A pharmaceutical composition for the repair of oral gingivitis and its preparation method

Abstract

This invention relates to a pharmaceutical composition for the repair of oral gingivitis and its preparation method, belonging to the field of biomedical technology. The pharmaceutical composition includes *Lactobacillus salivarius* fermentation extract, *Viola yedoensis* extract, cicada nymph extract peptide, guava polyphenol, and resveratrol. The *Lactobacillus salivarius* fermentation extract is obtained by aerobic and anaerobic fermentation of *Lactobacillus salivarius*, and purified by XAD7HP macroporous adsorption resin column. The *Viola yedoensis* extract is obtained by enzymatic hydrolysis of the whole *Viola yedoensis* herb by pectinase and cellulase, followed by enzymatic hydrolysis by a complex protease, and purified by a complex adsorption column. The cicada nymph extract peptide is obtained by stepwise enzymatic hydrolysis of cicada nymphs by papain and trypsin, yielding a 5kDa enzymatic hydrolysate, and purified by Q agarose gel FF adsorption column. The components are used in a specific ratio to achieve precise antibacterial activity, exhibiting highly effective anti-inflammatory and antioxidant properties, and are safe and reliable.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a pharmaceutical composition for the repair of oral gingivitis and its preparation method. Background Technology

[0002] Oral gingivitis repair medications aim to eliminate pathogens, relieve inflammation, and promote tissue repair. Existing technologies have formed a product system that integrates clinical treatment and daily care. Based on the application scenario and intensity, existing technologies can be divided into clinical treatment and daily care categories. These two categories have different focuses in terms of ingredient selection and mechanism of action, covering the treatment needs of mild to severe gingivitis.

[0003] Clinical treatment techniques must be used under professional guidance. For moderate to severe gingivitis or acute flare-ups, the core focus is on rapidly controlling inflammation and repairing damaged tissue. Local antibacterial techniques are central to clinical treatment, using high concentrations of antibiotics precisely targeted at the lesion. Mainstream techniques include: chlorhexidine mouthwash, which achieves broad-spectrum antibacterial activity by disrupting bacterial cell membranes and rapidly relieving gingival bleeding; minocycline gel and metronidazole gel, applied topically to the gingival sulcus, showing significant inhibitory effects against anaerobic bacteria, suitable for severe gingivitis or localized inflammatory lesions; ibuprofen gel and diclofenac sodium spray, which reduce prostaglandin synthesis by inhibiting cyclooxygenase, relieving gingival redness, swelling, and pain, suitable for symptomatic treatment during acute flare-ups; recombinant human epidermal growth factor, which promotes gingival fibroblast proliferation and accelerates wound healing; and hyaluronic acid gel, which forms a protective film over the gums while regulating the release of inflammatory factors, possessing both anti-inflammatory and reparative effects, suitable for moderate gingivitis accompanied by gingival defects.

[0004] Daily care techniques are designed for patient self-use, aiming to prevent and consolidate treatment effects. They are often integrated into oral care products, such as functional toothpaste. The core is the addition of low-concentration active ingredients, such as triclosan and zinc salts, which reduce plaque buildup by inhibiting bacterial metabolism; dipotassium glycyrrhizate to relieve mild gingival redness and swelling; and tea tree oil and peppermint oil for gentle antibacterial effects.

[0005] While current drug formulations can meet the basic treatment needs of gingivitis, improvements are still needed in efficacy, safety, and specificity to better meet patients' treatment requirements. Specifically, these improvements include the following aspects:

[0006] (1) Antibacterial technology: While killing pathogenic bacteria, broad-spectrum antibacterial drugs such as chlorhexidine and minocycline can also destroy the normal oral flora. Long-term use can easily lead to flora imbalance and increase the risk of other oral diseases.

[0007] (2) Inability to precisely target pathogens: Most existing antibacterial drugs lack specific targeting and have an inhibitory effect on all bacteria in dental plaque. They cannot be effective against core pathogens such as Porphyromonas gingivalis and Forsythia suspensa, resulting in low treatment efficiency.

[0008] (3) Anti-inflammatory repair techniques: mainly for short-term symptomatic relief, with weak long-term repair capabilities. Ibuprofen, diclofenac sodium, and other drugs can only suppress the inflammatory response and cannot eliminate pathogens or repair damaged tissues. Relapse is common after discontinuation of the drug, and long-term use can irritate the oral mucosa, leading to dryness and burning pain. Although recombinant human epidermal growth factor can promote the proliferation of gingival fibroblasts, it has poor availability and high cost.

[0009] (4) Some drugs have side effects and poor safety with long-term use. Summary of the Invention

[0010] To address the shortcomings of existing gingivitis treatments, such as poor antibacterial specificity, poor safety profiles in some drugs, and insufficient anti-inflammatory and restorative efficacy, this invention provides a pharmaceutical composition for gingivitis repair and its preparation method. The composition, prepared using a special method, combines *Lactobacillus salivarius* fermentation extract, *Viola yedoensis* extract, and cicada nymph extract peptides with guava polyphenols and resveratrol. This combination provides precise antibacterial action, highly effective anti-inflammatory and antioxidant properties, and is safe and reliable. It can be formulated into various dosage forms, including patches, gels, and mouthwashes, to suit different gingivitis flare-up scenarios. The specific technical solution is as follows:

[0011] A pharmaceutical composition for the repair of oral gingivitis, comprising Lactobacillus salivarius fermentation extract, Viola yedoensis extract, cicada nymph extract peptide, guava polyphenols and resveratrol in a mass ratio of (3-4.5):(2-3):(1.5-2.5):(1-1.5):(3-5);

[0012] The *Lactobacillus salivarius* fermentation extract is obtained by aerobic and anaerobic fermentation of *Lactobacillus salivarius*, and then purified and extracted using an XAD7HP macroporous adsorption resin column. The *Viola yedoensis* extract is obtained by enzymatic hydrolysis of the whole *Viola yedoensis* herb by pectinase and cellulase, followed by enzymatic hydrolysis by a complex protease, and then purified and extracted using a complex adsorption column. The complex protease includes bromelain and subtilisin. The packing material of the complex adsorption column includes HP-20 macroporous resin and polyamide resin. The cicada nymph extract peptide is obtained by stepwise enzymatic hydrolysis of cicada nymphs by papain and trypsin, yielding a 5kDa enzymatic hydrolysate, and then purified and extracted using a Q agarose gel FF adsorption column.

[0013] The preparation method of the Lactobacillus salivarius fermentation extract in the above-mentioned pharmaceutical composition includes: inoculating Lactobacillus salivarius into a fermentation medium, and performing aerobic fermentation and anaerobic fermentation in sequence; centrifuging, taking the supernatant, filtering for sterilization, concentrating the filtrate under reduced pressure, loading the sample onto an XAD7HP macroporous adsorption resin column, first rinsing with deionized water to remove impurities, then eluting with an ethanol aqueous solution, collecting the eluent, concentrating the eluent under reduced pressure to remove ethanol, and freeze-drying to obtain the Lactobacillus salivarius fermentation extract.

[0014] In the above method for preparing Lactobacillus salivarius fermentation extract, the Lactobacillus salivarius has a bacterial count of 1×10⁻⁶. 9 CFU / mL ~5×10 9 The inoculation volume of the *Lactobacillus salivarius* activated bacterial culture (CFU / mL) is 5%–8% of the fermentation medium. The fermentation medium is MRS medium supplemented with 1 wt%–1.5 wt% glucose. The aerobic fermentation is carried out at 36°C–38°C, 100 rpm–150 rpm, and with sterile air circulation for 8–12 hours. The aeration ratio of the sterile air is 0.5 vvm–1 vvm. During the aerobic fermentation, the pH is maintained at 6.0–6.4 using a 0.5 mol / L–0.8 mol / L sodium hydroxide aqueous solution. The anaerobic fermentation is carried out with aeration stopped and at 36°C–38°C, 50 rpm–100 rpm for 20–24 hours.

[0015] In the above method for preparing the fermented extract of *Lactobacillus salivarius*, the centrifugation is performed at 4℃~8℃ and 8000rpm~8500rpm for 15min~25min; the sterilization by filtration is performed through a 0.22μm filter membrane; the filtrate is concentrated under reduced pressure at 45℃~50℃ to 25%~35% of its volume; the elution volume of deionized water is 2BV~3BV, and the elution flow rate is 1.5BV / h~2.5BV / h; the volume concentration of the ethanol aqueous solution is 40%~50%; the elution volume of the ethanol aqueous solution is 3BV~4BV, and the elution flow rate is 1BV / h~1.5BV / h; and the temperature for concentrated under reduced pressure of the eluent is 45℃~50℃.

[0016] The preparation method of the Viola yedoensis extract in the above-mentioned pharmaceutical composition includes: taking dried Viola yedoensis whole herb powder, adding extractant at a material-to-liquid mass ratio of 1:(10-12), and reflux extraction; cooling to 50℃-55℃ and adjusting pH to 4.5-5.0, adding pectinase and cellulase, and enzymatically hydrolyzing at 50℃-55℃ for 2-3 hours; adjusting pH to 6.5-7.5, adding complex protease, and enzymatically hydrolyzing at 50℃-55℃ for 1.5-2 hours; inactivating enzymes, centrifuging, taking the supernatant and concentrating under reduced pressure, loading the sample onto a composite adsorption column, rinsing with deionized water and 30%-40% volume concentration ethanol aqueous solution to remove impurities, and finally eluting with 70%-75% volume concentration ethanol aqueous solution, collecting the eluent, concentrating under reduced pressure to remove ethanol, and freeze-drying to obtain Viola yedoensis extract.

[0017] In the above method for preparing Viola yedoensis extract, the particle size of the powder is sieved through a 40-60 mesh sieve; the extractant is a 70%-75% volume concentration ethanol aqueous solution containing 0.1wt%-0.15wt% citric acid; the reflux extraction is performed at 80℃-85℃ for 1-2 hours; the amount of pectinase added is 0.8%-1.5% of the powder mass; the amount of cellulase added is 0.8%-1.5% of the powder mass; the amount of complex protease added is 1.5%-2% of the powder mass; the complex protease is bromelain and subtilisin in a mass ratio of (2-3):(1-1.5); the stirring speed for enzymatic hydrolysis is 60-80 rpm; and the enzyme inactivation is performed by heating to 80℃-85℃. After 15-20 minutes, the solution is cooled to room temperature; the centrifugation is performed at 8000-8500 rpm for 10-15 minutes; the supernatant is concentrated under reduced pressure at 45-50°C to 30%-40% of its volume; the upper packing material of the composite adsorption column is HP-20 macroporous resin, and the lower packing material is polyamide resin; the volume ratio of HP-20 macroporous resin to polyamide resin is (1.5-2):1; the elution volume of deionized water is 2-3 BV, the elution volume of the 30%-40% volume concentration ethanol aqueous solution is 1-2 BV, the elution volume of the 70%-75% volume concentration ethanol aqueous solution is 3-4 BV; the reduced pressure concentration temperature of the eluent is 45-50°C.

[0018] The preparation method of the cicada nymph extract peptide in the above-mentioned pharmaceutical composition includes: taking dried cicada nymph powder, adding phosphate buffer at a material-to-liquid mass ratio of 1:(12-15), adding papain, enzymatically hydrolyzing at 55℃-60℃ for 1-1.5h, inactivating the enzyme, centrifuging, taking the supernatant, adjusting the pH to 8.0-8.5, adding trypsin, enzymatically hydrolyzing at 36℃-40℃ for 1-1.5h, inactivating the enzyme; ultrafiltration through an ultrafiltration membrane, taking the permeate and concentrating under reduced pressure, adjusting the pH to 8.0-8.5, loading the sample onto a Q agarose gel FF adsorption column, rinsing with Tris-HCl buffer at pH 8.0-8.5 to remove impurities, then eluting sequentially with 0.1mol / L-0.15mol / L sodium chloride aqueous solution and 0.2mol / L-0.3mol / L sodium chloride aqueous solution, combining the eluents and concentrating under reduced pressure, placing in a dialysis bag, dialyzing with deionized water, and freeze-drying to obtain the cicada nymph extract peptide.

[0019] In the above method for preparing cicada nymph extract peptides, the particle size of the freeze-dried cicada nymph powder is sieved through a 60-80 mesh sieve; the pH of the phosphate buffer is 6.5-7.0; the amount of papain added is 0.8%-1.5% of the dry powder mass; the amount of trypsin added is 0.8%-1.5% of the dry powder mass; the stirring speed for enzymatic hydrolysis is 60-80 rpm; the enzyme inactivation is performed by heating to 80-85℃ for 15-20 minutes and then cooling to room temperature; the centrifugation is performed at 4-6℃ and 5000-6000 rpm for 10-15 minutes; and the molecular weight cutoff of the ultrafiltration membrane is [not specified]. The permeate is 5 kDa; the permeate is concentrated under reduced pressure at 45°C to 50°C to 20% to 30% of its original volume; the elution volume of the Tris-HCl buffer solution with pH 8.0 to 8.5 is 2 BV to 3 BV; the elution volume of the 0.1 mol / L to 0.15 mol / L sodium chloride aqueous solution is 1.5 BV to 2 BV, and the elution volume of the 0.2 mol / L to 0.3 mol / L sodium chloride aqueous solution is 1.5 BV to 2 BV; the eluent is concentrated under reduced pressure at 45°C to 50°C to 10% to 15% of its volume; the dialysis bag has a molecular weight cutoff of 100 Da to 500 Da; and the dialysis time is 48 h to 52 h.

[0020] The preparation method of the above-mentioned pharmaceutical composition for repairing oral gingivitis includes: mixing Lactobacillus salivarius fermentation extract, Viola yedoensis extract, Cicada nymph extract peptide, guava polyphenol and resveratrol according to the formula mass ratio to obtain the pharmaceutical composition.

[0021] The above-mentioned pharmaceutical composition is mixed with pharmaceutically available excipients to prepare plasters, gels or mouthwashes.

[0022] The present invention provides a pharmaceutical composition for the repair of oral gingivitis and a method for its preparation, the beneficial effects of which include:

[0023] I. The drug of this invention has the following advantages: (1) Precise antibacterial and microbial balance protection: It can specifically inhibit the core pathogenic bacteria of gingivitis such as Porphyromonas gingivalis and Forsakenella, while having no significant inhibition on beneficial oral bacteria such as Streptococcus salivarius and Lactobacillus acidophilus, thus avoiding the risk of microbial imbalance caused by traditional broad-spectrum antibacterial drugs. (2) Anti-inflammatory, repair and antioxidant effects: It can reduce the release of inflammatory factors such as IL-6 and TNF-α to relieve inflammation, promote the survival of gingival fibroblasts, maintain a high cell survival rate to repair damaged tissue, and enhance antioxidant capacity by scavenging free radicals, thereby reducing the probability of inflammation recurrence. (3) High safety and good applicability: The drug components are extracted and purified by optimized process, with no significant cytotoxicity. Long-term use is not likely to irritate the oral mucosa. It can be prepared into various dosage forms such as plasters, gels, and mouthwashes, which are suitable for different gingivitis attack scenarios, such as local application during acute attacks and daily mouthwash.

[0024] II. The preparation of Lactobacillus salivarius fermentation extract involves a two-step fermentation process of aerobic and anaerobic fermentation to promote the full synthesis of active ingredients such as antimicrobial peptides and organic acids. Subsequent purification using XAD7HP macroporous adsorption resin (medium polarity) can efficiently enrich the target components, reduce impurities, and enhance targeted antibacterial and immunomodulatory capabilities.

[0025] III. In the preparation of Viola yedoensis extract, the enzymatic hydrolysis step employs a stepwise enzymatic hydrolysis using pectinase and cellulase, bromelain and subtilisin, effectively disrupting the cell wall structure. Pectinase and cellulase degrade polysaccharides, while the complex proteases degrade the protein backbone, increasing the dissolution rate of anti-inflammatory, antibacterial, and antioxidant components such as flavonoids, phenolic acids, and saponins. Purification utilizes a composite adsorption column of HP-20 macroporous resin and polyamide resin to achieve comprehensive enrichment of active ingredients, avoiding component loss caused by single resins and enhancing anti-inflammatory and antioxidant properties.

[0026] IV. In the preparation of peptides extracted from cicada nymphs, the enzymatic hydrolysis step uses papain and trypsin for stepwise hydrolysis to specifically generate 5kDa small molecule antimicrobial peptides; the purified peptides are then purified by using a 5kDa ultrafiltration membrane to remove macromolecular impurities and a Q agarose gel FF adsorption column (strong anion exchange) to enrich negatively charged antimicrobial peptides, thereby reducing inactive peptide residues and enhancing antimicrobial and bacterial adhesion inhibition capabilities.

[0027] V. Both guava polyphenols and resveratrol are natural polyphenols. Guava polyphenols enhance the ability to destroy the cell membranes of pathogenic bacteria, while resveratrol strengthens the free radical scavenging effect. Together, they improve the antibacterial and antioxidant properties of the drug, while reducing the risk of toxicity caused by excessive use of a single component. Detailed Implementation

[0028] The present invention will be further described below with reference to specific implementation examples, but the present invention is not limited to these embodiments.

[0029] Example 1

[0030] A pharmaceutical composition for the repair of oral gingivitis comprises Lactobacillus salivarius fermentation extract, Viola yedoensis extract, cicada nymph extract peptide, guava polyphenols and resveratrol in a mass ratio of 3.8:2.5:2:1.2:4.

[0031] The preparation method of Lactobacillus salivarius fermentation extract includes: inoculating Lactobacillus salivarius into MRS medium (formula: peptone 10 g / L, beef extract 10 g / L, yeast extract 5 g / L, glucose 20 g / L, sodium acetate 5 g / L, diammonium citrate 2 g / L, Tween-80 1 mL / L, MgSO4·7H2O 0.58 g / L, MnSO4·4H2O 0.25 g / L, pH 6.3), culturing at 37℃ in an anaerobic environment (85% N2, 10% H2, 5% CO2) for 18 h and subculturing twice to obtain a bacterial count of 3 × 10⁻⁶. 9 CFU / mL activated bacterial solution; inoculate the activated bacterial solution into fermentation medium (MRS medium supplemented with 1.2wt% glucose) at a 6% volume inoculation rate. Fermentation is carried out at 37℃, 120 rpm, and sterile air (0.8 vvm) for 10 h (the pH is maintained between 6.0 and 6.4 using 0.6 mol / L sodium hydroxide aqueous solution during this period), then aeration is stopped and anaerobic fermentation is carried out at 37℃, 80 rpm for 22 h; finally, centrifugation is performed at 6℃, 8200 rpm for 2 seconds. The supernatant was collected at 0 min, filtered through a 0.22 μm filter membrane for sterilization, concentrated under reduced pressure at 48 °C to 30% of its original volume, and then loaded onto an XAD7HP macroporous adsorption resin column (column diameter to height ratio 1:9, loading flow rate 1.5 BV / h). First, it was rinsed with 2.5 BV of deionized water at a flow rate of 2 BV / h to remove impurities, and then eluted with 45% ethanol aqueous solution at a flow rate of 1.2 BV / h for 3.5 BV. The eluent was collected and concentrated under reduced pressure at 48 °C to remove ethanol. The solution was then freeze-dried (pre-freezing temperature -45 °C, sublimation temperature 35 °C) to obtain the final product.

[0032] The preparation method of Viola yedoensis extract includes: pulverizing dried Viola yedoensis whole herb through a 50-mesh sieve to obtain powder; adding a 72% volume concentration ethanol aqueous solution containing 0.12wt% citric acid at a material-to-liquid mass ratio of 1:11; reflux extraction at 82℃ for 1.5h; cooling to 53℃ and adjusting the pH to 4.8; adding 1.2% pectinase and 1% cellulase by weight of the powder; enzymatic hydrolysis at 53℃ for 2.5h (stirring speed 70rpm); then adjusting the pH to 7.0 and adding 1.8% complex protease (pineapple) by weight of the powder. The enzyme was digested at 52℃ for 1.5 h with a ratio of protease to subtilisin (2:1.5) and stirred at 70 rpm. The enzyme was then inactivated at 82℃ for 18 min, cooled to room temperature, and centrifuged at 8200 rpm for 12 min. The supernatant was collected and concentrated under reduced pressure at 48℃ to 35% of its original volume. The supernatant was then loaded onto a composite adsorption column (HP-20 macroporous resin: polyamide resin = 1.8:1 volume ratio, column diameter to height ratio 1:9, loading flow rate 1.5 BV / h). The column was eluted sequentially with 2.5 BV deionized water at 2 BV / h, 1.5 BV 35% ethanol aqueous solution at 1.2 BV / h, and finally eluted with 3.5 BV 73% ethanol aqueous solution at 1.2 BV / h. The eluent was collected, concentrated under reduced pressure at 48℃ to remove ethanol, and then freeze-dried (pre-freezing temperature -45℃, sublimation temperature 35℃) to obtain the final product.

[0033] The preparation method of peptides extracted from cicada nymphs includes: pulverizing freeze-dried cicada nymphs through a 60-mesh sieve to obtain dry powder; adding phosphate buffer solution at pH 6.8 at a material-to-liquid mass ratio of 1:13; adding papain at 1.2% of the dry powder mass; and enzymatically hydrolyzing at 58℃ for 1 hour (stirring speed 70 rpm); then heating to 83℃ to inactivate the enzyme for 18 minutes, cooling to room temperature, and centrifuging at 5℃ and 5500 rpm for 12 minutes to collect the supernatant; adjusting the pH to 8.3; and adding trypsin at 1.3% of the dry powder mass. Enzymatic hydrolysis was performed at 37℃ for 1.5 h (stirring rate 70 rpm), followed by enzyme inactivation at 83℃ for 18 min, and then cooled to room temperature. The solution was ultrafiltered through a 5 kDa ultrafiltration membrane, and the permeate was collected and concentrated under reduced pressure at 48℃ to 25% of its original volume. The pH was adjusted to 8.0, and the solution was loaded onto a Q agarose gel FF column (column diameter to height ratio 1:11, loading flow rate 1 BV / h; the Q agarose gel FF column was equilibrated with Tris-HCl buffer at pH 8.0 before use). Initially, 2.5 BV of the solution was used. The solution was rinsed with pH 8.0 Tris-HCl buffer at a flow rate of 1.5 BV / h to remove impurities, followed by elution with 1.8 BV of 0.12 mol / L sodium chloride aqueous solution and 1.6 BV of 0.25 mol / L sodium chloride aqueous solution at a flow rate of 1 BV / h. The eluents were combined and concentrated under reduced pressure at 48°C to 12% of the original volume. The solution was then placed in a dialysis bag with a molecular weight cutoff of 100 Da to 500 Da and dialyzed with deionized water for 50 h (with the deionized water replaced every 8 h at a dialysis temperature of 5°C). Finally, the solution was freeze-dried (pre-freezing temperature -45°C, sublimation temperature 35°C) to obtain the final product.

[0034] Example 2

[0035] A pharmaceutical composition for the repair of oral gingivitis comprises Lactobacillus salivarius fermentation extract, Viola yedoensis extract, cicada nymph extract peptide, guava polyphenols and resveratrol in a mass ratio of 3:2:1.5:1:3.

[0036] The preparation method of Lactobacillus salivarius fermentation extract includes: inoculating Lactobacillus salivarius into MRS medium (formula: peptone 10 g / L, beef extract 10 g / L, yeast extract 5 g / L, glucose 20 g / L, sodium acetate 5 g / L, diammonium citrate 2 g / L, Tween-80 1 mL / L, MgSO4·7H2O 0.58 g / L, MnSO4·4H2O 0.25 g / L, pH 6.4), culturing at 37℃ in an anaerobic environment (85% N2, 10% H2, 5% CO2) for 18 h and subculturing twice to obtain a bacterial count of 5 × 10⁻⁶. 9 CFU / mL activated bacterial solution; inoculate the activated bacterial solution into fermentation medium (MRS medium supplemented with 1.5wt% glucose) at an 8% volume inoculation rate. Fermentation is carried out at 38℃, 150 rpm, and sterile air (1 vvm) for 12 h (the pH is maintained between 6.0 and 6.4 using 0.8 mol / L sodium hydroxide aqueous solution during this period), then aeration is stopped and anaerobic fermentation is carried out at 38℃, 100 rpm for 24 h; finally, centrifugation is performed at 8℃, 8500 rpm. The supernatant was collected after 25 min, filtered through a 0.22 μm filter membrane for sterilization, concentrated under reduced pressure at 50 °C to 35% of the original volume, and then loaded onto an XAD7HP macroporous adsorption resin column (column diameter to height ratio 1:10, loading flow rate 2 BV / h). First, it was rinsed with 3 BV of deionized water at a flow rate of 2.5 BV / h to remove impurities, and then eluted with 50% ethanol aqueous solution at a flow rate of 1.5 BV / h for 4 BV. The eluent was collected and concentrated under reduced pressure at 50 °C to remove ethanol. The solution was then freeze-dried (pre-freezing temperature -50 °C, sublimation temperature 40 °C) to obtain the final product.

[0037] The preparation method of Viola yedoensis extract includes: taking dried Viola yedoensis whole herb, pulverizing it through a 60-mesh sieve to obtain powder, adding a 75% volume concentration ethanol aqueous solution containing 0.15wt% citric acid at a material-to-liquid mass ratio of 1:12, and refluxing at 85℃ for 2 hours; cooling to 55℃ and adjusting the pH to 5.0, adding 1.5% pectinase and 1.5% cellulase by weight of the powder, and enzymatically hydrolyzing at 55℃ for 3 hours (stirring speed 80 rpm); then adjusting the pH to 7.5, and adding 2% compound protease by weight of the powder (… Bromelain: Subtilisin = 3:1), enzymatic hydrolysis at 55℃ for 2h (stirring rate 80rpm); then heated to 85℃ for 20min to inactivate the enzyme, cooled to room temperature, centrifuged at 8500rpm for 15min, and the supernatant was collected. The supernatant was concentrated to 40% of the original volume at 50℃ and loaded onto a composite adsorption column (HP-20 macroporous resin: polyamide resin = 2:1 volume ratio, column diameter to height ratio 1:10, loading flow rate 2BV / h). The column was eluted sequentially with 3BV deionized water at 2.5BV / h, 2BV 40% ethanol aqueous solution at 1.5BV / h to remove impurities, and finally eluted with 4BV 75% ethanol aqueous solution at 1.5BV / h. The eluent was collected, concentrated at 50℃ under reduced pressure to remove ethanol, and freeze-dried (pre-freezing temperature -50℃, sublimation temperature 40℃) to obtain the final product.

[0038] The preparation method of peptides extracted from cicada nymphs includes: pulverizing freeze-dried cicada nymphs through an 80-mesh sieve to obtain dry powder; adding phosphate buffer solution at pH 7.0 at a material-to-liquid mass ratio of 1:15; adding papain at 1.5% of the dry powder mass; enzymatically hydrolyzing at 60℃ for 1.5 h (stirring speed 80 rpm); heating to 85℃ to inactivate the enzyme for 20 min; cooling to room temperature; centrifuging at 6℃ and 6000 rpm for 15 min; collecting the supernatant; adjusting the pH to 8.5; and adding trypsin at 1.5% of the dry powder mass. Enzyme was hydrolyzed at 40℃ for 1.5 h (stirring rate 80 rpm), then heated to 85℃ for 20 min to inactivate the enzyme, and then cooled to room temperature. The solution was ultrafiltered through a 5 kDa ultrafiltration membrane, and the permeate was concentrated under reduced pressure at 50℃ to 30% of its original volume. The pH was adjusted to 8.2, and the solution was loaded onto a Q agarose gel FF column (column diameter to height ratio 1:12, loading flow rate 1.2 BV / h). The Q agarose gel FF column was equilibrated with Tris-HCl buffer at pH 8.2 before use. Initially, 3 BV was used. The solution was rinsed with Tris-HCl buffer at pH 8.2 at a flow rate of 2 BV / h to remove impurities. Then, it was eluted sequentially with 2 BV of 0.15 mol / L sodium chloride aqueous solution and 2 BV of 0.3 mol / L sodium chloride aqueous solution at a flow rate of 1.2 BV / h. The eluents were combined and concentrated under reduced pressure at 50°C to 15% of the original volume. The solution was then placed in a dialysis bag with a molecular weight cutoff of 100 Da to 500 Da and dialyzed with deionized water for 52 h (with the deionized water replaced every 8 h at a dialysis temperature of 6°C). Finally, it was freeze-dried (pre-freezing temperature -50°C, sublimation temperature 40°C) to obtain the final product.

[0039] Example 3

[0040] A pharmaceutical composition for the repair of oral gingivitis comprises Lactobacillus salivarius fermentation extract, Viola yedoensis extract, cicada nymph extract peptide, guava polyphenols and resveratrol in a mass ratio of 4.5:3:2.5:1.5:5.

[0041] The preparation method of Lactobacillus salivarius fermentation extract includes: inoculating Lactobacillus salivarius into MRS medium (formula: peptone 10 g / L, beef extract 10 g / L, yeast extract 5 g / L, glucose 20 g / L, sodium acetate 5 g / L, diammonium citrate 2 g / L, Tween-80 1 mL / L, MgSO4·7H2O 0.58 g / L, MnSO4·4H2O 0.25 g / L, pH 6.2), culturing at 37℃ in an anaerobic environment (85% N2, 10% H2, 5% CO2) for 18 h and subculturing twice to obtain a bacterial count of 1×10⁻⁶. 9 CFU / mL activated bacterial solution; inoculate the activated bacterial solution into fermentation medium (MRS medium with 1 wt% glucose) at a 5% volume inoculation rate. Fermentation is carried out at 36℃, 100 rpm, and sterile air (0.5 vvm) for 8 h (the pH is maintained between 6.0 and 6.4 during this period using 0.5 mol / L sodium hydroxide aqueous solution). Then, aeration is stopped and anaerobic fermentation is carried out at 36℃, 50 rpm for 20 h. Finally, centrifugation is performed at 4℃, 8000 rpm. Collect the supernatant after 15 min, filter it through a 0.22 μm filter membrane for sterilization, concentrate it under reduced pressure at 45 °C to 25% of the original volume, and then load it onto an XAD7HP macroporous adsorption resin column (column diameter to height ratio 1:8, loading flow rate 1 BV / h). First, rinse with 2 BV of deionized water at a flow rate of 1.5 BV / h to remove impurities, then elute with 3 BV of 40% ethanol aqueous solution at a flow rate of 1 BV / h. Collect the eluent, concentrate it under reduced pressure at 45 °C to remove ethanol, and freeze-dry it (pre-freezing temperature -40 °C, sublimation temperature 30 °C) to obtain the final product.

[0042] The preparation method of Viola yedoensis extract includes: pulverizing dried Viola yedoensis whole herb through a 40-mesh sieve to obtain powder; adding a 70% volume concentration ethanol aqueous solution containing 0.1wt% citric acid at a material-to-liquid mass ratio of 1:10; reflux extraction at 80℃ for 1 hour; cooling to 50℃ and adjusting the pH to 4.5; adding 0.8% pectinase and 0.8% cellulase by weight of the powder; enzymatic hydrolysis at 50℃ for 2 hours (stirring speed 60 rpm); then adjusting the pH to 6.5 and adding 1.5% complex protease (pineapple) by weight of the powder. The enzyme was digested at 50°C for 1.5 h with a ratio of protease to subtilisin (2:1.5). The enzyme was then inactivated at 80°C for 15 min and cooled to room temperature. The supernatant was collected by centrifugation at 8000 rpm for 10 min and concentrated under reduced pressure at 45°C to 30% of its original volume. The supernatant was then loaded onto a composite adsorption column (HP-20 macroporous resin: polyamide resin = 1.5:1 volume ratio, column diameter to height ratio 1:8, loading flow rate 1 BV / h). The column was eluted sequentially with 2 BV of deionized water at a flow rate of 1.5 BV / h, 1 BV of 30% ethanol aqueous solution at a flow rate of 1 BV / h, and finally eluted with 3 BV of 70% ethanol aqueous solution at a flow rate of 1 BV / h. The eluent was collected, concentrated under reduced pressure at 45°C to remove ethanol, and then freeze-dried (pre-freezing temperature -40°C, sublimation temperature 30°C) to obtain the final product.

[0043] The preparation method of peptides extracted from cicada nymphs includes: pulverizing freeze-dried cicada nymphs through a 60-mesh sieve to obtain dry powder; adding phosphate buffer solution at pH 6.5 at a material-to-liquid mass ratio of 1:12; adding papain at 0.8% of the dry powder mass; and enzymatically hydrolyzing at 55℃ for 1 hour (stirring speed 60 rpm); then heating to 80℃ to inactivate the enzyme for 15 minutes, cooling to room temperature, and centrifuging at 4℃ and 5000 rpm for 10 minutes to collect the supernatant; adjusting the pH to 8.0; and adding trypsin at 0.8% of the dry powder mass. Enzyme was hydrolyzed at 36℃ for 1 h (stirring rate 60 rpm), then heated to 80℃ for 15 min to inactivate the enzyme, and then cooled to room temperature. The solution was ultrafiltered through a 5 kDa ultrafiltration membrane, and the permeate was concentrated under reduced pressure at 45℃ to 20% of its original volume. The pH was adjusted to 8.5, and the solution was loaded onto a Q agarose gel FF column (column diameter to height ratio 1:10, loading flow rate 0.8 BV / h). The Q agarose gel FF column was equilibrated with Tris-HCl buffer at pH 8.5 before use. A 2 BV initial loading was used. The solution was rinsed with pH 8.5 Tris-HCl buffer at a flow rate of 1 BV / h to remove impurities, followed by elution with 1.5 BV 0.1 mol / L sodium chloride aqueous solution and 1.5 BV 0.2 mol / L sodium chloride aqueous solution at a flow rate of 0.8 BV / h. The eluents were combined and concentrated under reduced pressure at 45°C to 10% of the original volume. The solution was then placed in a dialysis bag with a molecular weight cutoff of 100 Da to 500 Da and dialyzed with deionized water for 48 h (with the deionized water replaced every 8 h at a dialysis temperature of 4°C). Finally, the solution was freeze-dried (pre-freezing temperature -40°C, sublimation temperature 30°C) to obtain the final product.

[0044] The preparation methods of the pharmaceutical compositions in the above embodiments involve mixing Lactobacillus salivarius fermentation extract, Viola yedoensis extract, cicada nymph extract peptide, guava polyphenols, and resveratrol according to the formula mass ratios of each embodiment to obtain the pharmaceutical composition. The pharmaceutical composition is then mixed with pharmaceutically available excipients to prepare plasters, gels, or mouthwashes.

[0045] Comparative Example 1

[0046] The difference from Example 1 is that the mass ratio is Lactobacillus salivarius fermentation extract: Viola yedoensis extract: Cicada nymph extract peptide: Guava polyphenol: Resveratrol = 1.5:4.8:2:1.2:4.

[0047] Comparative Example 2

[0048] The difference from Example 1 is that the mass ratio is Lactobacillus salivarius fermentation extract: Viola yedoensis extract: Cicada nymph extract peptide: Guava polyphenol: Resveratrol = 3.8:1:3.5:1.2:4.

[0049] Comparative Example 3

[0050] The difference from Example 1 is that the mass ratio is Lactobacillus salivarius fermentation extract: Viola yedoensis extract: Cicada nymph extract peptide: Guava polyphenol: Resveratrol = 1:2.5:4.8:1.2:4.

[0051] Comparative Example 4

[0052] The difference from Example 1 is that in the preparation of Lactobacillus salivarius fermentation extract, XAD7HP macroporous adsorption resin is replaced with HP-20 macroporous resin.

[0053] Comparative Example 5

[0054] The difference from Example 1 is that in the preparation of Viola yedoensis extract, bromelain is replaced with papain.

[0055] Comparative Example 6

[0056] The difference from Example 1 is that in the preparation of Viola yedoensis extract, Bacillus subtilis protease is replaced with papain.

[0057] Comparative Example 7

[0058] The difference from Example 1 is that no Bacillus subtilis protease is added in the preparation of Viola yedoensis extract.

[0059] Comparative Example 8

[0060] The difference from Example 1 is that in the preparation of Viola yedoensis extract, the composite adsorption column is only filled with HP-20 macroporous resin.

[0061] Comparative Example 9

[0062] The difference from Example 1 is that in the preparation of Viola yedoensis extract, the composite adsorption column is filled only with polyamide resin.

[0063] Comparative Example 10

[0064] The difference from Example 1 is that in the preparation of Viola yedoensis extract, HP-20 macroporous resin is replaced with XAD7HP macroporous adsorption resin.

[0065] Comparative Example 11

[0066] The difference from Example 1 is that trypsin is not used for enzymatic hydrolysis in the preparation of peptides extracted from cicada nymphs.

[0067] Comparative Example 12

[0068] The difference from Example 1 is that in the preparation of cicada nymph extract peptides, papain is replaced with bromelain.

[0069] Comparative Example 13

[0070] The difference from Example 1 is that in the preparation of peptides extracted from cicada nymphs, Q agarose gel FF is replaced with SP agarose gel FF.

[0071] The raw materials used in the above embodiments and comparative examples are as follows: Guava polyphenols were sourced from Shaanxi Guanchen Biotechnology Co., Ltd., with a mass content of 40%. Resveratrol was sourced from Taiyuan Bailong Biotechnology Co., Ltd., with a mass content of 98%. Lactobacillus salivarius was sourced from Wuhan Ruichen Standard Materials Technology Co., Ltd., strain number BNCC138618. Peptone (bovine bone), beef extract, and yeast extract in the MRS medium were sourced from Shandong Xinfuwo Bioengineering Co., Ltd. XAD7HP macroporous adsorption resin was sourced from Shanghai Yuanye Biotechnology Co., Ltd., model S14164. Pectinase was sourced from Shandong Shenchuang Biotechnology Co., Ltd., with an enzyme activity of 30,000 U / g. Cellulase was sourced from Nanjing Jingchang Biotechnology Co., Ltd., with an enzyme activity of 100,000 U / g. Bromelain was sourced from Peptide Biotechnology (Xi'an) Co., Ltd., with an enzyme activity of 100,000 U / g. Bacillus subtilis protease was sourced from Jiangsu Duoyang Bioengineering Technology Co., Ltd., with an enzyme activity of 20,000 U / g. HP-20 macroporous resin was sourced from Shanghai Yuanye Biotechnology Co., Ltd., model S27267. Polyamide resin was sourced from Shanghai Yuanye Biotechnology Co., Ltd., model S14143. Papain was obtained from Peptide Biotechnology (Xi'an) Co., Ltd., with an enzyme activity of 100,000 U / g. Trypsin was obtained from Xi'an Xinlu Biotechnology Co., Ltd., with an enzyme activity of 250,000 U / g. Q agarose gel FF was obtained from Shanghai Yuanye Biotechnology Co., Ltd., model S14074. SP agarose gel FF was obtained from Shanghai Yuanye Biotechnology Co., Ltd., model S14077.

[0072] I. Cytotoxicity test:

[0073] Sample preparation: The drug composition powders of each example and comparative example were vortexed with DMSO until completely dissolved to prepare a 100 mg / mL stock solution, which was then filtered through a 0.22 μm filter membrane for sterilization. When used, the samples were diluted to 10 μg / mL and 100 μg / mL concentrations with DMEM medium (containing 0.1% DMSO) containing 10% FBS and 1% penicillin-streptomycin.

[0074] The detection method included: human gingival fibroblasts (HGF-1) cells were cultured in a 37°C, 5% CO2 incubator in DMEM medium containing 10% FBS and 1% penicillin-streptomycin, at a concentration of 1×10⁻⁶ cells / mL. 4 Cells were seeded at a density of 0.5 g / well in 96-well plates and cultured for 24 hours until cell adhesion. The medium was then replaced with different concentrations of the sample (DMSO final concentration 0.1%), with a blank control (DMEM medium only) and a negative control (DMEM medium containing 0.1% DMSO, with cells). Cultured for another 24 hours, 20 μL of 5 mg / mL MTT solution was added to each well and incubated for 4 hours. After discarding the supernatant, 150 μL of DMSO was added and shaken for 10 minutes to dissolve the formazan. The absorbance (OD value) was measured at 570 nm using a microplate reader. Three replicates were performed for each assay.

[0075] Calculate cell viability: Cell viability (%) = (OD sample - OD blank) / (OD negative control - OD blank) × 100%.

[0076] II. Antibacterial activity test:

[0077] Sample preparation: Take the drug composition powder of each example and comparative example, dissolve it in sterile DMSO and vortex for 10 minutes to prepare a stock solution of 100 mg / mL. After sterilization by filtration through a 0.22 μm sterile filter membrane, store it in a sealed container at 4°C. Before use, dilute the stock solution with sterile physiological saline to a concentration of 4 mg / mL.

[0078] Strains selected: Pathogenic bacteria included *Porphyromonas gingivalis* (Pg, ATCC 33277), *Focusella foetida* (Fn, ATCC 43037), and *Prevotella intermedius* (Pi, ATCC 25611). Beneficial bacteria included *Streptococcus salivarius* (Ss, ATCC 13419), *Streptococcus griseus* (Sg, ATCC 10558), and *Lactobacillus acidophilus* (La, ATCC 4356). All strains were pre-cultured in appropriate media to the logarithmic growth phase (pathogenic bacteria cultured 48 hours anaerobic, beneficial bacteria cultured 18 hours CO2), and the bacterial suspension concentration was adjusted to 1.5 × 10⁻⁶ with sterile physiological saline. 8 CFU / mL.

[0079] The detection methods included: for pathogenic bacteria, BHI agar (pH 7.2) containing 5% defibrinated sheep blood and 0.001% vitamin K1 was used; for beneficial bacteria, BHI agar (pH 7.2) was used for *Streptococcus salivarius* and *Streptococcus griseus*, and MRS agar (pH 6.2) was used for *Lactobacillus acidophilus*. 20 mL of culture medium was poured into each plate and allowed to solidify at room temperature for 30 minutes. 100 μL of a 1.5 × 10⁻⁶ concentration was then collected. 8 A bacterial suspension of CFU / mL was evenly spread along different directions on an agar plate using a sterile L-shaped glass spreader, and allowed to stand for 5 minutes to absorb excess liquid. Sterile filter paper discs (6 mm in diameter, 1 mm thick, qualitative filter paper material) were precisely soaked with 10 μL of sample solution (4 mg / mL), and allowed to drain at room temperature for 2 minutes. The discs were then affixed to the agar surface using sterile forceps. Pathogenic bacteria plates were incubated at 37°C for 48 hours in an anaerobic environment of 85% N2, 10% H2, and 5% CO2; beneficial bacteria plates were incubated at 37°C for 24 hours in an anaerobic environment of 5% CO2 and 95% air. Three replicates were prepared for each method.

[0080] Evaluation index: Diameter of inhibition zone (mm).

[0081] III. Anti-inflammatory activity assay (LPS-induced RAW264.7 cell inflammation model):

[0082] Sample preparation: The stock solution was prepared in the same way as for cytotoxicity testing, and the sample was diluted to a concentration of 10 μg / mL with DMEM medium (containing 0.1% DMSO) containing 10% FBS and 1% penicillin-streptomycin.

[0083] The detection method included: RAW264.7 macrophages (ATCC TIB-71) were cultured in a 37°C, 5% CO2 incubator in DMEM medium containing 10% FBS and 1% penicillin-streptomycin, at a concentration of 5 × 10⁻⁶ cells / year. 4Cells were seeded at a density per well in 24-well plates and cultured for 24 hours. The medium was then replaced with medium containing 1 μg / mL LPS (E. coli O111:B4) and the sample. A blank control (DMEM medium only) and a model control (DMEM medium containing 1 μg / mL LPS) were included, and the plates were cultured for another 24 hours. The supernatant was collected, and the levels of inflammatory factors were detected using an IL-6 and TNF-α ELISA kit, following the manufacturer's instructions, and the absorbance was measured at 450 nm. Each assay was performed in triplicate.

[0084] Inhibition rate calculation: Inhibition rate of inflammatory factors (%) = [1 - (OD value of sample group - OD value of blank control group) / (OD value of model control group - OD value of blank control group)] × 100%.

[0085] IV. Detection of bacterial adhesion inhibition:

[0086] Sample preparation: The stock solution was prepared using the same method as for cytotoxicity testing, and the drug was diluted with serum-free culture medium to a concentration of 10 μg / mL.

[0087] The detection method includes: human gingival epithelial cells (HGEC, primary cells) are cultured in a 37°C, 5% CO2 incubator with serum-free medium, at a concentration of 1×10⁻⁶ cells / mL. 5 Cells were seeded at a density per well in 24-well plates and cultured for 24 hours; *Porphyromonas gingivalis* was cultured in an anaerobic chamber on BHI medium containing 5% defibrinated sheep blood and 0.001% vitamin K1 for 48 hours, and then adjusted to 1×10⁻⁶ cells / well with sterile PBS. 8 CFU / mL; discard the old HGEC culture medium, add an equal volume of sample culture medium (serum-free medium containing 10 μg / mL of drug) for 1 hour pretreatment, then add *Porphyromonas gingivalis* culture at MOI=100:1 (1 HGEC cell corresponds to 100 Pg cells) and co-culture for 2 hours; wash 3 times with sterile PBS to remove non-adhering bacteria, add 500 μL of 0.1% Triton X-100 to each well and shake at room temperature for 5 minutes to lyse the cells, serially dilute the lysis buffer 10-fold, and spread 100 μL onto BHI agar plates (containing 5% defibrinated sheep blood and 0.001% vitamin K1), and anaerobically incubate for 48 hours to count the colony count (CFU); set up a model control group (HGEC cell group with only bacterial culture and no drug). Each method has 3 replicates.

[0088] Calculate the adhesion inhibition rate: Inhibition rate (%) = [1 - (number of colonies in the sample group) / (number of colonies in the model control group)] × 100%.

[0089] V. Antioxidant Activity Assay (DPPH Free Radical Scavenging Rate):

[0090] Sample preparation: The drug composition powder was vortexed with methanol for 5 minutes until completely dissolved to prepare a 1 mg / mL stock solution, which was then diluted with methanol to a concentration of 10 μg / mL.

[0091] The detection method included: preparing a 0.1 mM DPPH methanol solution (weighing 4.08 mg DPPH, dissolving it in methanol, and bringing the volume to 100 mL) and storing it in the dark; adding 100 μL of sample solution and 100 μL of DPPH solution to each well of a 96-well plate, gently mixing, and reacting in the dark for 30 minutes; setting up a blank control (100 μL methanol + 100 μL DPPH solution) and a negative control (100 μL sample solution + 100 μL methanol), and measuring the absorbance (OD value) of each well at a wavelength of 517 nm using a microplate reader. Five replicates were prepared for each method.

[0092] Calculate the clearance rate: DPPH clearance rate (%) = [1 - (OD sample - OD negative control) / (OD blank control - OD negative control)] × 100%.

[0093] Table 1. Test Results (Average)

[0094]

[0095] The above test results show that the drugs in Examples 1 to 3 have optimized the component ratio and extraction and purification process, enriched the active ingredients (such as antimicrobial peptides, polyphenols, etc.), and have good effects in terms of safety, targeted antibacterial activity against harmful bacteria, anti-inflammation, and anti-oxidation.

[0096] Comparative Example 1 (decreased proportion of Lactobacillus salivarius fermentation extract, increased proportion of Viola yedoensis extract): Lactobacillus salivarius fermentation extract contains antimicrobial peptides and organic acids, which precisely inhibit pathogenic bacteria and regulate the oral mucosal immune response, but have no significant inhibitory effect on beneficial bacteria. Its decreased proportion leads to insufficient active ingredients targeting and inhibiting pathogenic bacteria, resulting in a weakened anti-inflammatory effect. While Viola yedoensis extract has broad-spectrum antibacterial activity, its selectivity for beneficial bacteria is poor; an increased proportion slightly enhances its inhibitory effect on beneficial bacteria. Simultaneously, the weakened immunomodulatory effect of Lactobacillus salivarius extract also affects its bacterial adhesion inhibition effect, and the imbalance in component proportions also leads to a decrease in antioxidant capacity.

[0097] Comparative Example 2 (decreased proportion of Viola yedoensis extract, increased proportion of Cicada nymph extract peptides): The flavonoids in Viola yedoensis extract reduce the release of inflammatory factors by inhibiting the cyclooxygenase-2 / lipoxygenase pathway, while saponins can disrupt the cell membranes of pathogenic bacteria. Flavonoids also scavenge free radicals, making them key to anti-inflammatory, antibacterial, and antioxidant effects. The decreased proportion of flavonoids directly leads to insufficient anti-inflammatory, antibacterial, and antioxidant components, weakening all related capabilities. Cicada nymph extract peptides can only exert limited antibacterial effects through antimicrobial peptides, lacking the anti-inflammatory and broad-spectrum antibacterial effects of Viola yedoensis extract. Furthermore, it has weak targeting of pathogenic bacteria and cannot compensate for the functional deficiencies of Viola yedoensis extract. It slightly enhances the inhibition of beneficial bacteria, resulting in a significant decrease in overall anti-inflammatory repair and antibacterial capabilities.

[0098] Comparative Example 3 (decreased proportion of Lactobacillus salivarius fermentation extract, significantly increased proportion of cicada nymph extract peptides): Lactobacillus salivarius fermentation extract has a much higher affinity for pathogenic bacteria than cicada nymph extract peptides, and can enhance anti-inflammatory effects by regulating the mitogen-activated protein kinase pathway, making it the core of maintaining oral flora balance and anti-inflammatory repair. A significant decrease in its proportion leads to a sharp drop in targeted antibacterial and anti-inflammatory capabilities. When the proportion of cicada nymph extract peptides is too high, it interferes with oral mucosal cell protein synthesis, increases cytotoxicity, and has a stronger inhibitory effect on beneficial bacteria than Lactobacillus salivarius extract, disrupting the flora balance. Simultaneously, its inhibitory ability on bacterial adhesion is weaker than that of Lactobacillus salivarius extract and Viola yedoensis extract. A severe imbalance in the component ratio also leads to a decrease in antioxidant capacity.

[0099] Comparative Example 4 (Purification resin for Lactobacillus salivarius fermentation extract: XAD7HP macroporous adsorption resin replaced with HP-20 macroporous resin): XAD7HP macroporous adsorption resin is moderately polar and has a high adsorption rate for moderately polar antimicrobial peptides and organic acids in Lactobacillus salivarius fermentation extract, effectively enriching active ingredients; HP-20 macroporous resin is weakly polar and has a low adsorption rate for these moderately polar active ingredients, leading to a significant loss of active ingredients and consequently reducing antibacterial and anti-inflammatory capabilities. The different components slightly affect cell survival rate and also weaken bacterial adhesion inhibition and antioxidant capacity.

[0100] Comparative Example 5 (Enzymatic hydrolysis of Viola yedoensis extract: bromelain replaced with papain): Bromelain specifically hydrolyzes the protein-polysaccharide complex in the cell wall of Viola yedoensis, disrupting the cell wall structure and significantly increasing the dissolution rate of intracellular active ingredients such as flavonoids and phenolic acids; papain's hydrolysis sites mainly target aromatic amino acid residues, resulting in low hydrolysis efficiency of the protein-polysaccharide complex, leading to insufficient dissolution of active ingredients and a decrease in anti-inflammatory, antibacterial, and antioxidant capabilities. Furthermore, incomplete hydrolysis leaves a small amount of macromolecular impurities in the extract, slightly enhancing the inhibition of beneficial bacteria and slightly affecting cell viability.

[0101] Comparative Example 6 (Enzymatic hydrolysis of Viola yedoensis extract: Subtilisin replaced with papain): Subtilisin efficiently degrades high-molecular-weight polysaccharides and structural proteins in the cell wall of Viola yedoensis, causing complete cell wall collapse and promoting the full dissolution of intracellular phenolic acids, saponins, and other active ingredients. Papain, on the other hand, has low degradation efficiency for high-molecular-weight polysaccharides and structural proteins, resulting in the residue of large molecules, which hinders the diffusion and release of active ingredients, and weakens anti-inflammatory and antibacterial abilities. The residue of large molecular impurities can also slightly enhance the inhibition of beneficial bacteria, affect cell survival rate, and lead to a decrease in bacterial adhesion inhibition and antioxidant capacity.

[0102] Comparative Example 7 (Enzymatic hydrolysis of Viola yedoensis extract: without the addition of Bacillus subtilis protease): Bacillus subtilis protease hydrolyzes the protein backbone of Viola yedoensis cell wall and works synergistically with pectinase and cellulase to ensure complete cell wall decomposition. Without Bacillus subtilis protease, pectinase and cellulase can only degrade some cell wall components and cannot destroy the protein backbone. Residual cell wall structure leads to a reduced dissolution rate of intracellular active ingredients such as flavonoids and phenolic acids, weakening anti-inflammatory and antibacterial capabilities. The large molecules remaining in the cell wall increase the impurity content of the extract, affecting cell survival rate and also reducing the antibacterial adhesion inhibition effect and antioxidant capacity.

[0103] Comparative Example 8 (Purification of Viola yedoensis extract: Replacing the composite adsorption column with a single HP-20 macroporous resin): In the composite adsorption column, the HP-20 macroporous resin adsorbs weakly polar saponins, while the polyamide resin specifically adsorbs highly polar phenolic acids through hydrogen bonding. The two work synergistically to achieve comprehensive enrichment of active ingredients. The single HP-20 macroporous resin has a low adsorption rate for phenolic acids, leading to a significant loss of these components. Since phenolic acids are key to enhancing anti-inflammatory and antibacterial effects, this resulted in a decrease in these capabilities. Simultaneously, HP-20 exhibits weaker adsorption capacity for some polar impurities than the composite column, slightly increasing the impurity content of the extract, affecting cell viability, and also weakening the bacterial adhesion inhibition effect and antioxidant capacity.

[0104] Comparative Example 9 (Purification of Viola yedoensis extract: Replacing the composite adsorption column with a single polyamide resin): The polyamide resin has a strong adsorption capacity for tannins in Viola yedoensis extract, but it also adsorbs target active ingredients such as flavonoids and phenolic acids, leading to competition for adsorption sites. In the composite adsorption column, the HP-20 macroporous resin can adsorb some impurities first, reducing competition. During purification with single polyamide resin, tannins occupy a large number of adsorption sites, resulting in a decrease in the elution rate of target active ingredients and an increase in tannin residue, which reduces anti-inflammatory and antibacterial capabilities. Tannin residue also increases cytotoxicity, affects cell survival rate, enhances inhibition of beneficial bacteria, and weakens bacterial adhesion inhibition and antioxidant capacity.

[0105] Comparative Example 10 (Purification of Viola yedoensis extract: HP-20 macroporous resin replaced with XAD7HP macroporous adsorption resin): The XAD7HP macroporous adsorption resin and HP-20 macroporous resin adsorbed and purified different substances. The unchanged composition led to the loss of saponins, resulting in decreased antibacterial and anti-inflammatory capabilities. The impurity content of the extract affected cell survival rate and also weakened the bacterial adhesion inhibition effect and antioxidant capacity.

[0106] Comparative Example 11 (Preparation of Peptides Extracted from Cicada Nymphs: Without Trypsin Enzymatic Hydrolysis): In the preparation of peptides extracted from cicada nymphs, papain initially hydrolyzes the protein to produce medium-molecular-weight peptides. Trypsin then specifically hydrolyzes the basic amino acid residues of these medium-molecular-weight peptides to generate small-molecule antimicrobial peptides. These small-molecule peptides can penetrate the cell membranes of pathogenic bacteria and inhibit bacterial adhesion. Without trypsin, papain hydrolysis alone only produces medium-molecular-weight peptides and cannot form active small-molecule peptides, leading to a decrease in antibacterial, anti-inflammatory, and bacterial adhesion-inhibiting abilities. Incompletely hydrolyzed protein residues increase cytotoxicity, affect cell survival, weaken antioxidant capacity, and slightly enhance inhibition of beneficial bacteria.

[0107] Comparative Example 12 (Preparation of Peptides Extracted from Cicada Nymphs: Papain Replaced with Bromelain): Papain hydrolyzes various amino acid residues in cicada nymph proteins, producing diverse peptides containing a large number of small molecule peptides with antibacterial and anti-inflammatory activities. Bromelain mainly hydrolyzes hydrophobic amino acid residues of proteins, showing poor targeting of the hydrolysis sites in cicada nymph proteins, resulting in a lower yield of active peptides and altered peptide composition. The proportion of antibacterial and anti-inflammatory active sequences decreases, thereby weakening the antibacterial, anti-inflammatory, and bacterial adhesion inhibition abilities. It also slightly affects cell viability and antioxidant capacity, and slightly enhances the inhibition of beneficial bacteria.

[0108] Comparative Example 13 (Purification of Cicada Nymph Extracted Peptides: Q-Agarose Gel FF Replaced with SP-Agarose Gel FF): Cicada nymph antimicrobial peptides are mostly negatively charged. Q-Agarose Gel FF, a strong anion exchange resin, can specifically adsorb negatively charged active peptides through electrostatic interactions, resulting in a high adsorption rate. SP-Agarose Gel FF, a strong cation exchange resin, can only adsorb positively charged substances and cannot bind to negatively charged antimicrobial peptides. This leads to a significant decrease in the recovery rate of the target active peptides, with most active peptides being lost, resulting in a significant reduction in antibacterial, anti-inflammatory, and bacterial adhesion inhibition capabilities. Furthermore, a slight increase in impurity content can mildly affect cell viability and antioxidant capacity.

Claims

1. A pharmaceutical composition for the repair of oral gingivitis, characterized in that, The pharmaceutical composition comprises Lactobacillus salivarius fermentation extract, Viola yedoensis extract, cicada nymph extract peptide, guava polyphenols and resveratrol in a mass ratio of (3-4.5):(2-3):(1.5-2.5):(1-1.5):(3-5); The preparation method of the Lactobacillus salivarius fermentation extract includes: using an inoculum volume of 5% to 8% of the fermentation medium, and adding bacteria with a concentration of 1×10⁻⁶. 9 CFU / mL ~5×10 9 CFU / mL of Lactobacillus salivarius (formerly classified as Lactobacillus salivarius, now classified as Ligilactobacillus) Activated Lactobacillus salivarius culture was inoculated into MRS medium supplemented with 1wt%–1.5wt% glucose and fermented aerobically for 8–12 h under sterile air conditions at 36℃–38℃, 100rpm–150rpm, and an aeration ratio of 0.5vvm–1vvm. During aerobic fermentation, the pH was maintained at 6.0–6.4 using 0.5mol / L–0.8mol / L sodium hydroxide aqueous solution. After aeration was stopped, anaerobic fermentation was carried out at 36℃–38℃ and 50rpm–100rpm for 20–24 h. After centrifugation, the supernatant was collected, filtered for sterilization, and the filtrate was concentrated under reduced pressure. The filtrate was then loaded onto an XAD7HP macroporous adsorption resin column, rinsed with deionized water to remove impurities, and then eluted with an ethanol aqueous solution. The eluent was collected, concentrated under reduced pressure to remove ethanol, and then freeze-dried to obtain the Lactobacillus salivarius fermentation extract. The preparation method of the Viola yedoensis extract includes: taking dried Viola yedoensis whole herb powder, adding a 70%–75% volume concentration ethanol aqueous solution containing 0.1 wt%–0.15 wt% citric acid at a material-to-liquid mass ratio of 1:(10–12), and reflux extraction at 80℃–85℃ for 1–2 hours; cooling to 50℃–55℃ and adjusting the pH to 4.5–5.0, adding 0.8%–1.5% of pectinase and 0.8%–1.5% of cellulase by weight of the powder, and enzymatically hydrolyzing at 50℃–55℃ for 2–3 hours; then adjusting the pH to... 6.5–7.5, add 1.5%–2% of the powder mass of a complex protease, the complex protease being bromelain and subtilisin in a mass ratio of (2–3):(1–1.5), and hydrolyze at 50–55℃ for 1.5–2 hours; inactivate the enzyme, centrifuge, collect the supernatant and concentrate under reduced pressure, load the sample onto a composite adsorption column, wash with deionized water and 30%–40% volume concentration ethanol aqueous solution to remove impurities, and finally elute with 70%–75% volume concentration ethanol aqueous solution, collect the eluent, concentrate under reduced pressure to remove ethanol, freeze dry, and obtain Viola yedoensis extract; The upper packing material of the composite adsorption column is HP-20 macroporous resin, and the lower packing material is polyamide resin. The volume ratio of HP-20 macroporous resin to polyamide resin is (1.5~2):

1. The preparation method of the cicada nymph extract peptides includes: taking dried cicada nymph powder, adding phosphate buffer solution with pH 6.5-7.0 at a material-to-liquid mass ratio of 1:(12-15), adding 0.8%-1.5% papain by weight of the dried powder, enzymatically hydrolyzing at 55℃-60℃ for 1-1.5 hours, inactivating the enzyme, centrifuging, taking the supernatant, adjusting the pH to 8.0-8.5, adding 0.8%-1.5% trypsin by weight of the dried powder, enzymatically hydrolyzing at 36℃-40℃ for 1-1.5 hours, inactivating the enzyme; and then ultrafiltration with a molecular weight cutoff of 5 kDa. Membrane ultrafiltration was performed, and the permeate was concentrated under reduced pressure. The pH was adjusted to 8.0–8.5, and the sample was loaded onto a Q agarose gel FF adsorption column. The column was eluted with Tris-HCl buffer at pH 8.0–8.5 to remove impurities, and then eluted sequentially with 0.1 mol / L–0.15 mol / L sodium chloride aqueous solution and 0.2 mol / L–0.3 mol / L sodium chloride aqueous solution. The eluents were combined and concentrated under reduced pressure. The eluent was then placed in a dialysis bag with a molecular weight cutoff of 100 Da–500 Da, dialyzed with deionized water, and freeze-dried to obtain cicada nymph extract peptides.

2. The pharmaceutical composition for repairing oral gingivitis according to claim 1, characterized in that, In the preparation method of Lactobacillus salivarius fermentation extract, the centrifugation is carried out at 4℃~8℃ and 8000rpm~8500rpm for 15min~25min; the sterilization by filtration is carried out through a 0.22μm filter membrane; the filtrate is concentrated under reduced pressure at 45℃~50℃ to 25%~35% of its volume; the elution volume of deionized water is 2BV~3BV, and the elution flow rate is 1.5BV / h~2.5BV / h; the volume concentration of the ethanol aqueous solution is 40%~50%; the elution volume of the ethanol aqueous solution is 3BV~4BV, and the elution flow rate is 1BV / h~1.5BV / h; the temperature for concentrated under reduced pressure of the eluent is 45℃~50℃.

3. The pharmaceutical composition for repairing oral gingivitis according to claim 1, characterized in that, In the preparation method of Viola yedoensis extract, the particle size of the powder is sieved through a 40-60 mesh sieve; the stirring speed for enzymatic hydrolysis is 60-80 rpm; the enzyme inactivation is performed by heating to 80-85℃ for 15-20 minutes and then cooling to room temperature; the centrifugation is performed at 8000-8500 rpm for 10-15 minutes; the supernatant is concentrated under reduced pressure at 45-50℃ to 30%-40% of its volume; the elution volume of deionized water is 2-3 BV; the elution volume of the 30%-40% volume concentration ethanol aqueous solution is 1-2 BV; the elution volume of the 70%-75% volume concentration ethanol aqueous solution is 3-4 BV; and the reduced pressure concentration temperature of the eluent is 45-50℃.

4. The pharmaceutical composition for repairing oral gingivitis according to claim 1, characterized in that, In the preparation method of peptides extracted from cicada nymphs, the particle size of the freeze-dried cicada nymph powder is sieved through a 60-80 mesh sieve; the stirring speed for enzymatic hydrolysis is 60-80 rpm; the enzyme inactivation is performed by heating to 80-85℃ for 15-20 minutes and then cooling to room temperature; the centrifugation is performed at 4-6℃ and 5000-6000 rpm for 10-15 minutes; and the permeate is concentrated under reduced pressure at 45-50℃ to 20%-30% of its original volume. The elution volume of the Tris-HCl buffer solution with pH 8.0–8.5 is 2 BV–3 BV; the elution volume of the 0.1 mol / L–0.15 mol / L sodium chloride aqueous solution is 1.5 BV–2 BV; the elution volume of the 0.2 mol / L–0.3 mol / L sodium chloride aqueous solution is 1.5 BV–2 BV; the eluent is concentrated under reduced pressure at 45°C–50°C to 10%–15% of its volume; the dialysis time is 48 h–52 h.

5. A method for preparing a pharmaceutical composition for repairing oral gingivitis as described in claim 1, characterized in that, The method includes: mixing Lactobacillus salivarius fermentation extract, Viola yedoensis extract, Cicada nymph extract peptide, guava polyphenols and resveratrol according to the formula mass ratio to obtain a pharmaceutical composition.

6. The method for preparing a pharmaceutical composition for repairing oral gingivitis according to claim 1, characterized in that, The pharmaceutical composition is mixed with pharmaceutically available excipients to prepare plasters, gels, or mouthwashes.

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