An anti-inflammatory polysaccharide oral bacteriostatic composition and its preparation process

Abstract

The application discloses an anti-inflammatory polysaccharide oral bacteriostatic composition and a preparation process thereof, and belongs to the technical field of biomedical materials. The preparation process comprises the following steps: dissolving chitosan in an acetic acid aqueous solution, stirring and treating to obtain a pre-preparation solution; preparing a sodium tripolyphosphate aqueous solution, uniformly and dropwise adding the chitosan solution, and uniformly mixing to obtain a nano core suspension; dissolving epigallocatechin gallate in a buffer solution, adding the nano core suspension for incubation, and centrifugally recovering to obtain polyphenol loaded particles; adding a sodium hyaluronate solution, and standing to obtain core-shell nanoparticles; mixing with a zinc ion solution; adding xylitol and a citric acid / citric acid sodium buffer salt, sterilizing and filtering; and spray drying to obtain a powder dosage form. The composition has excellent bacteriostatic rate, anti-inflammatory efficiency, stability and biocompatibility, and is suitable for oral care products.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biomedical materials, and in particular relates to an anti-inflammatory polysaccharide oral bacteriostatic composition and a preparation process thereof. BACKGROUND

[0002] Oral health is an important part of human overall health. Oral diseases such as dental caries, periodontal disease, oral ulcer and gingivitis have become a global public health problem. According to the data of the World Health Organization (WHO), about 3.5 billion people worldwide suffer from oral diseases, among which dental caries and periodontal disease are the most common chronic infectious diseases. The main cause of these diseases is the imbalance of oral microbial community, especially the formation and accumulation of bacterial biofilm. Oral microorganisms include hundreds of bacteria, fungi and viruses, among which pathogenic bacteria such as oral streptococcus, streptococcus mutans, staphylococcus aureus, proteus and porphyromonas gingivalis, etc. adhere to the surface of teeth to form plaque biofilm, leading to enamel demineralization, periodontal tissue inflammation and infection. Traditional oral care products such as mouthwash, toothpaste and oral spray mainly rely on chemical bacteriostatic agents to control these microorganisms, but there are many limitations in the existing technology, and innovative bacteriostatic materials and preparation processes are needed to improve efficacy and safety.

[0003] The research and development of existing oral bacteriostatic materials originated in the middle of the 20th century. With the discovery and application of antibiotics, early products mostly used broad-spectrum antibacterial agents such as chlorhexidine and fluoride. These materials exert their effects by directly killing bacteria or inhibiting their growth. For example, chlorhexidine, as a cationic surfactant, can destroy bacterial cell membranes, leading to leakage of cell contents, and has broad-spectrum antibacterial effect, which has been widely used in mouthwash and toothpaste. Studies have shown that chlorhexidine can effectively reduce oral bacterial load and inhibit plaque formation, but its long-term use can lead to bacterial resistance, oral mucosa coloring and taste changes. In addition, fluoride such as sodium fluoride can prevent and treat dental caries by promoting enamel remineralization and inhibiting bacterial acid production, but its antibacterial spectrum is narrow, mainly targeting acid-producing bacteria, and its effect on other pathogenic bacteria such as anaerobes is limited. In the 1980s, with the rise of natural materials, researchers began to explore plant extracts and polysaccharides as bacteriostatic agents. For example, tea polyphenols (such as epigallocatechin gallate, EGCG) are introduced into oral products due to their antioxidant and antibacterial activity, which can inhibit bacterial enzyme activity and interfere with biofilm formation. However, these single-component products often have low bacteriostatic efficiency, and EGCG is easily oxidized and degraded in the oral environment, resulting in unstable efficacy.

[0004] In the 21st century, the introduction of nanotechnology and drug delivery systems marked a major advance in the field of oral antimicrobial materials. Nanomaterials, with their high specific surface area and unique optical, electrical, and magnetic properties, can enhance the loading and release control of antimicrobial agents. For example, titanium dioxide (TiO2) nanoparticles, as photocatalysts, generate reactive oxygen species (ROS) under ultraviolet light, which can destroy bacterial cell walls and have broad-spectrum antibacterial effects. In recent years, TiO2 and its reduced form (TiO2-x) have attracted attention in oral medicine. A review suggests that TiO2 nanomaterials can be used for oral cancer diagnosis, periodontal disease treatment, and implant surface coating, selectively killing Streptococcus mutans through a photodynamic mechanism while promoting tissue regeneration. However, its clinical application is limited by light source dependence and potential cytotoxicity, which needs further optimization. Another important development is the use of nanodrug delivery systems, such as mesoporous silica nanoparticles (MSNs) loaded with chlorhexidine or antibiotics, which can achieve sustained-release effects. Studies have shown that this system can increase the loading rate of chlorhexidine to more than 60% and maintain antibacterial activity in composite resins for more than 2 weeks, significantly reducing the incidence of secondary caries. Similarly, chitosan, a natural polysaccharide, has been widely studied due to its biocompatibility and antibacterial properties. Chitosan can bind to the negatively charged cell wall of bacteria through positive charges, interfering with membrane permeability. However, the low solubility and poor loading efficiency of single chitosan materials limit their application in oral environments.

[0005] In terms of antimicrobial mode, existing technologies are mainly divided into release antimicrobial, contact antimicrobial, and combined antimicrobial. Release antimicrobial relies on the diffusion of antimicrobial agents from the material to the environment to actively kill bacteria, such as amoxicillin or sodium hypochlorite loaded in resin matrix, which can effectively kill residual bacteria and achieve long-term release. A study showed that adding 5% chlorhexidine directly to composite resin can inhibit the growth of Streptococcus mutans and Lactobacillus casei, but the material surface is prone to form cavities, affecting mechanical strength. Contact antimicrobial immobilizes antimicrobial agents on the material surface, such as quaternary ammonium salts or silver nanoparticles (AgNPs), which directly contact and destroy bacteria. Silver nanoparticles are popular due to their broad-spectrum antibacterial properties and low resistance, which can release silver ions to interfere with bacterial enzyme systems and destroy biofilms in root canal treatment. Studies have shown that AgNPs gel can effectively inhibit bacteria in root canals, but high concentrations may cause cytotoxicity. Combined antimicrobial mode combines multiple mechanisms, such as combining release and contact, or adding calcium compounds to repair demineralized tissue. For example, composite materials combining silver nanoparticles and hydroxyapatite not only kill bacteria but also promote dentin remineralization, making them suitable for minimally invasive dental restoration.

[0006] Antibacterial coating for dental implants is another hot area. Implant-related infections (such as peri-implantitis) are the main cause of implant failure. Existing technologies have developed various coating materials, such as composite coatings with TiO2, hydrogel or calcium phosphate as carriers. These coatings can load antibiotics and control release, reducing the risk of infection. A study using a degradable coating loaded with gentamicin using poly (lactic-co-glycolic acid) (PLGA) copolymer showed that it can extend the drug release time, but the coating strength is low and the brittleness is high, which cannot withstand the occlusal force in the oral cavity. Mesoporous bioactive glass (MBG) as a carrier has high specific surface area and drug carrying capacity, but the irregularity of the pore channel limits its clinical application. In addition, degradable polymers such as polyhydroxyalkanoate (PHA) are used in the form of microcapsules to regulate drug release kinetics, but they face the problem of uneven biodegradation rate.

[0007] In recent years, photodynamic therapy (PDT) and cold atmospheric plasma (CAP) have entered the field of oral bacteria inhibition as emerging non-invasive technologies. PDT uses photosensitizers (such as methyl blue) to generate ROS under light, selectively killing bacteria without damaging host tissues. A review pointed out that PDT is effective against periodontal disease-related bacteria, but the light source and dosage need to be optimized. CAP kills bacteria by generating plasma and has been used for root canal disinfection, showing better antibacterial effect than traditional methods. However, these technologies rely on equipment, are costly, and have insufficient clinical data.

[0008] The application of polysaccharide materials in oral bacteria inhibition is increasingly prominent. Chitosan, with a high degree of deacetylation (usually 80-95%) and a moderate molecular weight (5×10 4 -3×10 5 Da), has good biodegradability and mucosal adhesion, and is often used as a nanoparticle carrier. In existing technologies, chitosan is ionically cross-linked with tripolyphosphate (TPP) to form a nanocore, which can load polyphenols such as EGCG, enhancing antioxidant and anti-inflammatory effects. Hyaluronic acid sodium is used as a coating layer to improve the moisture retention and stability of the particles. Zinc ions (such as zinc gluconate) are introduced to synergistically inhibit bacteria and interfere with bacterial metabolism. Xylitol and citric acid buffer salts are used to adjust the pH and sweetness, improving the taste. However, existing chitosan-based products have low loading efficiency (usually <50%), particle aggregation (Zeta potential absolute value <15 mV), and poor storage stability (size change rate >10%). Spray drying technology is used for powder preparation, but parameter optimization is insufficient, resulting in non-uniform products.

[0009] Despite the progress made by the prior art, it still faces multiple challenges. First, narrow antibacterial spectrum: many materials are only effective against specific bacteria, failing to comprehensively address the complex microbial community in the oral cavity, leading to an increase in drug-resistant strains. Second, limited anti-inflammatory effect: existing products focus more on inhibiting bacteria and ignore the inhibition of inflammatory factors (such as TNF-α), which can easily trigger chronic inflammation. Third, poor biocompatibility: chemical agents such as chlorhexidine or silver ions can cause cytotoxicity and allergies, with a cell survival rate of <80%. Fourth, insufficient stability: nano materials are prone to aggregation, and their performance deteriorates after storage. Fifth, complex preparation process: it is difficult to control loading and release, and the production cost is high. These problems limit the clinical promotion of products and patient compliance.

[0010] To address these shortcomings, researchers are exploring multi-target synergistic mechanisms, such as combining nano-engineering and natural active ingredients. For example, selective antibacterial peptides (STAMP) such as C16G2 can target Streptococcus mutans and restore the balance of the oral microbiota, but the cost of synthesis is high and the safety needs to be verified. In the future, intelligent materials such as pH-responsive nanoparticles can release antibacterial agents in an acidic environment, suitable for caries prevention and treatment. Overall, although existing oral antibacterial materials have evolved from traditional chemical agents to nano and biological materials, further innovation is needed to achieve efficient, long-lasting, and safe comprehensive performance. This provides a basis for the present invention, which aims to solve the above problems by optimizing polysaccharide-based nanocomposites. SUMMARY

[0011] To solve the problems of narrow antibacterial spectrum, limited anti-inflammatory effect, poor biocompatibility, insufficient particle stability and low storage durability of oral antibacterial products in the prior art, the present application provides an anti-inflammatory polysaccharide oral antibacterial composition and its preparation process. The oral antibacterial compositions in the prior art mostly use traditional chemical antibacterial agents (such as chlorhexidine) or single natural extracts (such as simple chitosan or polyphenols), which have certain antibacterial activity, but have many defects: first, the antibacterial spectrum is narrow, only effective against a few bacteria, and cannot comprehensively inhibit common oral pathogenic bacteria such as oral streptococcus, staphylococcus aureus, proteus vulgaris, streptococcus mutans and porphyromonas gingivalis, resulting in an antibacterial rate usually less than 80%; second, the anti-inflammatory performance is weak, and it is difficult to effectively inhibit the release of inflammatory factors (such as TNF-α), which is easy to cause recurrence of oral inflammation or mucous membrane irritation; third, the material has poor biocompatibility, and long-term use may cause cytotoxicity or allergic reaction, and the cell survival rate is often less than 80%; in addition, the existing nanoscale materials are easy to aggregate and precipitate (Zeta potential absolute value < 15mV), have poor storage stability, and the particle size change rate is as high as 10%-20%; finally, it lacks a multi-target synergistic mechanism, is easy to produce bacterial resistance, and the preparation process is complex, the loading efficiency is low, and it cannot realize efficient controlled release and mucosal adhesion. These problems limit the clinical application and market promotion of the product. Through innovative nano core-shell structure design, polyphenol loading, zinc ion synergy and optimization of parameters, the present application realizes excellent performance of antibacterial rate 91.8%-93.5%, inflammation inhibition rate 77.9%-80.2%, Zeta potential absolute value 23.1-25.6mV, cell survival rate 94.2%-96.8% and storage change rate 2.7%-3.5%, significantly improving the comprehensive performance of the product.

[0012] The application adopts the technical scheme that a preparation process of an anti-inflammatory polysaccharide oral cavity bacteriostatic composition comprises the following steps: S1, acid dissolution and pre-dispensing: dissolving chitosan (CAS No. 9012-76-4) in 80-150 times the volume fraction of 0.5-1.5% acetic acid aqueous solution by mass, then stirring to obtain a pre-dispensed chitosan solution; S2, ion cross-linking nano nucleation: preparing a 0.05-0.6% tripolyphosphate (CAS No. 7758-29-4) aqueous solution by mass, uniformly adding the pre-prepared chitosan solution prepared in step S1 into the tripolyphosphate aqueous solution at a constant speed, then uniformly mixing to obtain a nano nucleus suspension; S3, polyphenol loading: dissolving epigallocatechin gallate (CAS No. 989-51-5) in a buffer solution, adding the nano nucleus suspension obtained in step S2 for incubation, then centrifuging to recover the precipitate to obtain polyphenol-loaded particles; S4, surface coating: adding 0.05-0.5 times the mass of sodium hyaluronate (CAS No. 9067-32-7) solution into the polyphenol-loaded particles of step S3, adjusting the pH to 5.8-6.5, and standing at room temperature for 10-30 min to obtain nano particles with a core-shell structure; S5, metal ion synergy: mixing zinc ion solution with the nano particles with a core-shell structure in step S4, wherein the zinc ion concentration is 30-50 mg / L, and the mass ratio between the zinc ion solution and the nano particles with a core-shell structure is 10:(5-8) to obtain a mixed solution; S6, final treatment: sequentially adding xylitol (CAS No. 87-99-0) and citric acid / sodium citrate buffer salt into the mixed solution of step S5, the ratio among the three is 5:(1-5):(1-5), adjusting the pH to 6.2-6.8, filtering through a 0.22 μm sterilization filter to obtain a preliminary product; S7, finished product: performing spray drying treatment on the preliminary product obtained in step S6, collecting the powder and storing at 4°C to obtain a powder dosage form. The parameters of the citric acid / sodium citrate buffer salt are as follows: 0.1M citric acid and 0.1M trisodium citrate are mixed at a ratio of 54:46, deionized water is added, the volume is adjusted to 100 mL, it is diluted to 0.1 mmol / L, and the pH is adjusted to 4.2 at 25°C.

[0013] Preferably, the parameters of chitosan in step S1 are as follows: deacetylation degree 80-95%, number average molecular weight 5×10 4 -3×10 5 Da; the parameters of the stirring treatment in step S1 are as follows: the pH is adjusted to 4.8-5.6, the temperature is 25-30°C, the rotation speed is 500-700 rpm, and the time is 20-40 min.

[0014] Preferably, the mass of the prepared chitosan solution in step S2 is 15-25 times the mass of the aqueous sodium tripolyphosphate solution; the parameters for uniform dropwise addition in step S2 are as follows: 0.2-1 mL / min; the parameters for mixing and uniform treatment in step S2 are as follows: temperature 45-55°C, rotation speed 500-800 rpm, and time 10-30 min.

[0015] Preferably, the parameters of the nanometer core in step S2 are as follows: particle size 120-200 nm, Zeta potential +15-+40 mV, and PDI≤0.3.

[0016] Preferably, the buffer in step S3 is a phosphate buffer with pH 6-7; the mass of the nanometer core suspension added in step S3 is 0.3-0.5 times the mass of epigallocatechin gallate; the parameters for incubation in step S3 are as follows: pH 5.5-6, temperature 45-60°C, and time 25-50 min; the rotation speed for centrifugation in step S3 is 8000-12000 rpm, and the centrifugation time is 30-45 min.

[0017] Preferably, the mass percentage of the sodium hyaluronate solution in step S4 is 2-6%; the relative molecular mass of the sodium hyaluronate in step S4 is 1×10 5 -1.5×10 6 Da; the parameters of the core-shell structure nanoparticles in step S4 are as follows: Zeta potential -15--25 mV, and particle size 150-250 nm.

[0018] Preferably, the zinc ion solution in step S5 is an aqueous zinc gluconate (CAS No.: 4468-02-4) solution; the parameters for blending and mixing in step S5 are as follows: temperature 45-55°C, and time 20-40 min.

[0019] Preferably, the storage temperature of the preliminary product in step S6 is 0-4°C.

[0020] Preferably, the parameters for spray drying in step S7 are as follows: inlet air temperature 120-150°C, and outlet air temperature 60-80°C.

[0021] Based on the preparation process of the anti-inflammatory polysaccharide oral bacteriostatic composition, the following lists the possible instruments, models, manufacturers, and related parameter information involved in each stage according to steps.

[0022] S1: acid dissolution and pre-dispensing (chitosan is dissolved in aqueous acetic acid, and stirring treatment)

[0023] Main instruments: magnetic stirrer or mechanical stirrer (for dissolving and stirring chitosan).

[0024] Model example: PM-100 ball mill (for wet grinding dissolution, combined with stirring function).

[0025] Vendor example: Retsch (Germany) or similar (e.g. Shanghai Bilon Instruments).

[0026] Parameter information: speed 500-700 rpm, temperature 25-30 °C, time 20-40 min, pH adjusted to 4.8-5.6. The equipment needs to support real-time pH monitoring and temperature control; can be equipped with a glass reaction kettle or a volumetric flask (volume adjusted according to the mass ratio of 80-150 times).

[0027] Auxiliary instrument: pH meter (for pH adjustment).

[0028] Model example: General Laboratory pH meter, such as PBS buffer pH adjustment equipment in the preparation of pH-responsive nanoparticles.

[0029] Vendor example: Mettler Toledo (Switzerland) or Haier Biomedical.

[0030] Parameter information: pH range 1-14, accuracy ±0.01, suitable for acidic solution (volume fraction of acetic acid aqueous solution 0.5-1.5%).

[0031] S2: Ion crosslinking nanonucleation (preparing a sodium tripolyphosphate solution, adding chitosan solution at a constant speed, mixing and processing)

[0032] Main instrument: dropping device / peristaltic pump (for adding chitosan solution at a constant speed).

[0033] Model example: high-pressure nozzle or peristaltic pump system (such as a spray dryer pump).

[0034] Vendor example: General Laboratory Pump, such as PerkinElmer or local vendors (e.g. Shanghai Keye Instruments).

[0035] Parameter information: dropping speed 0.2-1 mL / min, chitosan solution added mass is 15-25 times that of sodium tripolyphosphate aqueous solution. The equipment needs to accurately control the flow rate and support the preparation of solutions with mass fraction of 0.05-0.6%.

[0036] Auxiliary instrument: mechanical stirrer or nanoparticle generator (for mixing and processing and nanonucleation).

[0037] Model example: VSP-G1 nanoparticle generator (desktop type, used to generate 1-20 nm particles, supporting stirring and aerosol formation).

[0038] Vendor example: VSPARTICLE (Netherlands).

[0039] Parameter information: rotation speed 500-800 rpm, temperature 45-55℃, time 10-30 min; nano-core parameters: particle size 120-200 nm, Zeta potential +15-+40 mV, PDI≤0.3.

[0040] Measuring instrument: Zeta potential analyzer (for verifying nano-core parameters).

[0041] Model example: Microtrac STABINO ZETA or Zetasizer Advance.

[0042] Vendor example: Microtrac (Germany) or Malvern Panalytical (UK).

[0043] Parameter information: particle size range 0.3 nm-300 μm, potential range -3000 mV to +3000 mV, suitable for suspension analysis.

[0044] S3: Polyphenol loading (dissolve EGCG in buffer, add nano-core suspension for incubation, centrifugal recovery)

[0045] Main instrument: constant temperature incubator or stirring incubation equipment (for incubation).

[0046] Model example: stirring device for AIE nanoparticle preparation (with ultrasonic output probe, supporting stirring and dispersion).

[0047] Vendor example: Guangzhou Aige Technology (China).

[0048] Parameter information: pH 5.5-6, temperature 45-60℃, time 25-50 min; buffer is pH 6-7 phosphate buffer, and the added mass of nano-core suspension is 0.3-0.5 times that of EGCG.

[0049] Auxiliary instrument: centrifuge (for centrifugal recovery of precipitate).

[0050] Model example: Haier Biomedical LX-100L1000R (floor type large capacity refrigerated centrifuge) or LX-60T500-J.

[0051] Vendor example: Haier Biomedical (China).

[0052] Parameter information: rotation speed 8000-12000 rpm, time 30-45 min; maximum RCF up to 7325 g, sound level <65 dB, supporting biocompatible material processing.

[0053] S4: Surface coating (add sodium hyaluronate solution, adjust pH, and stand at room temperature)

[0054] Main instrument: stirring dissolving equipment (for sodium hyaluronate solution preparation and mixing).

[0055] Model example: magnetic stirrer or PM-100 ball mill (wet grinding dissolution).

[0056] Vendor example: Retsch (Germany) or Shanghai Bilang Instruments.

[0057] Parameter information: sodium hyaluronate solution mass percentage 2-6%, relative molecular mass 1×10 5 -1.5×10 6 Da; the added mass is 0.05-0.5 times the mass of the polyphenol-loaded particles, the pH is adjusted to 5.8-6.5, and it is left to stand for 10-30 min.

[0058] Auxiliary instrument: pH meter (for pH adjustment).

[0059] Model example: Microtrac STABINO ZETA with built-in pH module.

[0060] Vendor example: Microtrac (Germany).

[0061] Parameter information: pH range 1-14; core-shell nanoparticle parameters: Zeta potential -15--25 mV, particle size 150-250 nm.

[0062] Measuring instrument: Zeta potential analyzer (to verify core-shell structure parameters).

[0063] Model example: Zetasizer Advance.

[0064] Vendor example: Malvern Panalytical (UK).

[0065] Parameter information: supports simultaneous measurement of particle size and potential.

[0066] S5: metal ion synergy (zinc ion solution and nanoparticle blending mixing)

[0067] Main instrument: mechanical stirrer or mixer (for blending).

[0068] Model example: stirring device for AIE nanoparticle preparation (multi-blade stirring with temperature control).

[0069] Vendor example: Guangzhou Aige Science and Technology (China).

[0070] Parameter information: zinc ion concentration 30-50 mg / L (zinc gluconate aqueous solution), mass ratio 10:5-8; temperature 45-55°C, time 20-40 min.

[0071] S6: Final treatment (addition of xylitol and buffer salt, pH adjustment, sterile filtration)

[0072] Main instrument: pH adjustment and filtration system (for pH adjustment and 0.22 pm sterile filtration).

[0073] Model example: General laboratory filter (e.g. Millipore system).

[0074] Vendor example: Merck Millipore (Germany).

[0075] Parameter information: ratio 5: 1-5: 1-5, pH adjustment to 6.2-6.8; storage temperature 0-4°C.

[0076] Auxiliary instrument: pH meter (as above).

[0077] S7: Finished product (spray drying process, collection of powder, storage at 4°C)

[0078] Main instrument: spray dryer (for drying the preliminary product).

[0079] Model example: Small spray dryer BILON-6000YS or Shanghai Keyon Instruments.

[0080] Vendor example: Shanghai Billong Instruments (China) or Shanghai Keyon Instruments.

[0081] Parameter information: inlet air temperature 120-150°C, outlet air temperature 60-80°C; evaporated water volume 1500-2000 mL / H, nozzle diameter 0.5-2.0 mm optional, full stainless steel material supported, noise < 65 dB.

[0082] Auxiliary instrument: low-temperature storage device (e.g. refrigerator).

[0083] Model example: Haier Biomedical low-temperature refrigerator.

[0084] Vendor example: Haier Biomedical (China).

[0085] Parameter information: temperature 4°C, for storage of powder dosage forms.

[0086] The application discloses an anti-inflammatory polysaccharide oral bacteriostatic composition and a preparation process thereof. Compared with the prior art, the application has significant technical advantages in multiple dimensions. The existing oral bacteriostatic products mostly use traditional chemical bacteriostatic agents or simple natural extract formulations, such as chlorhexidine mouthwash or single polysaccharide-based materials. These products often have problems such as narrow bacteriostatic spectrum, limited anti-inflammatory effect, poor biocompatibility and insufficient storage stability. For example, although traditional chitosan-based products have certain bacteriostatic activity, they are easily affected by pH, resulting in low solubility, lack of synergistic mechanism for oral inflammation, and may cause mucous membrane irritation or drug resistance problems during long-term use. Through innovative nano core-shell structure design, polyphenol loading, zinc ion synergy and optimization of preparation parameters, the application realizes comprehensive improvement of bacteriostasis, anti-inflammatory, stability and biological safety. The technical effects are described in detail from the aspects of bacteriostatic performance, anti-inflammatory efficiency, particle stability, biocompatibility, storage stability and overall application potential.

[0087] Firstly, in terms of bacteriostatic performance, the product of the application shows excellent broad-spectrum bacteriostatic effect. Test results show that the bacteriostatic rate of the examples is between 91.8%-93.5%, and the inhibition rate for common oral pathogenic bacteria such as oral streptococcus is significantly higher than that of the prior art. In the prior art, the bacteriostatic rate of single chitosan or EGCG (epigallocatechin gallate) preparation is usually only 70%-80%, because these materials are difficult to achieve efficient loading and controlled release. The application adopts ion crosslinking nano nucleation technology to form a nano core with a particle size of 120-200 nm (zeta potential +15-+40 mV, PDI≤0.3) from chitosan and sodium tripolyphosphate, then load EGCG and coat the surface with sodium hyaluronate to form a core-shell structure (particle size 150-250 nm, zeta potential -15--25 mV). This structure not only improves the loading efficiency of EGCG (nano core suspension to EGCG mass ratio 0.3-0.5:1), but also enhances the bacteriostatic synergistic effect through zinc ion synergy (concentration 30-50 mg / L, mass ratio 10:5-8). As an auxiliary bacteriostatic agent, zinc ions can interfere with bacterial membrane permeability and enzyme activity, complement the polyphenol antioxidant effect of EGCG, and form a "multi-target" attack mechanism. In contrast, the comparative examples have a bacteriostatic rate of 64.1%-66.5% due to the absence of key components (such as sodium tripolyphosphate or zinc ions) or parameter deviations (such as a drop speed lower than 0.2 mL / min), proving the necessity of the process of the application. In practical applications, this high bacteriostatic rate can effectively prevent oral infections such as gingivitis and dental caries, reduce antibiotic dependence, and meet the green trend of modern oral care.

[0088] Secondly, the anti-inflammatory performance of the present application is far superior to the prior art. Oral inflammation is often accompanied by the need to inhibit bacteria, and existing products such as fluoride toothpaste or simple polyphenol mouthwash usually have an inhibition rate of inflammation factors (such as TNF-α) of not more than 60%, and are prone to cause secondary inflammation. The present application realizes the level of 77.9%-80.2% of the inhibition rate of inflammation factors through the polyphenol loading of EGCG and the coating of sodium hyaluronate. EGCG as a strong antioxidant can inhibit the NF-κB pathway and reduce the release of pro-inflammatory factors, and the coating of sodium hyaluronate not only improves the mucosal adhesion of the particles (pH adjusted to 5.8-6.5, and left standing for 10-30 min), but also provides moisturizing and repair functions, and cooperates with zinc ion to regulate immune response. The use of RAW264.7 cell model in the test confirms this effect: the TNF-α inhibition rate of the example group is significantly higher than that of the 50.9%-53.4% of the comparative examples. For example, the replacement of zinc source of Comparative Example 2 with zinc chloride leads to an increase in ionic toxicity and a decrease in inhibition rate; the pH of the buffer solution of Comparative Example 5 is lower than 6, which affects the stability of EGCG and further weakens the anti-inflammatory effect. The innovation of the present application lies in the multi-layer synergy: the nano core provides a carrier, the polyphenol loading targets oxidative stress, and the zinc ion and the buffer salt (xylitol and citric acid / sodium citrate ratio 5:1-5:1-5, pH 6.2-6.8) maintain the balance of the microenvironment. This design makes the product suitable for patients with chronic oral inflammation such as periodontal disease, and compared with the prior art, it can reduce the recurrence rate of inflammation and improve the quality of life.

[0089] In terms of particle stability, the product of the present application shows excellent physicochemical stability. Existing nanoscale oral materials often cause aggregation and precipitation due to low Zeta potential (absolute value <15 mV), affecting the use effect. The core-shell structure design of the present application increases the absolute value of Zeta potential to 23.1-25.6 mV, and through the charge balance of the positive charge nano core (+15-+40 mV) and the negative charge coating layer (-15--25 mV), the electrostatic repulsion force is enhanced. At the same time, the spray drying parameters (inlet air temperature 120-150℃, outlet air temperature 60-80℃) ensure the uniformity of the powder form, and PDI≤0.3 ensures the uniformity of particle distribution. This makes the product of the present application more resistant to saliva dilution and pH fluctuations in the oral environment, prolonging the action time.

[0090] Biocompatibility is another outstanding advantage of the present application. In the prior art, chemical bacteriostatic agents often cause cytotoxicity, and the survival rate of L929 cells is less than 80%. The present application uses natural polysaccharides (such as chitosan with a degree of deacetylation of 80-95% and a molecular weight of 5×10 4 -3×10 5Da) and biocompatible materials (such as sodium hyaluronate), MTT tests show that cell survival rates reach 94.2%-96.8%. Zinc ions are introduced in the form of zinc gluconate (temperature 45-55℃, mixing time 20-40min), avoiding heavy metal toxicity; xylitol as a sweetener and humectant, further enhancing safety. Comparative Example 7 pH is lower than 5.5, resulting in acid irritation, survival rate drops to 70.8%-74.2%. This effect ensures that the product is suitable for sensitive groups such as children and the elderly, reducing the risk of allergies.

[0091] In terms of storage stability, the product of the present application has a particle size change rate of only 2.7%-3.5% after 3 months of storage at 4℃, which is much lower than the 10%-20% of the prior art. This is due to 0.22μm sterilization filtration and spray drying process, as well as low temperature storage (0-4℃). Comparative Example 5 inlet temperature is lower than 120℃, resulting in incomplete drying, with a change rate of 18.1%-19.5%. This stability facilitates commercial production and transportation.

[0092] Overall, the technical effect of the present application not only lies in the improvement of quantitative indicators, but also in the comprehensive application potential. Through multi-step optimization (such as stirring pH 4.8-5.6, rotation speed 500-700rpm), the product realizes the balance of bacteriostasis, anti-inflammatory and safety, and is suitable for the form of mouthwash, toothpaste or spray. Compared with the prior art, the present application reduces production cost (without complex equipment), improves therapeutic efficacy persistence, and meets the concept of sustainable development. In the future, it can be extended to other biomedical fields, such as wound healing materials. In summary, these advantages are due to innovative nano-engineering and synergistic formulation, marking a major advance in oral care technology. BRIEF DESCRIPTION OF DRAWINGS

[0093] Figure 1 is a hemolysis chart of the anti-inflammatory polysaccharide oral bacteriostatic composition prepared in Example 1 of the present application.

[0094] Figure 2 is a HE staining chart of the chronic toxicity experiment of the anti-inflammatory polysaccharide oral bacteriostatic composition prepared in Example 1 of the present application, with a scale of 50μm. DETAILED DESCRIPTION

[0095] The present application will be described in detail below through specific examples, but the purpose and purpose of these exemplary embodiments are only used to exemplify the present application, and do not constitute any form of any limitation on the actual protection scope of the present application, nor limit the protection scope of the present application. For the range of parameters not mentioned, the intermediate value is selected. At the same time, for the mass ratio not explicitly stated or mentioned, it generally refers to the mass ratio after addition. In addition, in the present application, the unit of mass is gram (g).

[0096] Example 1

[0097] The preparation method of the anti-inflammatory polysaccharide oral bacteriostatic composition is as follows: acid dissolution and pre-dispersion: chitosan (degree of deacetylation 88%, number average molecular weight 1.75 x 10 5 Da) is dissolved in a volume fraction of 1% acetic acid aqueous solution with a mass of 115 times, then stirring treatment is performed, the pH is adjusted to 5.2, the temperature is 28°C, the rotation speed is 600 rpm, and the time is 30 min, to obtain a pre-dispersed chitosan solution. Ionic cross-linking nano nucleation: a mass fraction of 0.325% sodium tripolyphosphate aqueous solution is prepared, and the pre-prepared chitosan solution is added at a uniform speed to the sodium tripolyphosphate aqueous solution, the added mass of the pre-prepared chitosan solution is 20 times the mass of the sodium tripolyphosphate aqueous solution, the uniform speed addition parameter is 0.6 mL / min, then uniform mixing treatment is performed, the temperature is 50°C, the rotation speed is 650 rpm, and the time is 20 min, to obtain a nano nucleus suspension; the nano nucleus parameters are a particle size of 160 nm, a Zeta potential of +28 mV, and a PDI of 0.15. Polyphenol loading: epigallocatechin gallate is dissolved in a phosphate buffer with a pH of 6.5, and the nano nucleus suspension is added for incubation, the added mass of the nano nucleus suspension is 0.4 times the mass of the epigallocatechin gallate, the incubation parameters are a pH of 5.75, a temperature of 52°C, and a time of 38 min, then centrifugation is performed at a rotation speed of 10,000 rpm for 38 min, the precipitate is recovered, and polyphenol-loaded particles are obtained. Surface coating: 0.275 times the mass of a sodium hyaluronate solution (mass percentage 4%, relative molecular mass 7.5 x 10 5 Da) is added to the polyphenol-loaded particles, the pH is adjusted to 6.15, and the mixture is left to stand at room temperature for 20 min to obtain core-shell structured nanoparticles; the core-shell structured nanoparticles have a Zeta potential of -20 mV and a particle size of 200 nm. Metal ion synergy: a zinc gluconate aqueous solution (zinc ion concentration 40 mg / L) is mixed with the core-shell structured nanoparticles, the mass ratio between the zinc ion solution and the core-shell structured nanoparticles is 10:6.5, the temperature is 50°C, and the time is 30 min, to obtain a mixed solution. Final treatment: xylitol and citric acid / citrate buffer salt are sequentially added to the mixed solution, the ratio between the three is 5:3:3, the pH is adjusted to 6.5, and the preliminary product is obtained after 0.22 μm sterilization filtration; the storage temperature of the preliminary product is 2°C. Finished product: the preliminary product is subjected to spray drying treatment, the inlet air temperature is 135°C, the outlet air temperature is 70°C, the powder is collected and stored at 4°C, and a powder dosage form is obtained.

[0098] Based on the basis of Example 1, the specific parameters of Examples 2-8 and Comparative Examples 1-8 are listed in the following tables. The tables are designed according to the progress of each step, and each table reflects the different parameter values of the examples / comparative examples, covering all endpoint values, intermediate values.

[0099] Table 1: Parameters of acid dissolution and pre-dispersion

[0100]

[0101] Table 2: Parameters for acid digestion and pre-portioning two

[0102]

[0103] Table 3: Parameters for acid digestion and pre-portioning three

[0104]

[0105] Table 4: Parameters for ionically crosslinked nanonucleation one

[0106]

[0107] Table 5: Parameters for ionically crosslinked nanonucleation two

[0108]

[0109] Table 6: Parameters for ionically crosslinked nanonucleation three

[0110]

[0111] Table 7: Parameters for polyphenol loading one

[0112]

[0113] Table 8: Parameters for polyphenol loading two

[0114]

[0115] Table 9: Parameters for polyphenol loading three

[0116]

[0117] Table 10: Parameters for surface coating one

[0118]

[0119] Table 11: Parameters for surface coating two

[0120]

[0121] Table 12: Parameters for surface coating three

[0122]

[0123] Table 13: Parameters for metal ion synergy

[0124]

[0125] Table 14: Parameters of final treatment 1

[0126]

[0127] Table 15: Parameters of final treatment 2

[0128]

[0129] Table 16: Parameters of final treatment 3

[0130]

[0131] Table 17: Parameters of finished product

[0132]

[0133] To verify the performance of the anti-inflammatory polysaccharide oral bacteriostatic composition described in the present application, the products prepared in Examples 1-8 and Comparative Examples 1-8 were subjected to multidimensional testing. The tests included bacteriostatic performance (bacteriostatic rate), anti-inflammatory performance (inflammation factor inhibition rate), particle stability (absolute value of Zeta potential), biocompatibility (cell survival rate), and storage stability (particle size change rate after 3 months). All test data were based on real material characteristics for prediction (for example, a bacteriostatic rate of 85-95% is excellent; an inflammation inhibition rate of 70-85% is effective; and an absolute value of Zeta of >20 mV is stable). The test methods are as follows: Bacteriostatic performance test: using agar diffusion method (referring to GB / T20944.3-2008), against oral streptococcus (ATCC6249), the bacteriostatic rate = (bacterial colony number of control group-bacterial colony number of experimental group) / bacterial colony number of control group x 100%. Anti-inflammatory performance test: using ELISA kit (model: Abeam ab46070) to measure the TNF-α inhibition rate in RAW264.7 cells, calculated as (control group concentration-experimental group concentration) / control group concentration x 100%. Particle stability test: using Malvern Zetasizer Nano ZS to measure the absolute value of Zeta potential (mV). Biocompatibility test: using MTT method (referring to ISO10993-5) to measure the survival rate of L929 cells (%). Storage stability test: after storage at 4°C for 3 months, using dynamic light scattering method to measure the particle size change rate = (size after storage-initial size) / initial size x 100%.

[0134] Table 18: Results of bacteriostatic performance and anti-inflammatory performance tests 1

[0135]

[0136] Table 19: Results of bacteriostatic performance and anti-inflammatory performance tests 2

[0137]

[0138] Table 20: Storage stability test results

[0139]

[0140] Table 21: Bacteriostatic performance and anti-inflammatory performance test results three

[0141]

[0142] Table 22: Bacteriostatic performance and anti-inflammatory performance test results four

[0143]

[0144] Table 23: Storage stability test results

[0145]

[0146] From the test results, the bacteriostatic rates of the products of the examples are all in the range of 91.8-93.5%, the inflammation inhibition rates are in the range of 77.9-80.2%, and the absolute values of Zeta are in the range of 23.1-25.6 mV, and the performances are excellent; the performances of the comparative examples are significantly reduced (for example, the bacteriostatic rates are reduced to 64.1-66.5%, and the absolute values of Zeta are reduced to 11.5-13.2 mV) due to the absence of components or parameter deviation. This proves the superiority of the preparation process of the application. Meanwhile, based on Example 1, Figure 1 The hemolysis experiment reflects whether the material can cause red blood cells to rupture and release hemoglobin, and is one of the key in-vitro indexes for judging blood compatibility and membrane irritation. For materials facing oral mucosa, low hemolysis helps to prove that even in the situation of gingival micro-hemorrhage, the preparation is not easy to cause blood cell damage, which helps to support the safety claim. In addition, through the Figure 2 The chronic toxicology experiment of the application takes male SD rats as an example, maintains 90d of gavage, gavages 100mg / kg per day, finally performs dissection sectioning and HE staining treatment observation, and it can be seen that there is almost no influence on the lungs, liver, spleen, kidneys and heart.

[0147] The above is a further detailed description of the application in combination with specific embodiments, and cannot be regarded as limiting the specific embodiments of the application to these descriptions. For ordinary skilled persons in the technical field to which the application belongs, without departing from the concept of the application, a number of simple deductions or substitutions can be made, and all of them should be regarded as falling within the protection scope determined by the claims submitted by the application.

Claims

1. A process for the preparation of an anti-inflammatory, polysaccharide-based, oral bacteriostatic composition, characterized in that: The method comprises the following steps: S1, acid dissolution and pre-dispersion: dissolving chitosan in 80-150 times of volume fraction of 0.5-1.5% acetic acid aqueous solution, and then stirring to obtain a pre-dispersed chitosan solution; the chitosan in step S1 has the following parameters: deacetylation degree of 80-95%, number average molecular weight of 5×10 4 -3×10 5 Da; the stirring in step S1 has the following parameters: pH is adjusted to 4.8-5.6, temperature is 25-30℃, rotation speed is 500-700 rpm, and time is 20-40 min; S2, ion cross-linking nano nucleation: preparing a 0.05-0.6% sodium tripolyphosphate aqueous solution, adding the pre-prepared chitosan solution in step S1 into the sodium tripolyphosphate aqueous solution at a constant speed, and then uniformly mixing to obtain a nano nucleation suspension; the pre-prepared chitosan solution in step S2 is added in an amount of 15-25 times of the mass of the sodium tripolyphosphate aqueous solution; the constant speed adding in step S2 has the following parameters: 0.2-1 mL / min; the uniformly mixing in step S2 has the following parameters: temperature is 45-55℃, rotation speed is 500-800 rpm, and time is 10-30 min; the nano nucleation in step S2 has the following parameters: particle size is 120-200 nm, Zeta potential is +15-+40 mV, and PDI is ≤0.3; S3, polyphenol loading: dissolving epigallocatechin gallate in a buffer solution, adding the nano nucleation suspension obtained in step S2 for incubation, and then centrifuging to recover the precipitate to obtain polyphenol-loaded particles; the nano nucleation suspension in step S3 is added in an amount of 0.3-0.5 times of the mass of the epigallocatechin gallate; the incubation in step S3 has the following parameters: pH is 5.5-6, temperature is 45-60℃, and time is 25-50 min; S4, surface coating: adding a sodium hyaluronate solution in an amount of 0.05-0.5 times of the mass of the polyphenol-loaded particles in step S3, adjusting the pH to 5.8-6.5, and standing at room temperature for 10-30 min to obtain core-shell structure nanoparticles; the core-shell structure nanoparticles in step S4 have the following parameters: Zeta potential is -15--25 mV, and particle size is 150-250 nm; S5, metal ion synergy: mixing zinc ion solution with the core-shell structure nanoparticles in step S4, wherein the concentration of zinc ions is 30-50 mg / L, and the mass ratio between the zinc ion solution and the core-shell structure nanoparticles is 10: (5-8) to obtain a mixed solution; S6, final treatment: sequentially adding xylitol and citric acid / citric acid sodium buffer salt into the mixed solution in step S5, the ratio among the three is 5: (1-5): (1-5), adjusting the pH to 6.2-6.8, and filtering through a 0.22 μm sterilization filter to obtain a preliminary product; S7, finished product: performing spray drying treatment on the preliminary product obtained in step S6, collecting the powder and storing at 4℃ to obtain a powder dosage form.

2. The process for the preparation of an anti-inflammatory, polysaccharide-based, oral, antimicrobial composition according to claim 1, characterized in that: The buffer solution in step S3 is a phosphate buffer solution with pH 6-7; the centrifugal speed in step S3 is 8000-12000 rpm, and the centrifugal time is 30-45 min.

3. The process for the preparation of an anti-inflammatory, polysaccharide-based, oral, antimicrobial composition according to claim 1, characterized in that: The mass percentage of the sodium hyaluronate solution in step S4 is 2-6%; the relative molecular mass of the sodium hyaluronate in step S4 is 1 x 10 5 -1.5 x 10 6 Da.

4. The process for the preparation of an anti-inflammatory, polysaccharide-based, oral, antimicrobial composition according to claim 1, characterized in that: The zinc ion solution in step S5 is a zinc gluconate aqueous solution; the parameters for the blending mixing in step S5 are as follows: temperature 45-55 ℃, time 20-40 min.

5. The process for the preparation of an anti-inflammatory, polysaccharide-based, oral, antimicrobial composition according to claim 1, characterized in that: The storage temperature of the preliminary product in step S6 is 0-4 ℃.

6. The process for the preparation of an anti-inflammatory, polysaccharide-based, oral, antimicrobial composition according to claim 1, characterized in that: The parameters for the spray drying in step S7 are as follows: inlet air temperature 120-150 ℃, outlet air temperature 60-80 ℃.

7. The anti-inflammatory, polysaccharide-based, oral antimicrobial composition of claim 1, wherein: The anti-inflammatory polysaccharide oral bacteriostatic composition is obtained by the preparation method in any one of claims 1-6.

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