Natural plant polyphenol-peptide compound applied to mouth wash

Abstract

The invention discloses a natural plant polyphenol-peptide compound applied to mouth wash. The compound consists of plant polyphenols such as procyanidine and catechin in specific parts by weight, and five sections of small molecule peptides derived from soybeans, corns and the like. By optimizing extraction, separation and compounding processes, the polyphenol and the peptide have synergistic interaction. The prepared mouthwash has the functions of efficiently resisting bacteria (harmful bacteria are reduced by 85%-95%), resisting inflammation (inflammatory factors are remarkably reduced), repairing enamel (hardness is improved by 42%), freshening breath (VSC concentration is reduced to 18 ppb) and the like. Compared with traditional mouthwash, the mouthwash overcomes the defects of component irritation, single function and the like, adopts a green process to ensure activity, and is good in stability and good in taste.

Description

Technical Field

[0001] The present invention belongs to the technical field of oral care, and particularly relates to a multifunctional mouthwash with a natural plant polyphenol - peptide complex as the core component in a specific weight ratio. The preparation method, component combination and its applications in aspects such as oral antibacterial, anti - inflammatory, tooth enamel repair and fresh breath are elaborated in detail. Background Art

[0002] With the popularization of oral health awareness, mouthwash has become an important product for daily oral care. However, there are still many problems in the components, efficacy and technology of existing mouthwash products.

[0003] In terms of components, although alcohol - containing mouthwashes have significant antibacterial effects, alcohol can stimulate the oral mucosa, leading to oral dryness and burning pain. Long - term use may disrupt the oral micro - ecological balance, and about 40% of long - term users have experienced oral mucosal discomfort symptoms. Alcohol - free mouthwashes mostly rely on chemically synthesized antibacterial agents such as triclosan and quaternary ammonium salts. These components are prone to cause bacterial drug resistance and inhibit the growth of beneficial oral bacteria, affecting the stability of the oral micro - ecosystem.

[0004] In terms of efficacy, traditional mouthwashes have a single function, mostly focusing only on antibacterial, with poor relief and repair effects on inflammations such as oral ulcers and gingivitis, and it is difficult to achieve a long - term and gentle balance in tooth enamel protection and fresh breath.

[0005] In terms of technology, traditional extraction methods often cause inactivation of plant active ingredients due to factors such as high temperature, affecting the product efficacy; stability problems such as precipitation and stratification are prone to occur during the component mixing process, reducing the product quality and user experience. Therefore, it is of great significance to develop a new type of safe, efficient, multifunctional and stable mouthwash. Summary of the Invention

[0006] 1. Composition of the Natural Plant Polyphenol - Peptide Complex

[0007] The present invention provides a natural plant polyphenol - peptide complex applied to mouthwash, which is composed of the following components in parts by weight:

[0008] · Plant polyphenols: 15 - 30 parts of procyanidins derived from grape seeds, 10 - 25 parts of catechins in green tea, 10 - 20 parts of anthocyanins in blueberries, and 10 - 20 parts of ellagic acid in pomegranate peel. These plant polyphenols have various biological activities such as antioxidant, antibacterial and anti - inflammatory, and can effectively inhibit the growth of harmful oral bacteria and reduce the inflammatory response.

[0009] · Small molecule peptides:

[0010] Peptide sequence α: Glu-Lys-Pro-Gly-Ser-Thr-Ala-Val-Leu-Ile-Phe-Tyr-Trp-Arg, 10 - 25 parts, derived from soybean extract;

[0011] · Peptide sequence β: Gly-Asp-Glu-Pro-Arg-Ser-Thr-Val-Leu-Phe-Met-His-Asn-Lys, 8 - 20 parts, derived from corn extract;

[0012] · Peptide sequence γ: Thr-Arg-Glu-Asp-Pro-Gly-Ser-Ile-Val-Leu-Phe-Tyr-Trp-GIn-Ala, 10 - 22 parts, derived from wheat extract;

[0013] · Peptide sequence δ: Pro-Lys-Glu-Asp-Gly-Ser-Thr-Val-Ile-Leu-Phe-Tyr-Trp-Asn-Glu-Ser, 8 - 20 parts, derived from oat extract;

[0014] · Peptide sequence ε: Ser-Gly-Pro-Thr-Lys-Arg-Glu-Asp-Val-Leu-Ile-Phe-Tyr-Trp-His-GIn-Asn, 8 - 20 parts, derived from pea extract. These small molecule peptides have functions such as antibacterial, promoting repair, and regulating the microecology, and cooperate with plant polyphenols to enhance the comprehensive efficacy of the mouthwash.

[0015] 2. Preferred component content ratio

[0016] In a more preferred embodiment, the weight parts of each component in the natural plant polyphenol - peptide complex are as follows:

[0017] For the plant polyphenol part, procyanidin 20 - 28 parts, catechin 12 - 22 parts, anthocyanin 12 - 18 parts, ellagic acid 12 - 18 parts; for the small molecule peptide part, peptide sequence α 12 - 22 parts, peptide sequence β 10 - 18 parts, peptide sequence γ 12 - 20 parts, peptide sequence δ 10 - 18 parts, peptide sequence ε 10 - 18 parts. Under this ratio, the synergistic effect between plant polyphenols and small molecule peptides is more significant, and the antibacterial, anti-inflammatory, repair, and fresh breath functions of the mouthwash can be maximally exerted.

[0018] 3. Preparation method of the natural plant polyphenol - peptide complex

[0019] The preparation method of the natural plant polyphenol - peptide complex of the present invention is specifically as follows:

[0020] · Raw material pretreatment: Carefully select mature grape seeds, fresh green tea, blueberries, pomegranate peels, soybeans, corn, wheat, oats, and peas. Wash the grape seeds and pomegranate peels and dry them thoroughly; remove impurities from the green tea; wash the blueberries; remove impurities from the soybeans, corn, wheat, oats, and peas and then dry them. Subsequently, crush the dried grape seeds, pomegranate peels, soybeans, corn, wheat, oats, and peas and pass them through a 80-100 mesh sieve; make a homogenate of the green tea and blueberries to prepare for subsequent extraction.

[0021] · Extraction:

[0022] · Extraction of plant polyphenols:

[0023] · Grape seeds: Mix the grape seed powder with 60-80% ethanol at a solid-liquid ratio of 1:8-1:12 (g / mL), and perform ultrasonic extraction at a temperature of 40-60 °C for 2-4 hours with an ultrasonic frequency of 30-50 kHz. After extraction, centrifuge at a speed of 4000-6000 rpm for 10-20 minutes, collect the supernatant, and obtain grape seed extract by vacuum concentration.

[0024] · Green tea: Mix the green tea homogenate with 50-70% ethanol at a solid-liquid ratio of 1:6-1:10 (g / mL), and perform stirring extraction at a temperature of 35-55 °C for 1-3 hours with a stirring speed of 100-200 rpm. After extraction, filter, collect the filtrate, and obtain green tea extract by vacuum concentration.

[0025] · Blueberries: Mix the blueberry homogenate with 40-60% ethanol at a solid-liquid ratio of 1:5-1:8 (g / mL), and perform ultrasonic extraction at a temperature of 30-50 °C for 1-3 hours with an ultrasonic frequency of 30-50 kHz. After extraction, centrifuge, collect the supernatant, and obtain blueberry extract by vacuum concentration.

[0026] Pomegranate peels: Mix the pomegranate peel powder with 70-90% ethanol at a solid-liquid ratio of 1:7-1:11 (g / mL), and perform reflux extraction at a temperature of 45-65 °C for 2-4 hours. After extraction, cool, filter, collect the filtrate, and obtain pomegranate peel extract by vacuum concentration.

[0027] · Extraction of small molecule peptides:

[0028] · Soybeans: Mix the soybean powder with deionized water at a solid-liquid ratio of 1:6-1:10 (g / mL), add 0.5-1.5% alkaline protease at a temperature of 40-60 °C for enzymatic hydrolysis, maintain the pH value at 8-10 during the enzymatic hydrolysis process, and the enzymatic hydrolysis time is 2-4 hours. After enzymatic hydrolysis, inactivate the enzyme, centrifuge at a speed of 4000-6000 rpm for 10-20 minutes, collect the supernatant, and obtain soybean peptide extract by ultrafiltration (cut-off molecular weight 5000 Da).

[0029] · Corn: Mix corn flour with deionized water at a solid-liquid ratio of 1:5 - 1:8 (g / mL). At a temperature of 35 - 55 °C, add 0.4 - 1.2% neutral protease for enzymatic hydrolysis. During the hydrolysis process, maintain the pH value at 6 - 8, and the hydrolysis time is 2 - 4 hours. After the hydrolysis, inactivate the enzyme, centrifuge and ultrafilter (with a molecular weight cut-off of 3000 Da) to obtain the corn peptide extract.

[0030] · Wheat: Mix wheat flour with deionized water at a solid-liquid ratio of 1:6 - 1:10 (g / mL). At a temperature of 40 - 60 °C, add 0.6 - 1.4% complex protease for enzymatic hydrolysis. During the hydrolysis process, maintain the pH value at 7 - 9, and the hydrolysis time is 2 - 4 hours. After the hydrolysis, inactivate the enzyme, centrifuge and ultrafilter (with a molecular weight cut-off of 2000 Da)

[0031] to obtain the wheat peptide extract.

[0032] · Oat: Mix oat flour with deionized water at a solid-liquid ratio of 1:5 - 1:8 (g / mL). At a temperature of 35 - 55 °C, add 0.5 - 1.3% acidic protease for enzymatic hydrolysis. During the hydrolysis process, maintain the pH value at 4 - 6, and the hydrolysis time is 2 - 4 hours. After the hydrolysis, inactivate the enzyme, centrifuge and ultrafilter (with a molecular weight cut-off of 1500 Da) to obtain the oat peptide extract.

[0033] · Pea: Mix pea flour with deionized water at a solid-liquid ratio of 1:6 - 1:10 (g / mL). At a temperature of 40 - 60 °C, add 0.4 - 1.2% flavor protease for enzymatic hydrolysis. During the hydrolysis process, maintain the pH value at 6 - 8, and the hydrolysis time is 2 - 4 hours. After the hydrolysis, inactivate the enzyme, centrifuge and ultrafilter (with a molecular weight cut-off of 1000 Da) to obtain the pea peptide extract.

[0034] · Separation and purification:

[0035] · Plant polyphenols: Pass each plant extract through a macroporous adsorption resin column, and select a suitable macroporous adsorption resin according to the polarity and molecular size of polyphenolic components. First, rinse with deionized water to remove impurities, and then perform gradient elution with ethanol solutions of different concentrations (10%, 30%, 50%, 70%, 90%) to collect the eluate containing polyphenols. Then, further separate and purify the eluate by high-speed countercurrent chromatography to obtain high-purity procyanidins, catechins, anthocyanins, and ellagic acid.

[0036] · Small molecule peptides: Initially separate each peptide extract by gel filtration chromatography (using Sephadex G-25 or Sephacryl S-100 gel media) according to molecular size, and then perform fine separation by ion exchange chromatography (select cation or anion exchange resin according to the charge properties of peptide molecules). By adjusting the pH value and ionic strength of the eluent, collect the purified peptide solution.

[0037] · Complex formation: Precisely mix the purified plant polyphenols and small molecule peptides in proportion, add an appropriate amount of deionized water to make the total volume of the mixture reach an amount convenient for the reaction. Under the conditions of a temperature of 30 - 40 °C and a pH of 6 - 7, use ultrasonic assistance at 200 - 400 W for 10 - 20 minutes, while stirring at a speed of 200 - 400 rpm to promote the binding of polyphenols and peptides and form a stable natural plant polyphenol - peptide complex.

[0038] 4. Composition of the mouthwash

[0039] A mouthwash is composed of the following components in parts by weight: 2 - 8 parts of the above - mentioned natural plant polyphenol - peptide complex, 5 - 15 parts of glycerol, 3 - 10 parts of xylitol, 2 - 8 parts of sorbitol, 0.1 - 0.5 parts of menthol, 0.1 - 0.5 parts of essence, an appropriate amount of pH regulator (citric acid or sodium hydroxide), and deionized water is added to make up to 100 parts. Among them, glycerol, xylitol, and sorbitol are used as humectants to prevent the mouthwash from volatilizing and keep the oral cavity moist; menthol and essence give the mouthwash a fresh taste; the pH regulator is used to adjust the pH value of the mouthwash to 6.0 - 7.0 to ensure its stability and safety in the oral environment.

[0040] 5. Preparation method of the mouthwash

[0041] A preparation method of the mouthwash as described above includes the following steps:

[0042] · Add the natural plant polyphenol - peptide complex to deionized water and stir at a speed of 100 - 200 rpm for 10 - 20 minutes to fully dissolve it.

[0043] · Add glycerol, xylitol, and sorbitol in sequence and continue to stir for 10 - 20 minutes until completely dissolved.

[0044] · Add menthol and essence, stir and mix well for 10 - 20 minutes to evenly distribute the fragrance.

[0045] · Use a pH regulator (citric acid or sodium hydroxide solution) to adjust the pH value of the solution to 6.0 - 7.0, continuously stir and monitor the change of the pH value during the adjustment process.

[0046] · After making up to the required volume, filter and sterilize through a 0.22 - μm filter membrane to remove possible microorganisms and impurities to obtain the finished mouthwash.

[0047] 6. Innovation points

[0048] · Novel composite composition: For the first time, a variety of plant polyphenols and small molecule peptides are compounded in a specific weight ratio to form a unique combination of active ingredients. The plant polyphenols and small molecule peptides synergistically enhance their antibacterial, anti-inflammatory and repair capabilities through intermolecular forces such as hydrogen bonds and hydrophobic interactions.

[0049] · Multifunctional synergy: This mouthwash combines antibacterial, anti-inflammatory, tooth enamel repair and fresh breath functions. The plant polyphenols and small molecule peptides jointly inhibit harmful bacteria and reduce inflammation; the small molecule peptides promote tooth enamel repair; at the same time, it reduces volatile sulfides and improves breath, breaking through the limitation of the single function of traditional mouthwashes.

[0050] · Green preparation process: Green processes such as low-temperature enzymatic hydrolysis and ultrasonic-assisted extraction are used to avoid the destruction of active ingredients by high temperatures and retain the biological activities of plant polyphenols and small molecule peptides to the greatest extent. Chemical cross-linking agents are not used in the process of forming the complex, ensuring the safety and environmental friendliness of the product.

[0051] · Precise ingredient regulation: By precisely controlling the weight parts of each ingredient and the preparation conditions, the structure and performance of the natural plant polyphenol-peptide complex are precisely regulated, optimizing the efficacy of the mouthwash to meet different oral care needs.

[0052] · Good stability and taste: The special ingredient combination and preparation process ensure that the mouthwash is not prone to precipitation and stratification during storage, with good stability. At the same time, natural flavoring agents are added to give the product a fresh and pleasant taste, enhancing the user experience.

[0053] Beneficial effects

[0054] 1. High-efficiency antibacterial

[0055] The mouthwash of the present invention has a significant inhibitory effect on common harmful bacteria in the oral cavity such as Streptococcus mutans, Porphyromonas gingivalis, Candida albicans, etc. Verified by experiments, after using this mouthwash, the number of harmful bacteria in the oral cavity can be reduced by 85%-95%, effectively preventing the occurrence of oral diseases such as dental caries and periodontitis, and the antibacterial effect is better than that of traditional mouthwashes.

[0056] 2. Anti-inflammatory and repair

[0057] The natural plant polyphenol-peptide complex can reduce the expression levels of oral inflammatory factors (such as TNF-α, IL-6), alleviating inflammatory symptoms such as oral ulcers and gingivitis. The small molecule peptides can promote the proliferation and repair of oral mucosal cells and tooth enamel cells, accelerate the healing of oral ulcers, shorten the healing time by 3-6 days compared with the control group, and promote the remineralization of tooth enamel, enhancing the anti-caries ability of teeth.

[0058] 3. Fresh breath

[0059] By inhibiting the growth of odor-producing bacteria and reducing the production of volatile sulfides, this mouthwash can effectively improve bad breath problems. After use, it can keep the mouth fresh for up to 6 - 8 hours, which is significantly better than traditional mouthwashes.

[0060] 4. High safety

[0061] The ingredients of the mouthwash are all derived from natural plant extracts, without harmful chemically synthesized antibacterial agents and alcohol, and are non-irritating to the oral mucosa. Animal experiments and human trials have shown that long-term use will not cause oral flora imbalance and drug resistance problems, and it is suitable for all types of people, including children, the elderly, and those with sensitive oral mucosa.

[0062] 5. Good stability

[0063] The unique preparation process and ingredient combination ensure the stability of the mouthwash ingredients during storage. After being tested by an accelerated stability test (stored at 40°C and 75% relative humidity for 3 months), there is no precipitation or stratification phenomenon, the product quality is reliable, and the shelf life is long. Detailed implementation methods

[0064] Example 1: Preparation of natural plant polyphenol - peptide complex

[0065] 1. Raw material pretreatment: Weigh 200 g of grape seeds, 150 g of green tea, 180 g of blueberries, 120 g of pomegranate peels, 300 g of soybeans, 260 g of corn, 240 g of wheat, 200 g of oats, and 280 g of peas. Wash and dry the grape seeds and pomegranate peels, remove impurities from the soybeans, corn, wheat, oats, and peas and then dry them; remove impurities from the green tea and wash the blueberries. Crush the dried grape seeds, pomegranate peels, soybeans, corn, wheat, oats, and peas, and pass through a 90-mesh sieve; make the green tea and blueberries into homogenates.

[0066] 2. Extraction:

[0067] · Extraction of plant polyphenols:

[0068] · Grape seeds: Mix the grape seed powder with 2000 mL of 70% ethanol, extract at 50°C and 40 kHz

[0069] ultrasonic frequency for 3 hours, centrifuge at 5000 rpm for 15 minutes, and collect the supernatant and concentrate it under reduced pressure to obtain grape seed extract.

[0070] · Green tea: Mix the green tea homogenate with 1500 mL of 60% ethanol, extract at 45°C and 150 rpm

[0071] stirring speed for 2 hours, filter to obtain the filtrate and concentrate it under reduced pressure to obtain green tea extract.

[0072] · Blueberries: Mix the blueberry homogenate with 1500 mL of 50% ethanol, extract at 40°C and 40 kHz

[0073] Extract for 2 hours at ultrasonic frequency, centrifuge, take the supernatant, and concentrate it under reduced pressure to obtain blueberry extract.

[0074] · Pomegranate peel: Mix pomegranate peel powder with 1300 mL of 80% ethanol, reflux and extract at 55 °C for 3 hours, cool, filter, take the filtrate, and concentrate it under reduced pressure to obtain pomegranate peel extract.

[0075] · Small molecule peptide extraction:

[0076] · Soybean: Mix soybean powder with 2400 mL of deionized water, add 1.0% alkaline protease at 50 °C, maintain pH 9, and enzymatically hydrolyze for 3 hours. After inactivating the enzyme, centrifuge at 5000 rpm for 15 minutes, take the supernatant, and ultrafilter through a 5000 Da ultrafiltration membrane to obtain soybean peptide extract.

[0077] · Corn: Mix corn flour with 2000 mL of deionized water, add 0.8% neutral protease at 45 °C, maintain pH 7, and enzymatically hydrolyze for 3 hours. After inactivating the enzyme, centrifuge and ultrafilter through a 3000 Da ultrafiltration membrane to obtain corn peptide extract.

[0078] · Wheat: Mix wheat flour with 2400 mL of deionized water, add 1.0% complex protease at 50 °C, maintain pH 8, and enzymatically hydrolyze for 3 hours. After inactivating the enzyme, centrifuge and ultrafilter through a 2000 Da ultrafiltration membrane to obtain wheat peptide extract.

[0079] · Oat: Mix oat flour with 1600 mL of deionized water, add 0.9% acidic protease at 45 °C, maintain pH 5, and enzymatically hydrolyze for 3 hours. After inactivating the enzyme, centrifuge and ultrafilter through a 1500 Da ultrafiltration membrane to obtain oat peptide extract.

[0080] · Pea: Mix pea flour with 2200 mL of deionized water, add 0.8% flavor protease at 50 °C, maintain pH 7, and enzymatically hydrolyze for 3 hours. After inactivating the enzyme, centrifuge and ultrafilter through a 1000 Da ultrafiltration membrane to obtain pea peptide extract.

[0081] · Pea: Mix pea flour with 2200 mL of deionized water, add 0.8% flavor protease at 50 °C, maintain pH 7, and enzymatically hydrolyze for 3 hours. After inactivating the enzyme, centrifuge and ultrafilter through a 1000 Da ultrafiltration membrane to obtain pea peptide extract.

[0082] · Pea: Mix pea flour with 2200 mL of deionized water, add 0.8% flavor protease at 50 °C, maintain pH 7, and enzymatically hydrolyze for 3 hours. After inactivating the enzyme, centrifuge and ultrafilter through a 1000 Da ultrafiltration membrane to obtain pea peptide extract.

[0083] 3. Separation and purification

[0084] Pass each plant extract through a macroporous adsorption resin column, elute successively with deionized water, 10%, 30%, 50%, 70%, and 90% ethanol solutions in a gradient manner, collect the polyphenol-containing eluate, and further separate and purify it by high-speed countercurrent chromatography to obtain 25 g of high-purity procyanidin, 20 g of catechin, 15 g of anthocyanin, and 15 g of ellagic acid respectively.

[0085] First, each peptide extract was preliminarily separated by Sephadex G-25 gel filtration chromatography according to molecular size, and then finely separated by cation exchange resin (CM-Sepharose). By adjusting the pH to 6.5 and performing gradient elution with 0-1M NaCl, the purified peptide solutions were collected separately to obtain peptide sequence α20g, peptide sequence β18g, peptide sequence γ20g, peptide sequence δ18g, and peptide sequence ε16g.

[0086] 4. Complex formation

[0087] The purified plant polyphenols and small molecule peptides were mixed, 500 mL of deionized water was added, and under the conditions of 35 °C and pH 6.5, ultrasonic treatment was carried out for 15 minutes with an ultrasonic power of 300 W, while stirring at a speed of 300 rpm to form a natural plant polyphenol-peptide complex. Detection by high performance liquid chromatography (HPLC) and mass spectrometry (MS) confirmed that the polyphenols and peptides were successfully complexed through hydrogen bonds and hydrophobic interactions.

[0088] Example 2: Preparation of mouthwash

[0089] 1. Take 50 g of the natural plant polyphenol-peptide complex prepared in Example 1, add 800 mL of deionized water, and stir at 150 rpm for 15 minutes until completely dissolved.

[0090] 2. Add 100 g of glycerol, 60 g of xylitol, and 50 g of sorbitol in sequence, and continue to stir for 15 minutes until dissolved.

[0091] 3. Add 3 g of menthol and 3 g of essence, and stir well for 20 minutes to make the fragrance uniform.

[0092] 4. Adjust the pH value to 6.5 with 5% citric acid solution, make up the volume to 1000 mL, and filter and sterilize through a 0.22 μm filter membrane to obtain the finished mouthwash.

[0093] Example 3: Antibacterial performance test

[0094] 1. Experimental strains: Streptococcus mutans, Porphyromonas gingivalis, Candida albicans.

[0095] 2. Experimental method: The strains were cultured to the logarithmic growth phase, and the bacterial liquid concentration was adjusted to 10 6 CFU / mL. Take the mouthwash prepared in Example 2 and mix it with the bacterial liquid at a ratio of 1:1 (v / v), incubate at 37 °C for 30 minutes, and the control group uses an equal amount of normal saline. After incubation, it was serially diluted and spread on a solid medium, and the colonies were counted after culturing at 37 °C for 24 hours.

[0096] 3. Experimental results:

[0097] · Streptococcus mutans: The number of colonies in the mouthwash group of the present invention decreased by 92%, while that in the traditional chlorhexidine-containing mouthwash group decreased by 78%.

[0098] · Porphyromonas gingivalis: The number decreased by 95% in the mouthwash group of the present invention and by 75% in the traditional group.

[0099] · Candida albicans: The number decreased by 90% in the mouthwash group of the present invention and by 70% in the traditional group.

[0100] · The results showed that the antibacterial effect of the mouthwash of the present invention was significantly better than that of the traditional product (P < 0.05).

[0101] Example 4: Anti-inflammatory performance test

[0102] 1. Experimental animals: 60 male SD rats aged 6 - 8 weeks were randomly divided into 5 groups (12 rats in each group): control group, model group, positive control group (commercially available anti-inflammatory mouthwash), low-concentration group of the mouthwash of the present invention (2% complex), and high-concentration group of the mouthwash of the present invention (8% complex).

[0103] 2. Experimental model: An inflammatory model was established by injecting lipopolysaccharide (LPS) into the oral mucosa.

[0104] 3. Experimental treatment: After modeling, gargle 3 times a day for 7 days.

[0105] 4. Detection indexes:

[0106] · Inflammatory factors: ELISA was used to detect the levels of TNF-α and IL-6.

[0107] · Histological observation: HE staining was used to evaluate the degree of mucosal damage.

[0108] 5. Experimental results:

[0109] · In the high-concentration group of the mouthwash of the present invention, TNF-α decreased by 62% and IL-6 decreased by 58%, which was significantly better than

[0110] the positive control group (decreased by 45% and 40% respectively);

[0111] · Histology showed that the degree of mucosal epithelial repair in the group of the present invention was 30% higher than that in the positive control group.

[0112] Example 5: Enamel repair performance test

[0113] 1. Experimental method: Human exfoliated enamel blocks were randomly divided into 4 groups and immersed in: artificial saliva (control group), fluoride-containing mouthwash (positive control), mouthwash of the present invention (5% complex), and blank solution. The solution was changed daily for 28 days.

[0114] 2. Detection indexes:

[0115] · Microhardness test: The surface hardness of enamel was measured using a Vickers hardness tester;

[0116] · Calcium and phosphorus content: The calcium-phosphorus ratio of enamel was detected by X-ray fluorescence spectroscopy (XRF).

[0117] 3. Experimental results:

[0118] · The enamel hardness of the mouthwash group of the present invention increased by 42%, higher than 28% of the fluoride mouthwash group;

[0119] · The calcium-phosphorus ratio was restored to the natural enamel level, while only partial improvement was observed in the fluoride mouthwash group.

[0120] Example 6: Breath freshness performance test

[0121] 1. Experimental subjects: 30 healthy volunteers were randomly divided into 3 groups (10 people in each group), and they used the mouthwash of the present invention, a commercially available deodorant mouthwash, and physiological saline respectively.

[0122] 2. Experimental method: After gargling, the concentration of volatile sulfur compounds (VSCs) was detected using an electronic nose at 0, 1, 2, 4, 6, and 8 hours, and the subjective breath score was recorded.

[0123] 3. Experimental results:

[0124] The VSC concentration in the mouthwash group of the present invention was maintained below 20 ppb for 8 hours, while it increased to 45 ppb in the commercially available group after 4 hours;

[0125] · The subjective scores of the volunteers showed that the satisfaction with breath freshness in the group of the present invention reached 93%, significantly higher than 67% of the commercially available group.

[0126] Example 7: Safety test

[0127] 1. Acute toxicity test: 20 Kunming mice (10 males and 10 females) were gavaged with the mouthwash of the present invention at a dose of 50 mL / kg body weight. The control group used physiological saline, and they were observed for 7 days. The results showed that there were no poisoning symptoms or deaths in the experimental group, indicating extremely low acute toxicity.

[0128] 2. Mucosal irritation test: 6 rabbits were smeared with the mouthwash on the oral mucosa twice a day for 7 consecutive days. Histological examination found no mucosal redness, ulceration, or cell degeneration, confirming no irritation.

[0129] 3. Microecological safety test: Rats were continuously used the mouthwash for 28 days. 16S rRNA sequencing showed that the relative abundances of oral beneficial bacteria

[0130] (such as Bifidobacterium and Lactobacillus) did not decrease significantly, while the beneficial bacteria in the traditional antibacterial mouthwash group decreased by 35%.

[0131] Example 8: Verification of the Synergistic Effect of Peptide Sequence α

[0132] Experimental Design

[0133] Sixty rats infected with Streptococcus mutans were selected and randomly divided into 6 groups, with 10 rats in each group:

[0134] · Blank control group: No treatment

[0135] · Normal saline group: Gargle with normal saline daily

[0136] · Traditional chemical antibacterial mouthwash group: Use a mouthwash containing 0.2% cetylpyridinium chloride

[0137] · Low-concentration peptide sequence α group: Use a mouthwash containing 0.05% peptide sequence α

[0138] · Medium-concentration peptide sequence α group: Use a mouthwash containing 0.15% peptide sequence α

[0139] · High-concentration peptide sequence α group: Use a mouthwash containing 0.3% peptide sequence α

[0140] Gargle 3 times a day, 1 minute each time, for 14 consecutive days.

[0141] Detection Indicators and Methods

[0142] 1. Antibacterial effect: Collect saliva samples on the 14th day of the experiment and determine the number of Streptococcus mutans by colony counting

[0143] 2. Biofilm inhibition: Observe the formation of dental surface bacterial biofilms using a laser confocal microscope

[0144] 3. Microecological impact: Analyze the oral flora structure experimental results by 16S rRNA sequencing

[0145]

[0146]

[0147] Result Analysis

[0148] 1. Antibacterial performance: The antibacterial effect of the high-concentration peptide sequence α group was significantly better than that of the traditional mouthwash group, with the reduction in the number of bacteria reaching 71.4%, while that of the traditional mouthwash group was 62.2%

[0149] 2. Biofilm inhibition: The biofilm thickness of the peptide sequence α group was significantly lower than that of the traditional mouthwash group, and the inhibition effect was increased by about 46%

[0150] 3. Microecological protection: The traditional mouthwash caused a 32% decrease in the number of beneficial bacteria, while the peptide sequence α group increased the abundance of beneficial bacteria by 20%

[0151] It shows that peptide sequence α not only has better antibacterial effect, but also can effectively protect the oral microecology and overcome the defects of traditional chemical antibacterial agents.

[0152] Example 9: Verification of the synergistic effect of peptide sequence β

[0153] Experimental design

[0154] Thirty models of patients with oral ulcers were constructed and randomly divided into 3 groups:

[0155] · Control group: Gargle with normal saline

[0156] · Traditional anti-inflammatory mouthwash group: Gargle with mouthwash containing 0.1% chlorhexidine

[0157] · Peptide sequence β mouthwash group: Gargle with mouthwash containing 0.2% peptide sequence β

[0158] Use it 3 times a day for 7 days.

[0159] Detection indexes and methods

[0160] 1. Inflammation relief: Evaluate the pain degree through Visual Analogue Scale (VAS)

[0161] 2. Ulcer healing: Measure the change of ulcer area

[0162] 3. Inflammatory factors: Detect the levels of TNF-α and IL-6 in saliva by ELISA

[0163] Experimental results

[0164]

[0165] Result analysis

[0166] 1. Pain relief: The pain score in the peptide sequence β group decreased by 66.2%, which was significantly better than 33.8% in the traditional mouthwash group

[0167] 2. Healing speed: The ulcer healing rate increased by 49% compared with the traditional mouthwash group

[0168] 3. Inflammation inhibition: The inhibitory effects on TNF-α and IL-6 increased by 45.1% and 41.7% respectively compared with the traditional group

[0169] It shows that peptide sequence β is significantly better than traditional anti-inflammatory mouthwash in terms of anti-inflammation and promoting ulcer healing.

[0170] Example 10: Verification of the synergistic effect of peptide sequence γ

[0171] Experimental design

[0172] Sixty extracted teeth with early demineralization were selected and randomly divided into 3 groups:

[0173] · Control group: Soaked in artificial saliva

[0174] · Traditional fluoridated mouthwash group: Used mouthwash containing 0.05% sodium fluoride

[0175] · Peptide sequence γ mouthwash group: Used mouthwash containing 0.2% peptide sequence γ

[0176] Treated 2 times a day for 28 days.

[0177] Detection indicators and methods

[0178] 1. Hardness recovery: Measured the surface hardness of enamel using a microhardness tester

[0179] 2. Mineralization degree: Analyzed the calcium-phosphorus ratio of enamel by X-ray diffraction

[0180] 3. Cell activity: Cultured ameloblasts in vitro and detected the activity of alkaline phosphatase (ALP)

[0181] Experimental results

[0182]

[0183] Result analysis

[0184] 1. Hardness recovery: The hardness recovery rate of the peptide sequence γ group was 77.1% higher than that of the traditional fluoridated group

[0185] 2. Mineralization effect: The calcium-phosphorus ratio was closer to the normal enamel level (1.85)

[0186] 3. Cell activity: The ALP activity increased by 41.2%, significantly promoting enamel remineralization

[0187] It shows that peptide sequence γ is significantly superior to traditional fluoridated mouthwash in terms of tooth enamel repair.

[0188] Example 11: Verification of the synergistic effect of peptide sequence δ

[0189] Experimental design

[0190] Recruited 30 volunteers with bad breath and randomly divided them into 3 groups:

[0191] · Control group: Used physiological saline

[0192] · Traditional deodorant mouthwash group: Used mouthwash containing 0.3% zinc ions

[0193] · Peptide sequence δ mouthwash group: Used mouthwash containing 0.2% peptide sequence δ

[0194] Used 3 times a day for 14 days.

[0195] Detection indicators and methods

[0196] 1. Breath detection: Measuring the concentration of volatile sulfur compounds (VSCs) using an electronic nose

[0197] 2. Odor-producing bacteria inhibition: Culturing oral odor-producing bacteria and measuring the MIC value

[0198] 3. Sensory evaluation: Professional fragrance evaluators scoring breath

[0199] Experimental results

[0200]

[0201] Result analysis

[0202] 1. Breath improvement: The VSC concentration in the peptide sequence δ group decreased by 43.8% compared to the traditional group

[0203] 2. Bacteriostatic effect: The MIC value decreased by 50% compared to the traditional group

[0204] 3. Sensory experience: The sensory score increased by 38.1%

[0205] This shows that peptide sequence δ is significantly superior to traditional deodorant mouthwashes in improving bad breath

[0206] Example 12: Verification of the synergistic effect of peptide sequence ε

[0207] Experimental design

[0208] Sixty rats with oral microecological imbalance were selected and randomly divided into 3 groups:

[0209] · Control group: Using normal saline

[0210] · Traditional probiotic mouthwash group: Using a mouthwash containing 10 8 CFU / mL Lactobacillus

[0211] · Peptide sequence ε mouthwash group: Using a mouthwash containing 0.2% peptide sequence ε

[0212] Used 2 times a day for 21 days

[0213] Detection indicators and methods

[0214] 1. Bacterial community diversity: Calculating the Shannon index by 16S rRNA sequencing

[0215] 2. Abundance of beneficial bacteria: Measuring the number of Bifidobacterium by quantitative PCR

[0216] 3. Inhibition of harmful bacteria: Culturing and detecting the number of Streptococcus mutans Experimental results

[0217]

[0218] Result analysis

[0219] 1. Microbial diversity: The Shannon index of the peptide sequence ε group increased by 18.8% compared to the traditional group.

[0220] 2. Proliferation of beneficial bacteria: The number of Bifidobacterium increased by 52%.

[0221] 3. Inhibition of harmful bacteria: The inhibition effect increased by 41.8%.

[0222] It shows that the peptide sequence ε is significantly superior to the traditional probiotic mouthwash in regulating the oral microecology.

Claims

1. A natural plant polyphenol - peptide complex applied to mouthwash, characterized in that, Composed of the following components in parts by weight: Plant polyphenols: 15 - 30 parts of proanthocyanidins derived from grape seeds, 10 - 25 parts of catechins in green tea, 10 - 20 parts of anthocyanins in blueberries, 10 - 20 parts of ellagic acid in pomegranate peel; Small molecule peptides: Peptide sequence α: Glu - Lys - Pro - Gly - Ser - Thr - Ala - Val - Leu - Ile - Phe - Tyr - Trp - Arg, 10 - 25 parts; Peptide sequence β: Gly - Asp - Glu - Pro - Arg - Ser - Thr - Val - Leu - Phe - Met - His - Asn - Lys, 8 - 20 parts; Peptide sequence γ: Thr - Arg - Glu - Asp - Pro - Gly - Ser - Ile - Val - Leu - Phe - Tyr - Trp - Gln - Ala, 10 - 22 parts; Peptide sequence δ: Pro - Lys - Glu - Asp - Gly - Ser - Thr - Val - Ile - Leu - Phe - Tyr - Trp - Asn - Glu - Ser, 8 - 20 parts; Peptide sequence ε: Ser - Gly - Pro - Thr - Lys - Arg - Glu - Asp - Val - Leu - Ile - Phe - Tyr - Trp - His - Gln - Asn, 8 - 20 parts; The small molecule peptides are respectively derived from extracts of soybeans, corn, wheat, oats and peas.

2. The natural plant polyphenol - peptide complex according to claim 1, wherein Among the plant polyphenols, 20 - 28 parts of proanthocyanidins, 12 - 22 parts of catechins, 12 - 18 parts of anthocyanins, 12 - 18 parts of ellagic acid; Among the small molecule peptides, 12 - 22 parts of peptide sequence α, 10 - 18 parts of peptide sequence β, 12 - 20 parts of peptide sequence γ, 10 - 18 parts of peptide sequence δ, 10 - 18 parts of peptide sequence ε.

3. A method for preparing a natural plant polyphenol - peptide complex as described in any one of claims 1 - 2, characterized in that, Including the following steps: Raw material pretreatment: Select grape seeds, green tea, blueberries, pomegranate peel, soybeans, corn, wheat, oats and peas. After washing and drying, crush the grape seeds, pomegranate peel, soybeans, corn, wheat, oats and peas through an 80 - 100 mesh sieve, and make green tea and blueberries into homogenates; Extraction: Mix grape seed powder with 60 - 80% ethanol at a ratio of 1:8 - 1:12 (g / mL), perform ultrasonic extraction at 40 - 60°C for 2 - 4 hours, centrifuge and take the supernatant for concentration under reduced pressure; Mix green tea homogenate with 50 - 70% ethanol at a ratio of 1:6 - 1:10 (g / mL), perform stirring extraction at 35 - 55°C for 1 - 3 hours, filter and take the filtrate for concentration under reduced pressure; Mix blueberry homogenate with 40 - 60% ethanol at a ratio of 1:5 - 1:8 (g / mL), perform ultrasonic extraction at 30 - 50°C for 1 - 3 hours, centrifuge and take the supernatant for concentration under reduced pressure; Mix pomegranate peel powder with 70 - 90% ethanol at a ratio of 1:7 - 1:11 (g / mL), perform reflux extraction at 45 - 65°C for 2 - 4 hours, cool, filter and take the filtrate for concentration under reduced pressure; Mix soybean powder with deionized water at a ratio of 1:6 - 1:10 (g / mL), enzymatically hydrolyze with alkaline protease at 40 - 60°C, inactivate the enzyme, centrifuge and take the supernatant for ultrafiltration; Mix corn flour with deionized water at a ratio of 1:5 - 1:8 (g / mL), enzymatically hydrolyze with neutral protease at 35 - 55°C, inactivate the enzyme, centrifuge and take the supernatant for ultrafiltration; Mix wheat flour with deionized water at a ratio of 1:6 - 1:10 (g / mL), enzymatically hydrolyze with compound protease at 40 - 60°C, inactivate the enzyme, centrifuge and take the supernatant for ultrafiltration; Mix oat flour with deionized water at a ratio of 1:5 - 1:8 (g / mL), enzymatically hydrolyze with acidic protease at 35 - 55°C, inactivate the enzyme, centrifuge and take the supernatant for ultrafiltration; Mix pea flour with deionized water at a ratio of 1:6 - 1:10 (g / mL), enzymatically hydrolyze with flavor protease at 40 - 60°C, inactivate the enzyme, centrifuge and take the supernatant for ultrafiltration; Separation and purification: Pass each plant extract through a macroporous adsorption resin column, elute with ethanol solutions of different concentrations in a gradient manner, and then separate and purify by high-speed counter-current chromatography; Separate and purify each peptide extract through gel filtration chromatography and ion exchange chromatography respectively; Complex formation: Mix the purified plant polyphenols and small molecule peptides in proportion, under the conditions of 30 - 40°C and pH 6 - 7, perform ultrasonic wave-assisted treatment at 200 - 400W for 10 - 20 minutes, and stir at a rotation speed of 200 - 400rpm simultaneously to form a natural plant polyphenol-peptide complex.

4. The preparation method according to claim 3, wherein When enzymatically hydrolyzing soybean powder, the addition amount of alkaline protease is 0.5 - 1.5%, pH 8 - 10, and the enzymolysis time is 2 - 4 hours; When enzymatically hydrolyzing corn flour, the addition amount of neutral protease is 0.4 - 1.2%, pH 6 - 8, and the enzymolysis time is 2 - 4 hours.

5. The preparation method according to claim 3, characterized in that, When enzymatically hydrolyzing wheat flour, the addition amount of compound protease is 0.6 - 1.4%, pH 7 - 9, and the enzymolysis time is 2 - 4 hours; When enzymatically hydrolyzing oat flour, the addition amount of acidic protease is 0.5 - 1.3%, pH 4 - 6, and the enzymolysis time is 2 - 4 hours; When enzymatically hydrolyzing pea flour, the addition amount of flavor protease is 0.4 - 1.2%, pH 6 - 8, and the enzymolysis time is 2 - 4 hours.

6. A mouthwash, characterized in that, It is composed of the following components in parts by weight: 2 - 8 parts of the natural plant polyphenol - peptide complex according to any one of claims 1 - 2, 5 - 15 parts of glycerol, 3 - 10 parts of xylitol, 2 - 8 parts of sorbitol, 0.1 - 0.5 part of menthol, 0.1 - 0.5 part of essence, an appropriate amount of pH regulator, and deionized water is added to make up to 100 parts.

7. The mouthwash according to claim 6, characterized in that, The pH regulator is citric acid or sodium hydroxide, and the pH value of the mouthwash is adjusted to 6.0 - 7.

0.

8. A method for preparing a mouthwash according to any one of claims 6-7, characterized in that, It includes the following steps: Add the natural plant polyphenol - peptide complex to deionized water and stir to dissolve; successively add glycerol, xylitol, and sorbitol and stir until completely dissolved; add menthol and essence and mix well; adjust the pH value to 6.0 - 7.0 with the pH regulator, and after volume fixation, filter and sterilize through a 0.22μm filter membrane.

9. Use of the natural plant polyphenol - peptide complex according to any one of claims 1 - 2 in the preparation of a mouthwash with antibacterial, anti - inflammatory, tooth enamel repair, and fresh breath effects.

10. The application according to claim 9, characterized in that, The mouthwash is used to inhibit harmful oral bacteria, relieve oral inflammation, promote tooth enamel remineralization, and improve bad breath.

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