Lycium barbarum polysaccharide spray as well as preparation method and application thereof
By preparing a Lycium barbarum polysaccharide-gelatin microsphere spray, the problem of inconvenient local oral administration of Lycium barbarum polysaccharide was solved, achieving continuous and targeted drug release and improving the therapeutic effect.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- UNIV OF ELECTRONICS SCI & TECH OF CHINA
- Filing Date
- 2026-04-09
- Publication Date
- 2026-05-19
AI Technical Summary
Existing methods of administering Lycium barbarum polysaccharides are inconvenient, have low comfort levels, are difficult to maintain effective concentrations in the oral cavity, have poor patient compliance and bioavailability, and cannot achieve sustained and targeted protective effects.
Using partially oxidized dialdehyde polysaccharide as a matrix, wolfberry polysaccharide-gelatin microspheres were prepared, and wolfberry polysaccharide spray was prepared by emulsion template method to achieve uniform dispersion and stable release of wolfberry polysaccharide in the oral cavity.
The wolfberry polysaccharide spray can adhere closely to the oral mucosa, continuously and controllably release the active pharmaceutical ingredients, prolong the local action time, improve the therapeutic effect, and enhance the targeted delivery efficiency.
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Figure CN122056834A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedical technology, specifically relating to a wolfberry polysaccharide spray, its preparation method and application. Background Technology
[0002] The smoke produced by burning tobacco contains thousands of harmful and carcinogenic substances, including polycyclic aromatic hydrocarbons, benzene, and aldehydes. Nicotine, as the most significant toxic component, has a wide range of negative impacts on oral health, with damage to oral soft tissues being particularly prominent. Long-term smoking exposes the oral mucosa to continuous high temperatures and chemical toxins, leading to a state of chronic stress. This not only directly burns the mucosal tissue but also inhibits the self-repair and regeneration capabilities of oral soft tissues, delaying the healing process of oral wounds. Simultaneously, smoking exacerbates periodontal inflammation, reduces local resistance, and makes the tissues more susceptible to bacterial and other pathogenic invasion, thus significantly increasing the risk of periodontal disease.
[0003] Lycium barbarum polysaccharides are active ingredients obtained from Lycium barbarum through isolation, extraction, and purification. Further purification yields Lycium barbarum glycopeptides, which possess significant immunomodulatory, anti-inflammatory, and antioxidant activities. During cellular oxidative damage repair, Lycium barbarum polysaccharides can alleviate oxidative stress damage by reducing the number of free radicals and enhancing the activity of antioxidant enzymes. In vitro experiments have confirmed that Lycium barbarum polysaccharides can effectively scavenge superoxide anions, DPPH free radicals, and hydroxyl free radicals, and inhibit the excessive generation of reactive oxygen species (ROS) under inflammatory conditions. At the molecular level, Lycium barbarum glycopeptides can effectively control inflammatory responses by regulating signaling pathways such as Toll-like receptor 4, NF-κB, and JNK MAPK, inhibiting the release of pro-inflammatory mediators such as TNF-α, IL-1β, IL-6, and iNOS from immune cells, while upregulating the expression of the anti-inflammatory factor IL-10. Furthermore, Lycium barbarum glycopeptides can also exert cell protection and oxidative damage repair effects by scavenging free radicals and enhancing the expression and activity of antioxidant enzymes. The applicant's previous research has also demonstrated that Lycium barbarum glycopeptides have a protective effect on periodontal ligament cells and keratinocytes, and can promote their proliferation and differentiation. In summary, Lycium barbarum polysaccharides have good biocompatibility, immunomodulatory capacity, and antioxidant properties, making them potentially valuable in the field of oral biomedicine.
[0004] However, there are still obvious limitations when using wolfberry polysaccharides directly to prevent or treat oral injuries: conventional forms of administration (such as oral liquids, mouthwashes, etc.) are not easy to maintain an effective concentration in the oral cavity, are inconvenient to use, and have low patient compliance and comfort, making it difficult to achieve a continuous and targeted protective effect.
[0005] Therefore, providing a Lycium barbarum polysaccharide delivery system that is convenient for local oral administration, has good patient compliance and bioavailability, and can stably release active ingredients and target them to oral soft tissues has become a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention
[0006] One of the objectives of this invention is to provide a wolfberry polysaccharide spray that solves the problems of inconvenient administration, low comfort, and limited effect in the use of wolfberry polysaccharides in the prior art.
[0007] The second objective of this invention is to provide a method for preparing the wolfberry polysaccharide spray.
[0008] A third objective of this invention is to provide the application of the wolfberry polysaccharide spray.
[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect of this invention discloses a wolfberry polysaccharide spray, which includes a partially oxidized dialdehyde polysaccharide aqueous solution and wolfberry polysaccharide-gelatin microspheres dispersed therein; The wolfberry polysaccharide-gelatin microspheres use cross-linked gelatin as a matrix and uniformly encapsulate wolfberry polysaccharides inside; The mass ratio of partially oxidized dialdehyde polysaccharide, gelatin, and wolfberry polysaccharide is 1~3:0.5~5:0.1~0.5; The concentration of the partially oxidized dialdehyde polysaccharide aqueous solution is 0.1–2 g / mL.
[0010] In some embodiments of the present invention, the partially oxidized dialdehyde polysaccharide is prepared by specific oxidation with sodium periodate, including at least one of dialdehyde sodium alginate, dialdehyde hyaluronic acid, dialdehyde starch, dialdehyde cellulose and dialdehyde chitosan; its degree of oxidation is 10%-80%.
[0011] The second aspect of this invention discloses a method for preparing the above-mentioned wolfberry polysaccharide spray, which includes the following steps: preparing wolfberry polysaccharide-gelatin microspheres using an emulsion template method, and then dispersing the wolfberry polysaccharide-gelatin microspheres in a partially oxidized dialdehyde polysaccharide solution to obtain the wolfberry polysaccharide spray.
[0012] In some preferred embodiments of the present invention, when dispersing Lycium barbarum polysaccharide-gelatin microspheres in a partially oxidized dialdehyde polysaccharide solution, the stirring speed is controlled at 100-300 rpm and the stirring time is 15-30 minutes to ensure that the microspheres are uniformly dispersed without aggregation. In some embodiments of the present invention, the preparation of Lycium barbarum polysaccharide-gelatin microspheres using the emulsion template method specifically includes the following steps: Lycium barbarum polysaccharide and gelatin were dissolved together in water to form an aqueous phase; the oil phase material was mixed with a surfactant to form an oil phase; the two phases were mixed and emulsified, and then a crosslinking agent was added for crosslinking; after the reaction was completed, the precipitate was collected by solid-liquid separation to obtain gelatin microspheres loaded with Lycium barbarum polysaccharide.
[0013] In some embodiments of the present invention, the oil phase material comprises liquid paraffin; Or / and the surfactant includes at least one of Span-80, Span-20, Span-40, Span-60, Span-85, polyglycerol monostearate, and polyglycerol-3-diisostearate; Or / and the crosslinking agent includes at least one of genipin, gallic acid, gallic acid epoxide, tea polyphenols, fucoidan, polysaccharide crosslinking agents, tannic acid, and trehalose.
[0014] In some embodiments of the present invention, the concentration of gelatin in the aqueous phase is 40-60 wt%; The volume ratio of oil phase material to surfactant is 6~25:1; The volume ratio of the aqueous phase to the oil phase is 5~7:21~27; The amount of crosslinking agent used is 0.1~0.5% of the gelatin mass.
[0015] In some embodiments of the present invention, the two phases are mixed and stirred at 30-40°C for 20-50 minutes to obtain an emulsion; then a crosslinking agent is added and stirring continues.
[0016] The third aspect of this invention discloses the use of the above-mentioned wolfberry polysaccharide spray in the preparation of a medicament for preventing and / oral tissue damage.
[0017] In some embodiments of the present invention, the oral tissue damage includes oral tissue damage caused by tobacco.
[0018] In some embodiments of the present invention, the oral tissue damage includes oral diseases related to immune regulation.
[0019] Preferably, the oral diseases related to immune regulation include at least one of periodontitis, peri-implantitis, oral mucositis, or oral ulcers.
[0020] The wolfberry polysaccharide described in this invention is the glycopeptide component of wolfberry polysaccharide.
[0021] Compared with the prior art, the present invention has the following beneficial effects: This invention utilizes modern medical technology to apply the active ingredient of traditional Chinese medicine, wolfberry polysaccharide, to the field of oral medicine, providing a novel solution with significant clinical value for the prevention and treatment of oral soft tissue injuries, especially those caused by tobacco.
[0022] Specifically, Lycium barbarum polysaccharides can effectively protect periodontal ligament cells and keratinocytes and promote their proliferation and differentiation, thereby directly exerting pharmacological effects on tissue damage caused by tobacco. Simultaneously, a spray formulation based on partially oxidized dialdehyde polysaccharides achieves uniform dispersion of Lycium barbarum polysaccharide-loaded microsphere gelatin through a Schiff base reaction, forming a stable suspension system. This system, with its excellent adhesion, can closely adhere to the oral mucosa or periodontal tissues, and continuously and controllably releases the active pharmaceutical ingredients during degradation. This not only significantly prolongs the drug's retention time in the local area of action but also further enhances the overall therapeutic effect by improving targeted delivery efficiency. Attached Figure Description
[0023] Appendix Figure 1 This is a process flow diagram of the present invention.
[0024] Appendix Figure 2 The FITR spectra of sodium alginate (SA) and dialdehyde sodium alginate (OSA) are shown; the horizontal axis is Wavelength (cm). 1 ) represents the wave number (cm) 1 The vertical axis T (%) represents the light transmittance (%).
[0025] Appendix Figure 3 The images show fluorescence micrographs of gelatin microspheres and statistical analysis results of their particle size. The left image shows the fluorescence micrograph of the gelatin microspheres, while the right image shows the statistical analysis results of their particle size. The vertical axis "Frequency" represents the frequency, i.e., the number of particles.
[0026] Appendix Figure 4 The figures show the standard curve of wolfberry polysaccharide and the release curve of wolfberry polysaccharide spray; the left figure is the standard curve of wolfberry polysaccharide, and the right figure is the release curve of wolfberry polysaccharide spray.
[0027] Appendix Figure 5 The graph shows the CCK-8 results for different concentrations of CSE.
[0028] Appendix Figure 6 The CCK-8 results for the biocompatibility test of the wolfberry polysaccharide spray are shown in the figure.
[0029] Appendix Figure 7 The images show fluorescent staining of live and dead cells. The labels in the figures indicate: 1: blank control group, 2: CSE treatment group, 3: sodium alginate pretreatment group, 4: LbGP-OSA spray pretreatment group, a and c: live cell staining (green indicates live cells); b and d: dead cell staining (red indicates dead cells).
[0030] Appendix Figure 8Microscopic images of the scratch test.
[0031] Appendix Figure 9 This is a statistical chart showing the area of scratch healing.
[0032] Appendix Figure 10 The images show ROS immunofluorescence staining. The labels in the figures indicate: 1: blank control group, 2: CSE group, 3: sodium alginate pretreatment group, 4: LbGP-OSA spray pretreatment group, a: ROS immunofluorescence staining 12 hours after CSE induction, b: live cell staining 24 hours after CSE induction.
[0033] Appendix Figure 11 The figure shows the statistical results of ROS immunofluorescence staining.
[0034] Appendix Figure 12 This is a graph showing the results of a GSH test.
[0035] Appendix Figure 13 Diagrams showing ulcer models induced by different CSE concentrations.
[0036] Appendix Figure 14 The figures show the experimental results of the effect of wolfberry polysaccharide spray on ulcer healing. Figure A is a gross morphological observation (circles mark the ulcer areas), and Figure B is a bar chart of quantitative analysis of ulcer area in each group.
[0037] Appendix Figure 15 Figure 1 shows the pathological morphology of ulcer tissue and the detection results of collagen deposition in different treatment groups of Experiment Example 3; Figure A is the pathological staining image of ulcer tissue, and the inset is a magnified view of a local area; Figure B is a bar chart of quantitative analysis of collagen deposition rate in ulcer tissue of each group. Detailed Implementation
[0038] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.
[0039] The wolfberry polysaccharide described in this embodiment of the invention was provided by the Ningxia Zhongning Wolfberry (Tianren) Academician Workstation, with a purity ≥90% (determined by high performance liquid chromatography).
[0040] The GSH reagent kit described in this embodiment of the invention is a reduced glutathione (GSH) content detection kit provided by Beijing Solarbio Science & Technology Co., Ltd.
[0041] Example 1 This embodiment discloses a method for preparing the wolfberry polysaccharide spray of the present invention, the process flow of which is shown in the attached figure. Figure 1As shown, the specific steps are as follows: 650 mg of wolfberry polysaccharide was dissolved in 30 ml of 40 wt% gelatin aqueous solution to obtain the aqueous phase; Mix 25 ml of liquid paraffin with 1 ml of surfactant and stir to obtain the oil phase; Take 5 ml of the aqueous phase and add it dropwise to the oil phase. Stir at 30°C for 25 min to obtain an emulsion. Then, add the crosslinking agent genipin (19.2 mg, 0.16 wt% based on the mass of gelatin in the aqueous phase) to the emulsion and continue stirring for 45 min. Centrifuge, collect the precipitate, and wash three times with anhydrous ethanol. Then, disperse the precipitate in 10 mL of a partially oxidized dialdehyde sodium alginate solution with a concentration of 1 g / mL to obtain the wolfberry polysaccharide spray. The partially oxidized dialdehyde sodium alginate in this embodiment has an oxidation degree of 29.17 ± 2.46%.
[0042] The preparation method of partially oxidized dialdehyde sodium alginate in this embodiment is as follows: 5g of sodium alginate and 1.75g of sodium periodate are dissolved in 50ml of deionized water, the pH is adjusted to 3, and the mixture is stirred in the dark for 4 hours. Then, the mixture is purified by dialysis and freeze-dried to obtain partially oxidized dialdehyde sodium alginate with an oxidation degree of 29.17±2.46%.
[0043] Example 2 This embodiment discloses a method for preparing the wolfberry polysaccharide spray of the present invention, the process flow of which is shown in the attached figure. Figure 1 As shown, the specific steps are as follows: 900 mg of wolfberry polysaccharide was dissolved in 30 ml of 60 wt% gelatin aqueous solution to obtain the aqueous phase; Mix 20 ml of liquid paraffin with 2 ml of surfactant and stir to obtain the oil phase; Take 7 ml of the aqueous phase and add it dropwise to the oil phase. Stir at 40°C for 35 min to obtain an emulsion. Then add the crosslinking agent genipin (28.8 mg, based on the mass of gelatin in the aqueous phase, the addition ratio is 0.16 wt%) to the emulsion and continue stirring for 45 min. Centrifuge, collect the precipitate, and wash it three times with anhydrous ethanol. Then disperse the precipitate in 15 mL of a partially oxidized dialdehyde polysaccharide solution with a concentration of 1 g / mL to obtain the wolfberry polysaccharide spray.
[0044] The partially oxidized dialdehyde polysaccharide in this embodiment is dialdehyde hyaluronic acid with an oxidation degree of 80%.
[0045] Example 3 This embodiment discloses a method for preparing the wolfberry polysaccharide spray of the present invention, the process flow of which is shown in the attached figure. Figure 1 As shown, the specific steps are as follows: 1400 mg of wolfberry polysaccharide was dissolved in 30 ml of 50 wt% gelatin aqueous solution to obtain the aqueous phase; 18 ml of liquid paraffin was mixed with 3 ml of surfactant and stirred to obtain the oil phase; Take 6 ml of the aqueous phase and add it dropwise to the oil phase. Stir at 30°C for 35 min to obtain an emulsion. Then add the crosslinking agent genipin (24 mg, based on the mass of gelatin in the aqueous phase, the addition ratio is 0.16 wt%) to the emulsion and continue stirring for 45 min. Centrifuge, collect the precipitate, and wash it three times with anhydrous ethanol. Then disperse the precipitate in 12 mL of a partially oxidized dialdehyde polysaccharide solution with a concentration of 1 g / mL to obtain the wolfberry polysaccharide spray.
[0046] The partially oxidized dialdehyde polysaccharide in this embodiment is a dialdehyde chitosan with an oxidation degree of 50%.
[0047] Experimental Example 1 This test example examines the physicochemical properties of the wolfberry glycopeptide spray of Example 1 of the present invention.
[0048] 1. Characterization of partially oxidized sodium alginate (OSA) The Fourier transform infrared (FTIR) characterization results of the partially oxidized sodium alginate in Example 1 of this invention are as follows: Figure 2 As shown: Partially oxidized sodium alginate (OSA) at 1721.64 cm⁻¹ -1 The presence of a distinct aldehyde characteristic absorption peak indicates that sodium periodate has successfully oxidized sodium alginate.
[0049] Furthermore, the aldehyde content of OSA was determined to be 29.17±2.46% by the alkali consumption method, further confirming its good cross-linking ability.
[0050] The wolfberry glycopeptide spray in this experiment was prepared according to the method in Example 1.
[0051] 2. Morphological observation The particle size distribution of gelatin microspheres in wolfberry glycopeptide spray was determined using a laser particle size analyzer, and the morphology and structure of the microspheres were observed using a fluorescence microscope.
[0052] Laser particle size analyzer measurement results are as follows Figure 3 As shown in the right figure: the gelatin microspheres have a uniform particle size distribution, with an average diameter of 13.3 ± 2.3 μm; under a fluorescence microscope, the microspheres show complete morphology and clear outlines. Figure 3 (Left image).
[0053] 3. Stability Test The wolfberry glycopeptide spray sample was sealed and stored in a constant temperature and humidity chamber in the dark. The color, transparency, and precipitation or stratification of the sample were observed at set target times. The pH value was measured with a precision pH meter, the morphology was observed and analyzed using a fluorescence microscope, and its rheological properties were tested using a rotational rheometer.
[0054] The results showed that after storage at 25℃ and 60% relative humidity for one month, the appearance, pH value (6.7±0.2), and microstructure of the wolfberry polysaccharide spray of the present invention remained stable. Its viscosity remained within the range of 6.4-6.56 cP, and the viscosity and shear stress remained essentially constant at a constant shear rate, with viscosity fluctuations of only 2.5% during this period, indicating that the sample has good physical stability.
[0055] 4. Drug release performance and mucosal adhesion Four experimental groups were set up: ① LbGP-OSA gelatin microsphere spray group: wolfberry glycopeptide spray prepared according to the method of Example 1.
[0056] ② LbGP-OSA solution spray group: Lycium barbarum polysaccharide (LbGP) is directly dissolved in partially oxidized sodium alginate (OSA) solution to prepare the solution (without microsphere carrier); the concentrations of Lycium barbarum polysaccharide and oxidized sodium alginate are the same as those of the LbGP-OSA gelatin microsphere spray group.
[0057] ③ LbGP solution spray group: LbGP is dissolved in pure water; the concentration of LbGP is the same as that of LbGP-OSA gelatin microsphere spray group.
[0058] ④ OSA solution control group: blank OSA solution (without drug loading), the degree of OSA oxidation and concentration were consistent with the LbGP-OSA gelatin microsphere spray group.
[0059] 4.1 Drug release performance evaluation One mL of sample was placed into a dialysis bag with a molecular weight cutoff of 3.5 kDa and immersed in 10 mL of PBS (pH 6.8) release medium at 37°C to simulate the intraoral environment. One mL samples were taken at preset time points (0, 1, 2, 4, 6, 8, 12, and 24 h), and an equal volume of fresh release medium was immediately added to maintain the leak conditions. The LbGP concentration was measured using a UV spectrophotometer (λ=280 nm), and the cumulative drug release rate was calculated based on the standard curve. Q t (%,%), the formula is as follows: (1); In formula (1), C t The concentration at time t is expressed in mg / ml. V total The total volume of the released medium (10 mL); V sample The volume of a single sample (1 mL); W initial Initial drug loading, in mg / ml; Standard curve such as Figure 4 As shown in the left figure, the in vitro drug release curve is as follows: Figure 4 As shown in the right figure, the results indicate that LbGP in the LbGP-OSA gelatin microsphere spray is released rapidly in the initial half hour, with a cumulative release of less than 40%, and a cumulative release rate of 83.7% ± 3.2% after 3 hours. In contrast, both the LbGP-OSA spray and the LbGP spray reach release equilibrium at approximately 1 hour. The LbGP-OSA gelatin microsphere spray exhibits significant sustained-release characteristics, with a more prolonged and controllable drug release behavior, demonstrating a significant advantage over ordinary sprays.
[0060] 4.2 Mucosal adhesion performance evaluation 0.5 mL of sample was evenly spread on the isolated porcine oral mucosa (area 2 cm²). 2 After standing for 2 minutes, rinse gently 3 times with 5 mL of physiological saline (37℃) and collect the rinsing fluid.
[0061] The residual amount of LbGP in the rinsing solution was determined by ultraviolet spectrophotometry, and the mucosal retention rate on the mucosal surface was measured. A retention The adhesion performance was evaluated using the formula: (%) (2); In formula (2), W washed The amount of medicine in the rinsing solution is expressed in mg / ml. W initial Initial drug loading, in mg / ml; Mucosal adhesion experiments showed that LbGP-OSA gelatin microsphere spray exhibited the best adhesion performance, with a mucosal retention rate >80%.
[0062] Experimental Example 2: In vitro biological detection and mechanism exploration of wolfberry glycopeptide spray formulation 1. Preparation of Cigarette Smoke Extract (CSE) The smoke from 10 cigarettes was completely passed into 10 mL of physiological saline, and after filtration, a CSE stock solution with an initial concentration of 100% was obtained. The absorbance (OD) was then measured at 320 nm using a UV spectrophotometer. The concentration corresponding to OD = 17.5 was defined as 100% CSE, and subsequent dilutions were performed based on this.
[0063] Tobacco smoke extract was diluted to the required concentration by volume fraction using serum-free complete culture medium for cell intervention. Gingival fibroblasts (HGF) cultured to the logarithmic growth phase were co-cultured with different concentrations of CSE (0%, 5%, 10%, 15%, 20%, 25%, 50%, 100%) for 24, 48, and 72 hours, with replicates at each concentration and time point. After culture, cell viability was assessed using the CCK-8 assay to determine the optimal experimental concentration of CSE.
[0064] The results are as follows Figure 5 As shown: Cell viability was detected using the CCK-8 assay, and the half-maximal inhibitory concentration (IC50) was calculated. 50 After treatment with 20% CSE for 48 hours, cell viability decreased to 48.2 ± 6.8% (P < 0.01), which is close to the half-maximal inhibitory concentration (IC50). 50 Therefore, it was determined as the intervention concentration for subsequent experiments.
[0065] 2. Biocompatibility testing of Lycium barbarum polysaccharide spray This experiment aimed to evaluate the in vitro biocompatibility of the Lycium barbarum polysaccharide spray of the present invention and its protective effect against human gingival fibroblast (HGF) toxicity induced by cigarette smoke extract (CSE). The experiment was divided into four groups: (1) Blank control group (Control); (2) CSE treatment group (CSE); using 20% CSE prepared according to the section "1. Preparation of Cigarette Smoke Extract (CSE)" in this test example; (3) Sodium alginate pretreatment group (OSA): The partially oxidized sodium alginate solution with a concentration of 1 g / mL as in Example 1 was used; (4) LbGP-OSA spray pretreatment group (LbGP-OSA): using the wolfberry polysaccharide spray prepared according to the method of Example 1.
[0066] HGF cells were administered at a rate of 3 × 10 3Cells were seeded at a density of [number] cells / ml in culture plates and cultured at 37°C and 5% CO2 until adherence. Afterward, each group underwent the following treatments: the blank control group continued culture in serum-free complete medium; the CSE-treated group received medium containing 20% CSE; the sodium alginate pretreatment group and the LbGP-OSA spray pretreatment group had Transwell chambers containing the corresponding material (sodium alginate solution or LbGP-OSA spray) placed above the cell culture wells for co-culture pretreatment for 24 hours. After pretreatment, the Transwell chambers and original medium in these two pretreatment groups were removed, and the medium was replaced with medium containing 20% CSE along with the CSE-treated group. Culture was terminated for all groups 12 and 24 hours after CSE treatment. Cell viability was assessed using the CCK-8 assay, and morphological observation was performed using live / dead cell fluorescence staining to comprehensively evaluate cytotoxicity and the protective effect of the material.
[0067] Cell viability results as follows Figure 6 As shown in the figure, the cell survival rate of each group was close to 100% at 0h, and the material itself was non-toxic. After CSE treatment, the survival rate of the CSE group decreased significantly, the protection of the OSA group was limited, and the survival rate of the LbGP-OSA group was always close to that of the blank control group.
[0068] Cell morphology and viability results are as follows Figure 7 As shown in the figure, the cells in the blank control group are normal spindle-shaped with few dead cells; the cells in the CSE group have abnormal morphology and many dead cells; the protective effect of the OSA group is average; the cell morphology of the LbGP-OSA group is no different from that of the blank control group, and there are very few dead cells.
[0069] The above results indicate that 20% cigarette smoke extract (CSE) is significantly toxic to human gingival fibroblasts (HGF), leading to a sharp decrease in cell survival, abnormal morphology (loss of spindle structure), and an increase in dead cells. Pretreatment with partially oxidized sodium alginate (OSA) alone can only slightly alleviate this toxicity, with limited protective effect. In contrast, Lycium barbarum polysaccharide spray (LbGP-OSA) not only has good in vitro biocompatibility but also significantly antagonizes the toxic effects of CSE, restoring the survival rate of HGF after CSE exposure to the level of the blank control group. Furthermore, the cells maintain normal spindle morphology, proliferate and increase in density over time, and the number of dead cells is extremely small. Its protective effect against HGF damage caused by CSE is significantly better than that of sodium alginate alone.
[0070] 3. Scratch assay to alleviate the inhibition of HGFs cell migration by tobacco using wolfberry polysaccharide spray. This experiment aimed to investigate the alleviating effect of the Lycium barbarum polysaccharide spray of the present invention on the inhibition of human gingival fibroblast (HGF) migration ability by tobacco extract (CSE). The experiment was divided into four groups: (1) Blank control group (control); (2) CSE treatment group (CSE); using 20% CSE prepared according to the section "1. Preparation of Cigarette Smoke Extract (CSE)" in this test example; (3) Sodium alginate pretreatment group (OSA): The partially oxidized sodium alginate solution with a concentration of 1 g / mL as in Example 1 was used; (4) LbGP-OSA spray pretreatment group (LbGP-OSA): using the wolfberry polysaccharide spray prepared according to the method of Example 1.
[0071] HGFs were used at 2×10 4 Cells were seeded at a density of 100 cells / mL in 12-well plates, with 2 mL of cell suspension added to each well, and three replicates per group. During the pretreatment phase, the blank control group and CSE group received 100 μL of PBS, the sodium alginate pretreatment group received 100 μL of aerosol matrix, and the LbGP-OSA aerosol pretreatment group received 100 μL of aerosol. All groups were co-cultured for 2 days. Subsequently, scratches were made using pipette tips, followed by washing with PBS, and then 2 mL of serum-free DMEM medium was added to each well. Next, the blank control group received 100 μL of PBS, while the other three groups received 100 μL of 20% CSE solution. Observations and photographs were taken under a microscope at 0, 12, and 24 hours after scratching.
[0072] ImageJ software was used to analyze the images. The percentage of scratch closure for cell migration was calculated by measuring the scratch area at each time point. The formula is: (initial scratch area - scratch area at a specified time point) / initial scratch area × 100%.
[0073] The results are as follows Figure 8 and Figure 9 As shown: there was no difference in scratch area among the groups at 0h. At 12 and 24h, the scratch healing area of the LbGP-OSA group (64.3±6.2% at 24h) was significantly higher than that of the CSE group (40.3±5.1%) and the OSA group. The CSE group had the worst healing.
[0074] The above results indicate that LbGP-OSA gelatin microsphere spray can effectively alleviate the inhibitory effect of tobacco extract on HGF cell migration.
[0075] 4. ROS immunofluorescence staining experiment on the effect of wolfberry polysaccharide spray on alleviating oxidative damage of tobacco to HGFs This experiment aimed to investigate the protective effect of the Lycium barbarum polysaccharide spray of this invention against oxidative damage to human gingival fibroblasts (HGFs) induced by tobacco extract (CSE) using ROS immunofluorescence staining. The experiment consisted of four groups: (1) Blank control group (Control); (2) CSE group (CSE); 20% CSE prepared according to the "1. Preparation of Cigarette Smoke Extract (CSE)" section of this test example; (3) Sodium alginate pretreatment group (OSA): The partially oxidized sodium alginate solution with a concentration of 1 g / mL as in Example 1 was used; (4) LbGP-OSA spray pretreatment group (LbGP-OSA): using the wolfberry polysaccharide spray prepared according to the method of Example 1.
[0076] HGFs were used at 2×10 4 Cells were seeded at a density of 1 mL / well in 24-well plates, with 1 mL of cell suspension added to each well, and three replicates per group. After well-adhered culture at 37°C and 5% CO2, cells were treated as follows: 50 μL PBS was added to each well in the blank control and CSE groups; 50 μL 1 g / mL LOSA solution was added to each well in the sodium alginate pretreatment group; and 50 μL of the LbGP-OSA spray pretreatment group was added. Pretreatment was maintained at 37°C and 5% CO2 for 48 hours, followed by gentle washing with PBS and replacement with fresh complete culture medium. Next, 50 μL PBS was added to the blank control group, and 50 μL 20% CSE solution was added to each of the other groups, and stimulation was continued under the same conditions for 6 hours. After stimulation, the culture medium was discarded, and staining was performed according to the ROS kit instructions. Finally, the cells were observed and images were acquired under an immunofluorescence microscope.
[0077] ROS immunofluorescence staining and live cell staining after CSE induction are shown in the following figures. Figure 10 As shown in the figure, the blank control group exhibited weak fluorescence and intact cell morphology; the CSE group showed strong fluorescence and severe cell damage; the OSA group showed slight relief; and the LbGP-OSA group showed fluorescence close to the blank control group, indicating good cell condition. These results demonstrate that LbGP-OSA spray can alleviate CSE-induced oxidative damage to HGFs.
[0078] Quantitative results of relative ROS levels within 24 hours of HGFs are as follows: Figure 11 As shown in the figure, the ROS level in the CSE group was significantly increased, while that in the OSA group was slightly decreased, and the ROS level in the LbGP-OSA group was close to that of the blank control group. These results indicate that the antioxidant protective effect of LbGP-OSA spray is significantly better than that of OSA.
[0079] 5. Detection experiment on GSH protein content in wolfberry polysaccharide spray to alleviate oxidative damage of tobacco to HGFs. This experiment aimed to investigate the protective effect of the Lycium barbarum polysaccharide spray of the present invention against oxidative damage to human gingival fibroblasts (HGFs) induced by tobacco extract (CSE), and to evaluate its antioxidant effect by detecting the intracellular glutathione (GSH) content. The experiment was divided into 4 groups: (1) Blank control group (control); (2) CSE group (cse); 20% CSE prepared according to the section “1. Preparation of Cigarette Smoke Extract (CSE)” in this test example; (3) Sodium alginate pretreatment group (OSA): The partially oxidized sodium alginate solution with a concentration of 1 g / mL as in Example 1 was used; (4) LbGP-OSA spray pretreatment group (LbGP-OSA): using the wolfberry polysaccharide spray prepared according to the method of Example 1.
[0080] HGFs were used at 2×10 4 Cells were seeded at 1 / 2 mL / well in 12-well plates, with 2 mL of cell suspension added to each well. Three replicates were used per group. Different pretreatments were applied to each group: the blank control group and CSE group received 100 μL PBS; the sodium alginate pretreatment group received an equal volume of partially oxidized sodium alginate solution; and the LbGP-OSA spray pretreatment group received 100 μL of Lycium barbarum polysaccharide spray. After pretreatment at 37°C and 5% CO2 for 2 days, cells were washed with PBS and the culture medium was replaced. The blank control group received 100 μL PBS, while the other groups received 50 μL of 20% CSE solution. Stimulation was continued for 6 hours under the same conditions to simulate the oxidative damage environment caused by tobacco. Finally, the GSH content in each group was measured strictly according to the GSH detection kit instructions.
[0081] GSH protein content test results are as follows: Figure 12 As shown, under 20% CSE induction conditions, there was no significant difference in intracellular GSH levels between the CSE group and the blank spray pretreatment group, but both were significantly lower than the control group (P<0.001). However, HGFs pretreated with LbGP-OSA gelatin microspheres showed significantly higher intracellular GSH levels than the CSE group (P<0.01) and also significantly higher than the blank spray pretreatment group (P<0.001), but still lower than the control group (P<0.01). These results indicate that LbGP-OSA spray pretreatment can significantly enhance the ability of HGFs to resist oxidative damage induced by 20% CSE.
[0082] Experimental Example 3 This study evaluated the healing effect of a wolfberry sugar spray on oral mucosal ulcers induced by CSE combined with glacial acetic acid in rats. After determining the modeling conditions through preliminary experiments, the effects of this preparation on ulcer area, histopathology, local inflammatory factors, and systemic toxicity were observed in the formal experiment.
[0083] 1. Preliminary experiment: Screening of CSE-induced concentration First, SD rats were anesthetized by isoflurane gas inhalation. Different concentration gradients of CSE solution (0%, 10%, 20%, 50%, 75%) were injected locally into the oral mucosa (below the submucosal layer of the lower lip) once daily, 0.5 ml each time, for 7 consecutive days as chronic pre-stimulation. Subsequently, filter paper discs approximately 5 mm in diameter, soaked in 35% glacial acetic acid, were attached to the surface of the rat's lower lip mucosa for 30 seconds to induce acute ulceration.
[0084] On the second day after glacial acetic acid stimulation, the ulcer area was measured to assess the model's effectiveness. Preliminary experimental results are attached. Figure 13 As shown, in a rat oral mucositis model induced by glacial acetic acid combined with CSE, the CSE concentration affected the ulcer extent. After injection of 20% CSE, the ulcer area on day 2 after glacial acetic acid stimulation was 8.16±1.67 mm², significantly larger than that in the control group (P<0.01). While 40% CSE could lead to more severe ulcers, it also increased animal mortality, resulted in excessively large ulcers, and prolonged healing time. Therefore, 20% CSE was ultimately chosen to balance efficacy and safety.
[0085] 2. Formal pharmacodynamic experiments 2.1 Drugs and Grouping Seventy-two SD rats were randomly divided into four groups (n=18): (1) Blank control group: given physiological saline; (2) Positive control group: given recombinant human epidermal growth factor gel; (3) OSA blank spray group: given a sodium alginate solution of partially oxidized seaweed with a concentration of 1 g / mL as in Example 1; (4) LbGP-OSA gelatin microsphere spray group: the wolfberry polysaccharide spray prepared in Example 1 was applied.
[0086] 2.2 Drug administration intervention All rats were used to establish an oral mucosal ulcer model according to the method determined in the preliminary experiment (pre-stimulation with 20% CSE for 7 days followed by chemical cauterization with glacial acetic acid). The day of ulcer formation was recorded as day 0 of the intervention. From day 0, rats in each group were administered a local spray of 100 µl of the drug at two fixed times in the morning and evening for 9 consecutive days. After each spray administration, the rats' heads were gently held still for about 20 seconds to prevent them from licking the drug.
[0087] 2.3 Evaluation Indicators (1) Ulcer healing dynamics: The ulcer area (mm²) was measured and recorded using digital calipers on days 0, 3, 5, 7 and 9.
[0088] (2) Histopathological evaluation: At the end of the experiment, tissue from the ulcer site was taken, embedded in paraffin, sectioned, and then subjected to HE staining (to observe epithelial regeneration and inflammatory infiltration) and Masson staining (to observe collagen fiber deposition).
[0089] (3) Detection of local inflammatory factors: Take ulcer tissue to prepare homogenate, and use immunofluorescence staining to detect the protein expression levels of TNF-α, IL-6 and IL-10.
[0090] (4) Preliminary safety evaluation: After the experiment, tissues from the heart, liver, spleen, lungs and kidneys were taken for HE staining to observe whether there were any organic lesions.
[0091] 2.3 Results As attached Figure 14 As shown, after LbGP-OSA spray intervention, the ulcer area significantly decreased to 5.63±1.19 mm on day 5. 2 Compared with the control group (9.03±1.23 mm) 2 The difference was significant (P<0.01), and complete healing was achieved on day 9. (See attached image) Figure 15 Further analysis showed that the epithelial structure of the LbGP group was intact, the basal cells were tightly arranged, and the infiltration of inflammatory cells was significantly less than that of the control group. Masson staining results indicated that the collagen fiber density of the LbGP group was 1.4 times higher than that of the control group (P<0.01), indicating that the quality of tissue repair was significantly improved.
[0092] The above results indicate that the Lycium barbarum polysaccharide spray of the present invention can significantly accelerate ulcer healing, inhibit inflammatory response, promote epithelial regeneration, and enhance collagen deposition.
[0093] The above description is merely a preferred embodiment of the present invention and is illustrative rather than restrictive. Those skilled in the art will understand that many changes, modifications, and even equivalent alterations can be made within the spirit and scope defined by the claims of the present invention, but all such changes will fall within the protection scope of the present invention.
Claims
1. A wolfberry polysaccharide spray, characterized in that, Includes partially oxidized dialdehyde polysaccharide aqueous solution and wolfberry polysaccharide-gelatin microspheres dispersed therein; The wolfberry polysaccharide-gelatin microspheres are based on cross-linked gelatin and uniformly encapsulated with wolfberry polysaccharides inside. The mass ratio of partially oxidized dialdehyde polysaccharide, gelatin, and wolfberry polysaccharide is 1~3:0.5~5:0.1~0.5; The concentration of the partially oxidized dialdehyde polysaccharide aqueous solution is 0.1–2 g / mL.
2. The wolfberry polysaccharide spray according to claim 1, characterized in that, The partially oxidized dialdehyde polysaccharide is prepared by specific oxidation with sodium periodate, including at least one of dialdehyde sodium alginate, dialdehyde hyaluronic acid, dialdehyde starch, dialdehyde cellulose and dialdehyde chitosan; its degree of oxidation is 10%-80%.
3. The method for preparing the wolfberry polysaccharide spray according to claim 1 or 2, characterized in that, The process includes the following steps: preparing Lycium barbarum polysaccharide-gelatin microspheres using the emulsion template method, and then dispersing the Lycium barbarum polysaccharide-gelatin microspheres in a partially oxidized dialdehyde polysaccharide solution to obtain the Lycium barbarum polysaccharide spray.
4. The method for preparing the wolfberry polysaccharide spray according to claim 3, characterized in that, The preparation of Lycium barbarum polysaccharide-gelatin microspheres using the emulsion template method includes the following steps: Lycium barbarum polysaccharide and gelatin were dissolved together in water to form an aqueous phase; the oil phase material was mixed with a surfactant to form an oil phase; the two phases were mixed and emulsified, and then a crosslinking agent was added for crosslinking; after the reaction was completed, the precipitate was collected by solid-liquid separation to obtain gelatin microspheres loaded with Lycium barbarum polysaccharide.
5. The method for preparing the wolfberry polysaccharide spray according to claim 4, characterized in that, The oil phase material includes liquid paraffin; Or / and the surfactant includes at least one of Span-80, Span-20, Span-40, Span-60, Span-85, polyglycerol monostearate, and polyglycerol-3-diisostearate; The crosslinking agent includes at least one of genipin, gallic acid, gallic acid epoxide, tea polyphenols, fucoidan, polysaccharide crosslinking agents, tannic acid, and trehalose.
6. The method for preparing the wolfberry polysaccharide spray according to claim 4, characterized in that, The concentration of gelatin in the aqueous phase is 40-60 wt%; The volume ratio of oil phase material to surfactant is 6~25:1; The volume ratio of the aqueous phase to the oil phase is 5~7:21~27; The amount of crosslinking agent used is 0.1~0.5% of the gelatin mass.
7. The method for preparing the wolfberry polysaccharide spray according to claim 4, characterized in that, After mixing the two phases, stir at 30-40℃ for 20-50 minutes to obtain an emulsion; then add a crosslinking agent and continue stirring.
8. The application of the wolfberry polysaccharide spray according to claim 1 or 2, characterized in that, Use in the preparation of medicaments for the prevention and / oral tissue damage.
9. The application of the wolfberry polysaccharide spray according to claim 8, characterized in that, The oral tissue damage includes oral tissue damage caused by tobacco.
10. The application of the wolfberry polysaccharide spray according to claim 8 or 9, characterized in that, The oral tissue damage includes oral diseases related to immune regulation.