dried tuberous root of the plant Ophiopogon japonicus (family Liliaceae), is a traditional Chinese medicine for nourishing Yin. Modern research shows that Ophiopogon japonicus polysaccharides are one of its main active ingredients, possessing various pharmacological effects such as immunomodulation, anti-inflammation, anti-oxidation, and hypoglycemia. However, the biological activity of natural Ophiopogon japonicus polysaccharides is relatively limited, and improving their activity through structural modification is a worthwhile direction to explore.

[0004] Selenium is an essential trace element for the human body, playing a vital role in antioxidation and immune regulation. Inorganic selenium, such as sodium selenite, while highly active, suffers from significant toxicity and a narrow safety window, limiting its direct application. In recent years, the strategy of combining inorganic selenium with polysaccharides to prepare selenized polysaccharides has attracted widespread attention. Selenized polysaccharides possess the dual activities of both selenium and polysaccharides, with significantly reduced toxicity. However, systematic structural characterization of selenized Ophiopogon japonicus polysaccharides and their application in the treatment of skin burns and inflammation have not yet been reported. Summary of the Invention

[0005] The purpose of this invention is to provide a simple and mild method for preparing selenized Ophiopogon japonicus polysaccharide.

[0006] Another object of the present invention is to provide selenized Ophiopogon japonicus polysaccharide prepared by the method and to systematically characterize its structure.

[0007] Another object of the present invention is to provide the use of the selenized Ophiopogon japonicus polysaccharide in the preparation of anti-inflammatory drugs, particularly drugs for treating skin burns and inflammation. Technical solution

[0008] This invention first provides a method for preparing selenized Ophiopogon japonicus polysaccharide, which specifically includes the following steps.

[0009] The first step is to add the extracted and purified Ophiopogon japonicus polysaccharide to a 0.1% to 0.5% nitric acid solution, stir to completely dissolve the polysaccharide, and prepare a sugar solution.

[0010] In the second step, sodium selenite is added at a mass ratio of 1:0.4–1.2 (Ophiopogon japonicus polysaccharide to sodium selenite), and the reaction is carried out at 50–100°C for 4–12 hours. After the reaction is completed, the mixture is cooled to room temperature, and the pH of the reaction solution is adjusted to neutral using sodium bicarbonate solution.

[0011] The third step involves dialyzing the reaction solution using a dialysis bag with a molecular weight cutoff of 3500 for 48–72 hours to remove free selenium and salts. After dialysis, the retained solution is concentrated under reduced pressure (60°C) to 5 mL, and then freeze-dried to obtain selenized Ophiopogon japonicus polysaccharide.

[0012] The present invention also provides a systematic structural characterization method for the above-mentioned selenized Ophiopogon japonicus polysaccharide, including ultraviolet spectroscopy, infrared spectroscopy, molecular weight analysis and scanning electron microscopy.

[0013] This invention further provides a method for evaluating the in vitro anti-inflammatory activity of selenized Ophiopogon japonicus polysaccharide, including the recovery and passage of RAW264.7 macrophages, CCK-8 assay for cell viability, LPS-induced NO release inhibition assay, TNF-α and IL-6 inflammatory factor release inhibition assay, and a method for analyzing cell metabolites based on GC-MS technology.

[0014] This invention further protects the use of the above-mentioned selenized Ophiopogon japonicus polysaccharide in the preparation of anti-inflammatory drugs, especially in the preparation of drugs for treating skin burns and inflammation. Beneficial effects

[0015] Compared with the prior art, the present invention has the following advantages.

[0016] 1. This invention employs the nitric acid-sodium selenite method to selenize Ophiopogon japonicus polysaccharides, clearly defining key parameters such as nitric acid concentration, polysaccharide to sodium selenite ratio, reaction temperature, and time. The process is simple, the conditions are mild, and the reproducibility is good.

[0017] 2. This invention provides a systematic structural characterization of selenized Ophiopogon japonicus polysaccharide, including ultraviolet spectroscopy, infrared spectroscopy, molecular weight determination, and scanning electron microscopy analysis, comprehensively revealing the influence of selenization modification on the structure of Ophiopogon japonicus polysaccharide.

[0018] 3. The prepared selenized Ophiopogon japonicus polysaccharide was confirmed by infrared spectroscopy to contain O-Se=O characteristic bonds.

[0019] 4. Cell experiments showed that selenized Ophiopogon japonicus polysaccharide had no significant toxicity to RAW264.7 cells in the concentration range of 25–100 μg / mL, and could significantly inhibit the release of LPS-induced NO, TNF-α and IL-6 in a concentration-dependent manner.

[0020] 5. Metabolomics analysis based on GC-MS can reveal the anti-inflammatory mechanism of selenized Ophiopogon japonicus polysaccharide at the metabolic level.

[0021] 6. Animal experiments have confirmed that this selenized Ophiopogon japonicus polysaccharide has a clear therapeutic effect on animal models of skin burns. Attached Figure Description

[0022] Figure 1 is a single-factor experimental diagram of the preparation method of selenized Ophiopogon japonicus polysaccharide.

[0023] Figure 2 is a response surface methodology diagram for the preparation of selenium-enriched Ophiopogon japonicus polysaccharide.

[0024] Figure 3 is a comparison of the ultraviolet spectra of crude Ophiopogon japonicus polysaccharide and refined Ophiopogon japonicus polysaccharide.

[0025] Figure 4 is a comparison of the infrared spectra of Ophiopogon japonicus polysaccharide and selenized Ophiopogon japonicus polysaccharide.

[0026] Figure 5 shows the molecular weight spectrum of Ophiopogon japonicus polysaccharides.

[0027] Figure 6 shows the molecular weight spectrum of selenium-enriched Ophiopogon japonicus polysaccharide.

[0028] Figure 7 shows scanning electron microscope images of Ophiopogon japonicus polysaccharide and selenized Ophiopogon japonicus polysaccharide.

[0029] Figure 8 shows the cytotoxic effects of selenium-enriched Ophiopogon japonicus polysaccharide on RAW264.7 cells.

[0030] Figure 9 shows the inhibitory effect of selenized Ophiopogon japonicus polysaccharide on LPS-induced NO release in RAW264.7 cells.

[0031] Figure 10 shows the inhibitory effect of selenized Ophiopogon japonicus polysaccharide on LPS-induced IL-6 release in RAW264.7 cells.

[0032] Figure 11 shows the inhibitory effect of selenized Ophiopogon japonicus polysaccharide on LPS-induced TNF-α release in RAW264.7 cells.

[0033] Figure 12 shows the total ion chromatogram (GC-MS) of the control group cells.

[0034] Figure 13 is a chromatogram (GC-MS) of total metabolic ions in the model group (LPS) cells.

[0035] Figure 14 shows the total ion chromatogram of cell metabolism (GC-MS) in the high-dose administration group.

[0036] Figure 15 shows a photograph of the therapeutic effect of selenized Ophiopogon japonicus polysaccharide on a mouse skin burn model. Detailed Implementation

[0037] The present invention will be further described in detail below with reference to specific embodiments. It should be noted that these embodiments are only for illustrating the present invention and are not intended to limit the present invention.

[0038] Part 1: Optimization of preparation method and structural characterization of selenized Ophiopogon japonicus polysaccharide

[0039] Example 1: Extraction of Ophiopogon japonicus polysaccharides

[0040] Dried Ophiopogon japonicus tuberous roots were pulverized and passed through a 60-mesh sieve. 10 g of Ophiopogon japonicus powder was weighed, added to 200 mL of distilled water, stirred evenly, and soaked for 2 hours. The mixture was ultrasonically extracted at 70℃ for 40 minutes, and the filtrate was collected by filtration. The filtrate was concentrated to an appropriate volume under reduced pressure, centrifuged, and the supernatant was collected. Four times the volume of 95% ethanol was added, and the mixture was allowed to stand overnight at 4℃. The precipitate was collected by centrifugation, washed twice with anhydrous ethanol, and dried under vacuum to obtain crude Ophiopogon japonicus polysaccharide. The crude polysaccharide was deproteinized using the Sevage method (chloroform: n-butanol = 5:1), repeated several times until no white precipitate was observed. The mixture was then dialyzed against distilled water for 48 hours, and freeze-dried to obtain purified Ophiopogon japonicus polysaccharide (hereinafter referred to as OJP).

[0041] Example 2 Preparation of selenized Ophiopogon japonicus polysaccharide

[0042] Weigh 0.1 g of the *Ophiopogon japonicus* polysaccharide prepared in Example 1 and place it in a 50 mL round-bottom flask. Add 10 mL of 0.3% nitric acid solution and heat and stir until the polysaccharide is completely dissolved. Add 0.8 g of sodium selenite and react at 70 °C for 8 hours. After the reaction is complete, cool to room temperature and adjust the pH to 7.0 with 1 mol / L sodium bicarbonate solution. Transfer the reaction solution to a dialysis bag with a molecular weight cutoff of 3500 and dialyze in deionized water for 48 hours, changing the water every 8 hours, until the dialysate shows no red color when tested with ascorbic acid. Collect the liquid in the dialysis bag, concentrate under reduced pressure, and freeze-dry for 48 hours to obtain a white, spongy, selenized *Ophiopogon japonicus* polysaccharide (hereinafter referred to as Se-OJP). The selenium content of this polysaccharide was determined to be 2.1 mg / g by ultraviolet spectroscopy.

[0043] Example 3: Effect of sodium selenite dosage on selenization modification of Ophiopogon japonicus polysaccharides

[0044] Following the method of Example 2, under the conditions of nitric acid concentration of 0.3%, reaction temperature of 70°C, and reaction time of 8 h, the ratio of sodium selenite to OJP was changed to 0.4:1, 0.6:1, 0.8:1, 1:1, and 1.2:1 to prepare selenized Ophiopogon japonicus polysaccharide, and the selenium content and yield of the selenized polysaccharide were determined.

[0045] Example 4: Effect of nitric acid concentration on selenization modification of Ophiopogon japonicus polysaccharides

[0046] Following the method of Example 2, under the conditions of reaction temperature 70℃, reaction time 8 h, and sodium selenite to OJP ratio of 1:1, the nitric acid concentration was changed to 0.1%, 0.2%, 0.3%, 0.4%, and 0.5% to prepare selenized Ophiopogon japonicus polysaccharide, and the selenium content and yield of the selenized polysaccharide were determined.

[0047] Example 5: Effect of reaction temperature on selenization modification of Ophiopogon japonicus polysaccharides

[0048] Following the method of Example 2, under the conditions of sodium selenite to OJP ratio of 1:1, nitric acid concentration of 0.3%, and reaction time of 8 h, the reaction temperature was changed to 50, 60, 70, 80, and 90 °C to prepare selenized Ophiopogon japonicus polysaccharide, and the selenium content and yield of the selenized polysaccharide were determined.

[0049] Example 6: Effect of reaction time on selenization modification of Ophiopogon japonicus polysaccharides

[0050] Following the method of Example 2, under the conditions of sodium selenite to OJP ratio of 0.8:1, reaction temperature of 70°C, and nitric acid concentration of 0.3%, the reaction time was changed to 4, 6, 8, 10, and 12 h to prepare selenized Ophiopogon japonicus polysaccharide, and the selenium content and yield of the selenized polysaccharide were determined.

[0051] Example 7 Response Surface Experiment

[0052] Based on the results of single-factor experiments, response surface methodology (BBD) was employed. Four factors were selected: the ratio of sodium selenite to OJP (A), nitric acid concentration (B), reaction time (C), and reaction temperature (D). The selenium content of selenized Ophiopogon japonicus polysaccharide was used as the response value for process optimization experiments using response surface methodology.

[0053] Table 1. Factors and Levels of Response Surface Design

[0054] Table 2. Response Surface Experimental Design and Results Test No. A B (%) C (h) D (℃) Selenium content (μg / g) 1 1 0 1 0 610 2 0 0 0 0 2080 3 -1 1 0 0 1150 4 0 0 0 0 1950 5 0 0 -1 -1 1065 6 -1 0 -1 0 730 7 0 1 -1 0 1320 8 0 1 0 -1 1320 9 0 1 0 1 1180 10 0 1 1 0 550 11 0 0 1 1 750 12 0 0 1 -1 540 13 -1 -1 0 0 1050 14 1 1 0 0 1230 15 -1 0 0 1 735 16 1 0 -1 0 1760 17 0 -1 0 1 1400 18 0 -1 0 -1 1010 19 0 -1 1 0 950 20 0 0 -1 1 1355 21 -1 0 0 -1 1150 22 1 0 0 -1 1150 23 0 -1 -1 0 1520 24 0 0 0 0 1885 25 1 0 0 1 1420 26 0 0 0 0 2065 27 0 0 0 0 1830 28 -1 0 1 0 880 29 1 -1 0 0 1790

[0055] Table 3 Analysis of Variance Source Sum of Squares df Mean Square F-value P-value Model 5.676E+06 14 4.054E+05 30.58 <0.0001 significant A 4.275E+05 1 4.275E+05 32.24 <0.0001 ** B(%) 78408.33 1 78408.33 5.91 0.0290 * C(h) 1.003E+06 1 1.003E+06 75.68 <0.0001 ** D(℃) 30502.08 1 30502.08 2.30 0.1516 AB 1.089E+05 1 1.089E+05 8.21 0.0125 * AC 4.225E+05 1 4.225E+05 31.86 <0.0001 ** AD 1.173E+05 1 1.173E+05 8.85 0.0100 ** BC 10000.00 1 10000.00 0.7542 0.3998 BD 70225.00 1 70225.00 5.30 0.0373 * CD 1600.00 1 1600.00 0.1207 0.7335 <![CDATA[A 2 ]]> 9.519E+05 1 9.519E+05 71.79 <0.0001 ** <![CDATA[B 2 ]]> 5.129E+05 1 5.129E+05 38.68 <0.0001 ** <![CDATA[C 2 ]]> 2.229E+06 1 2.229E+06 168.11 <0.0001 ** <![CDATA[D 2 ]]> 1.346E+06 1 1.346E+06 101.54 <0.0001 ** Residual 1.856E+05 14 13259.43 Lack of Fit 1.376E+05 10 13760.21 1.15 0.4867 Not significant Pure Error 48030.00 4 12007.50 Cor Total 5.862E+06 28 Note:** P <0.01, the difference is highly significant;* P <0.05, the difference is significant.

[0056] Analysis of response surface methodology and contour lines revealed that the optimal preparation method was as follows: sodium selenite to OJP ratio of 0.8:1, preparation time of 8 hours, temperature of 70℃, and nitric acid concentration of 0.3%.

[0057] Example 8 Ultraviolet Spectroscopy Analysis

[0058] Prepare 4 mg / mL crude and refined Ophiopogon japonicus polysaccharides, use distilled water as a blank, and scan and determine in the range of 200-800 nm.

[0059] The results showed that the absorption peaks of the purified Ophiopogon japonicus polysaccharide at 280 nm and 260 nm were significantly reduced, indicating that the protein had been basically removed.

[0060] Example 9 Infrared Spectroscopy Analysis

[0061] Weigh approximately 2 mg each of dried, constant-weight OJP and Se-OJP, mix and grind them with dried potassium bromide powder, compress them into tablets, and perform infrared spectral scanning in the range of 4000–400 cm⁻¹.

[0062] The results showed that a region located at 3407 cm was observed in OJP. -1 The strong and broad peak at 2939 cm⁻¹ is attributed to the vibrational stretching of OH. -1 The peak at 1641 cm⁻¹ is attributed to the stretching vibration of CH₄, and is caused by the stretching of CH₄ in -CH₂-. -1 The peak at 936 cm⁻¹ is caused by the angular vibration of the water of crystallization or amino NH in OJS. Additionally, the peak at 936 cm⁻¹... -1 The peak at that position corresponds to the stretching vibration of β-glucan. However, the intensity and size of the important characteristic absorption peak of Se-OJP changed; the intensity of the hydroxyl peak decreased in the selenized Ophiopogon japonicus polysaccharide, possibly because the hydroxyl groups in the polysaccharide underwent esterification, forming O-Se=O bonds. This indicates that selenium successfully combined with Ophiopogon japonicus polysaccharide in the form of selenite to form selenized Ophiopogon japonicus polysaccharide, and the selenization modification process did not destroy the main structure of the Ophiopogon japonicus polysaccharide.

[0063] Example 10 Molecular weight determination

[0064] The molecular weights of OJP and Se-OJP were determined using high-performance gel permeation chromatography (HPGPC). The injection conditions were as follows: 1. Column: YMC-Pack Diol-200 (8.0 × 300 mm, 5 μm); 2. Detector: RID differential detector; 3. Mobile phase: 0.7% sodium sulfate solution; 4. Flow rate: 0.7 mL / min; 5. Injection volume: 20 µL; 6. Column temperature: 40℃. A molecular weight standard curve was plotted with the retention time (TR) of the chromatographic peaks of each standard dextran as the x-axis and the logarithm of the standard molecular weight (lg Mw) as the y-axis. The relative molecular weights of OJP and Se-OJP were calculated based on the molecular weight standard curve.

[0065] The results showed that the retention time of OJP was 15.277 minutes, and the weight-average molecular weight was approximately 9.0 × 10⁻⁶ when substituted into the standard curve. 3 Da, with a number-average molecular weight of 6.1 × 10⁻⁶. 3 The chromatographic peaks of Da were symmetrical and single, indicating good purification and good homogeneity of the obtained polysaccharides. The retention time of Se-OJP was 15.248 minutes, and the weight-average molecular weight was approximately 9.3 × 10⁻⁶ when incorporated into the standard curve. 3 Da, with a number-average molecular weight of 6.5 × 10⁻⁶. 3 This indicates that the main structure of Ophiopogon japonicus polysaccharide did not change significantly during the selenization modification process.

[0066] Example 11 Scanning Electron Microscopy Analysis

[0067] Dry OJP and Se-OJP were uniformly dispersed on an aluminum plate and gold-plated under vacuum. After gold sputtering, the samples were scanned under a scanning electron microscope at room temperature with magnifications of 1000x and 200x to observe the surface morphology of the polysaccharides before and after selenization.

[0068] The results showed that OJP had a smoother, more regular surface with an angular structure. Se-OJP, on the other hand, had a rougher surface with dense, spherical aggregates of varying sizes. This change may be due to the significant impact of selenization modification on the higher-order structure of Ophiopogon japonicus polysaccharides, altering their microscopic morphology.

[0069] Part 2: Evaluation of the in vitro anti-inflammatory activity of Se-OJP

[0070] Example 12 Cell Resuscitation and Passaging

[0071] Frozen RAW264.7 macrophages were removed from liquid nitrogen and rapidly thawed in a 37°C water bath. The thawed cells were transferred to centrifuge tubes containing DMEM medium and centrifuged at low speed for 5 minutes. The supernatant was discarded, and 5 mL of fresh complete culture medium (high-glucose DMEM medium containing 10% fetal bovine serum and 1% penicillin and streptomycin) was added. The mixture was gently pipetted and then transferred to T25 cell culture flasks and incubated overnight at 37°C with 5% CO2. The following day, cell growth was observed under a microscope, and fresh medium was added after confirming good cell adhesion. After 2 days of culture, when the cells reached 80% confluence, they were passaged. After three passages, cells in the logarithmic growth phase were used for subsequent experiments.

[0072] Example 13: Investigating the effect of Se-OJP on the viability of RAW264.7 macrophages using the CCK-8 assay.

[0073] RAW264.7 macrophages that had been revived for three generations were observed under a microscope, confirming that the cells were in good condition. The cell concentration was adjusted to 5 × 10⁶ cells / mL using a cell counting method. 4 Cells were cultured overnight in a standard cell culture incubator at a rate of 100 μL per well in a 96-well plate. The following day, the plated cells were divided into a resting control group and a drug group, with three replicates per group. The final drug concentrations were adjusted to 6.25, 12.5, 25, 50, 100, 200, and 500 μg / mL, respectively. After 24 hours of incubation, the culture medium was removed, and 100 μL of complete culture medium containing 10 μL of CCK-8 assay reagent was added to each well. The cells were then incubated for another 2 hours. The absorbance of each well was measured at 450 nm using a microplate reader, and cell viability was calculated using the OD value.

[0074] The results showed that cell viability was significantly reduced at Se-OJP solution concentrations of 200 μg / mL and 500 μg / mL; however, cell viability remained unchanged compared to the control group at concentrations of 50 μg / mL and 100 μg / mL. Therefore, this study selected Se-OJP solutions at concentrations of 25, 50, and 100 μg / mL for subsequent anti-inflammatory activity experiments.

[0075] Example 14: Effect of Se-OJP on LPS-induced NO production in RAW264.7 macrophages

[0076] RAW264.7 macrophages in the logarithmic growth phase were harvested at a concentration of 5 × 10⁻⁶ cells / cell. 4 Cells were seeded per well in 96-well plates and cultured overnight. The following groups were set up: blank control group, LPS model group (1 μg / mL LPS), and Se-OJP low, medium, and high dose groups (25, 50, and 100 μg / mL, with 1 μg / mL LPS added simultaneously). A dexamethasone positive control group (DEX) was also included. Each group had three replicates. After 24 hours of culture, the cell supernatant was collected, and the NO content was measured according to the NO detection kit instructions.

[0077] The results showed that NO release was significantly increased in the LPS model group compared with the blank control group. Compared with the model group, all Se-OJP dosage groups inhibited NO release to varying degrees, showing a clear concentration-dependent effect. At a concentration of 100 μg / mL, the inhibition rate of Se-OJP on NO was significantly increased, and it had a better inhibitory effect compared with the positive control group (DEX).

[0078] Example 15 Effect of Se-OJP on LPS-induced TNF-α release from RAW264.7 macrophages

[0079] RAW264.7 macrophages in the logarithmic growth phase were harvested at a concentration of 5 × 10⁻⁶ cells / cell.4 Cells were seeded per well in 96-well plates and cultured overnight. The following groups were set up: blank control group, LPS model group (1 μg / mL LPS), and low, medium, and high dose Se-OJP groups (25, 50, and 100 μg / mL, with 1 μg / mL LPS added simultaneously). A positive control group (DEX) was also set up. After 24 hours of culture, the cell supernatant was collected, and the TNF-α content was measured according to the TNF-α detection kit.

[0080] The results showed that LPS treatment significantly increased TNF-α secretion in the cell culture supernatant of RAW264.7 macrophages. Compared with the model group, the positive control group (DEX) significantly reduced TNF-α levels. The TNF-α levels in the culture supernatant of RAW264.7 macrophages treated with low, medium, and high concentrations of Se-OJP were significantly lower than those in the model group, showing a good concentration-dependent relationship. These results indicate that Se-OJP can inhibit LPS-induced TNF-α release from RAW264.7 macrophages.

[0081] Example 16: Effect of Se-OJP on LPS-induced IL-6 release from RAW264.7 macrophages

[0082] RAW264.7 macrophages in the logarithmic growth phase were harvested at a concentration of 5 × 10⁻⁶ cells / cell. 4 Cells were seeded per well in 96-well plates and cultured overnight. The following groups were set up: blank control group, LPS model group (1 μg / mL LPS), and low, medium, and high dose Se-OJP groups (25, 50, and 100 μg / mL, with 1 μg / mL LPS added simultaneously). A positive control group (DEX) was also set up. After 24 hours of culture, the cell supernatant was collected, and the IL-6 content was measured according to the IL-6 detection kit.

[0083] The results showed that LPS treatment significantly increased IL-6 secretion in the cell culture supernatant of RAW264.7 macrophages. Compared with the model group, the positive control group (DEX) significantly reduced IL-6 levels. The IL-6 levels in the culture supernatant of RAW264.7 macrophages treated with low, medium, and high concentrations of Se-OJP were significantly lower than those in the model group, showing a good concentration-dependent relationship. These results indicate that Se-OJP can inhibit LPS-induced IL-6 release from RAW264.7 macrophages.

[0084] Part 3: GC-MS-based analysis of lactate, a cellular metabolite

[0085] Example 17 GC-MS Analysis Conditions and Sample Preparation

[0086] Metabolite experiments were conducted using a blank control group, an LPS model group, and a high-dose Se-OJP group (100 μg / mL), with six samples repeated for each group.

[0087] Sample pretreatment: Collect the cell pellet, add an appropriate amount of extraction buffer, vortex to mix, and centrifuge to collect the supernatant. After drying the supernatant with nitrogen, add n-decanoic acid as an internal standard, and then add N,O-bis(trimethylsilyl)trifluoroacetamide for derivatization. After derivatization, store at 4°C for analysis.

[0088] Detection conditions: Column: Elite-5MS (30 m × 250 μm, 0.25 μm. PerkinElmer, USA); Column temperature program: Initial temperature 50℃, hold for 2 min, increase to 280℃ at 10℃ / min, hold for 5 min. Inlet temperature 280℃; Injection volume 1.0 μL; Full injection; Carrier gas: High-purity helium, constant flow rate 1.0 mL / min. Ion source temperature 300℃, interface temperature 280℃, collision energy 70 eV, full scan mode, mass range (m / z): 50~650, solvent removal time 4 min.

[0089] The samples were tested according to the detection conditions. Decanoic acid was used as an internal standard, and the peak area ratio of the metabolite lactic acid to the internal standard was used as the lactic acid expression level. By comparing the differences in lactic acid among the groups, the anti-inflammatory mechanism of Se-OJP was revealed at the metabolic level.

[0090] The results showed that when macrophages transformed into the M2 anti-inflammatory type, they relied more on oxidative phosphorylation and fatty acid oxidation, with relatively weak glycolysis. The production of lactate as a metabolite was usually reduced. Therefore, compared with the blank control group, the lactate content in both the high-dose Se-OJP group and the LPS model group changed significantly. Compared with the LPS model group, the lactate content in the high-dose Se-OJP group was further reduced, indicating that the anti-inflammatory effect was enhanced and the intracellular lactate level tended to decrease. Macrophages showed anti-inflammatory activity after administration.

[0091] Table 4. Differences in the metabolite lactic acid Group lactate expression level Significance (P<0.01) Trend of change Blank control group 0.48±0.01 / / LPS model group 0.23±0.03 ** decline Se-OJP high-dose group 0.19±0.01 ** decline Note: In the table, "**" indicates that P < 0.01, and the difference is significant.

[0092] Part 4: Therapeutic effect of Se-OJP on skin scald inflammation in mice

[0093] Example 18: Treatment Experiment of a Mouse Skin Burn Model

[0094] Thirty SPF-grade Kunming mice, weighing 35–40 g, were randomly divided into three groups: a model control group, a positive control group (dexamethasone, 0.5 mg / kg), and a Se-OJP group (100 μg / mL), with 10 mice in each group. Hair was removed from the backs of the mice. The following day, a metal rod heated to 80°C was used to contact the back skin for 10 seconds, creating a second-degree burn model with a diameter of approximately 1 cm. Drug administration began 24 hours after the burn. The model control group received an equal volume of physiological saline, the positive control group received a subcutaneous injection of dexamethasone, and the Se-OJP group received a subcutaneous injection of 50 μg / mL Se-OJP solution, once daily for 14 consecutive days. Wound healing was observed daily, and photographs were taken on days 3, 7, 10, and 14.

[0095] The results showed that the control group mice exhibited significant redness and swelling of the wound, with considerable exudate remaining on day 7, and incomplete wound healing by day 14. Compared to the control group, the Se-OJP group mice showed significantly reduced redness and swelling of the wound, with scab formation beginning on day 7, a significantly improved wound healing rate by day 10 (P < 0.01), and near-complete wound healing by day 14. These results indicate that Se-OJP has a significant therapeutic effect on burn inflammation in mice. Summarize

[0096] This invention successfully prepared selenized Ophiopogon japonicus polysaccharide via the nitrate-sodium selenite method. Systematic structural characterization using ultraviolet spectroscopy, infrared spectroscopy, and scanning electron microscopy confirmed that selenium was successfully linked to the OJP molecule in the form of selenite ester, and that the selenization modification preserved the triple helix and branched structure of the polysaccharide.

[0097] Cytotoxicity assays showed that Se-OJP had no significant toxicity to RAW264.7 cells within the concentration range of 25–100 μg / mL. In vitro anti-inflammatory assays showed that this polysaccharide significantly inhibited LPS-induced release of NO, TNF-α, and IL-6 in a concentration-dependent manner, and at a concentration of 100 μg / mL, its inhibitory effect on NO was superior to that of the positive control dexamethasone.

[0098] Analysis of the metabolite lactate by GC-MS can further reveal its anti-inflammatory mechanism at the metabolic level.

[0099] Animal experiments have shown that Se-OJP can reduce the inflammatory response of mice after skin burns and promote wound healing.

[0100] In summary, the Se-OJP prepared by this invention has good anti-inflammatory activity and has the potential to be developed into a drug for treating skin burns and inflammation.

[0101] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing selenium-enriched Ophiopogon japonicus polysaccharide, characterized in that, Includes the following steps: Step 1: Weigh an appropriate amount of the extracted and purified Ophiopogon japonicus polysaccharide and place it in a container. Add a certain concentration of nitric acid solution and stir to dissolve it, preparing a sugar solution with a mass-to-volume ratio of 1-5 mg / mL. Step 2: Add Na₂SeO₃ according to the mass ratio, control the reaction temperature at 50-90℃, and the reaction time at 4-12 hours. After the reaction is complete, cool to room temperature and add NaHCO₃ solution to adjust the pH of the reaction solution. Step 3: After sequentially dialysis, vacuum concentration, and freeze-drying, obtain selenized Ophiopogon japonicus polysaccharide.

2. The preparation method according to claim 1, characterized in that, The concentration of the nitric acid solution mentioned in step one is 0.1% to 0.5% (v / v).

3. The preparation method according to claim 1, characterized in that, In step two, the mass ratio of Ophiopogon japonicus polysaccharide to sodium selenite is 1:0.4 to 1.

2.

4. The preparation method according to claim 1, characterized in that, In step two, the reaction temperature is 50–90℃ and the reaction time is 4–12 hours.

5. The preparation method according to claim 1, characterized in that, In step two, the pH is adjusted to 6.5–7.5 using a sodium bicarbonate solution (5.6%).

6. The preparation method according to claim 1, characterized in that, In step three, a semi-permeable membrane is used as the dialysis material. Dialysis is performed at room temperature for 48 hours, with the water changed every 8 hours until the dialysate shows no red color when tested with ascorbic acid.

7. The preparation method according to claim 1, characterized in that, In step three, the temperature for vacuum concentration is 60°C. The retained liquid is concentrated to half of its original volume. The concentrated liquid is then dried under vacuum to obtain a white solid powder.

8. A selenium-enriched Ophiopogon japonicus polysaccharide, characterized in that, It is prepared by the method described in any one of claims 1 to 7.

9. The application of selenized Ophiopogon japonicus polysaccharide according to claim 8 in the preparation of anti-inflammatory drugs, characterized in that, The anti-inflammatory drug is used to treat inflammation caused by skin burns.

Images