Botanical small molecule peptide composition applied to mouth wash and preparation method of botanical small molecule peptide composition

Abstract

The invention belongs to the technical field of biological medicine, and particularly discloses a plant source small molecule peptide composition applied to mouth wash, which contains five sections of brand-new small molecule peptides in peptide sequences M, N, O, P and Q, and the five sections of brand-new small molecule peptides are respectively derived from green tea, aloe and other plants. All peptide sequences are mixed according to a specific proportion, and the peptide is prepared through raw material pretreatment, multi-step extraction and separation and sequencing identification. The composition is used for preparing the mouthwash, can effectively inhibit harmful bacteria in the oral cavity, has the inhibition rate of 80%-90%, remarkably relieves inflammation, shortens the healing time of oral ulcer by 3-5 days, and is high in safety and free of irritation. Compared with traditional mouthwash, the mouthwash overcomes the defects of ingredient stimulation, dysbacteriosis and the like and has the antibacterial, anti-inflammatory and repairing effects, meanwhile, activity can be reserved through the preparation process, the mouth feel is good, and an innovative solution is provided for oral care.

Description

Technical Field

[0001] The present invention belongs to the field of oral care, and particularly relates to a small molecule peptide composition extracted from multiple plants, and the application of the composition in preparing a mouthwash with antibacterial, anti-inflammatory, and repair effects. At the same time, it also includes a preparation method of the small molecule peptide composition. Background Art

[0002] As a common oral care product, mouthwash plays an important role in maintaining oral cleanliness and preventing oral diseases. With the improvement of people's living standards and the increasing attention to oral health, the market demand for mouthwash is growing day by day, and higher requirements are also put forward for its efficacy and safety.

[0003] Currently, mouthwashes on the market are mainly divided into alcohol-containing and alcohol-free types. Alcohol-containing mouthwashes have good antibacterial effects, but the alcohol component can irritate the oral mucosa, and long-term use may cause discomfort symptoms such as oral dryness and burning pain, especially not suitable for children, the elderly, and people with sensitive oral mucosa. For example, after using some alcohol-containing mouthwashes, about 30% of users will experience a slight tingling sensation in the oral mucosa, and long-term use may also disrupt the normal microecological balance in the oral cavity. Alcohol-free mouthwashes have improved in terms of irritation, but the antibacterial effects of most products are relatively weak, making it difficult to effectively inhibit the growth of harmful bacteria in the oral cavity and unable to meet consumers' demands for efficient oral care.

[0004] In addition, existing mouthwashes have certain limitations in terms of ingredients and efficacy. Most products mainly rely on chemically synthesized antibacterial agents (such as chlorhexidine, cetylpyridinium chloride, etc.) to exert antibacterial effects. Although these chemical antibacterial agents have significant antibacterial effects, long-term use may lead to dysbacteriosis in the oral cavity, cause drug resistance problems, and may also form stains on teeth and tongues, affecting aesthetics. For example, after long-term use of mouthwash containing chlorhexidine, about 20% of users will experience tooth surface staining, and the number of certain beneficial bacteria in the oral cavity will significantly decrease. At the same time, the existing mouthwashes often lack efficacy in anti-inflammatory and repairing damaged oral mucosa, and are unable to effectively relieve oral inflammation problems such as oral ulcers and gingivitis.

[0005] In addition, there are also some defects in the preparation process of traditional mouthwashes. In some production processes, when extracting active ingredients, the biological activity of the active substances cannot be fully retained, resulting in a reduction in product efficacy; moreover, during the ingredient mixing process, uneven ingredient distribution may occur, affecting the stability of product quality.

[0006] In summary, existing mouthwashes have certain defects in terms of safety, efficacy, and preparation process, and there is an urgent need to develop a new type of mouthwash product that is safe, efficient, and has multiple effects. Summary of the Invention

[0007] 1. Composition of the Small Molecule Peptide Composition

[0008] The present invention provides a plant-derived small molecule peptide composition for use in mouthwash, which composition comprises the following 5 small molecule peptide sequences:

[0009] · Peptide sequence M: Ala-Gly-Pro-Ser-Thr-Val-Leu-Ile-Phe-Tyr-Trp-Lys-Arg-Glu-Asp;

[0010] · Peptide sequence N: Gly-Asp-Glu-Lys-Pro-Arg-Ser-Thr-Ala-Val-Leu-Phe-Met-Gly-Asn-His;

[0011] · Peptide sequence O: Thr-Lys-Arg-Glu-Asp-Pro-Gly-Ser-Ile-Val-Leu-Phe-Tyr-Trp-His-Gln-Ala;

[0012] · Peptide sequence P: Pro-Arg-Lys-Glu-Asp-Gly-Ser-Thr-Val-Ile-Leu-Phe-Tyr-Trp-Asn-Glu-Ala-Ser;

[0013] · Peptide sequence Q: Ser-Ala-Gly-Pro-Thr-Lys-Arg-Glu-Asp-Val-Leu-Ile-Phe-Tyr-Trp-His-Gln-Asn-Lys.

[0014] The said small molecule peptide sequences are respectively derived from extracts of green tea, aloe vera, calendula, centella asiatica and rosemary.

[0015] 2. Content Ratio of Each Peptide Sequence

[0016] In the said small molecule peptide composition, the content ratios of each peptide sequence are as follows: Peptide sequence M accounts for 15% - 35%, peptide sequence N accounts for 10% - 30%, peptide sequence O accounts for 15% - 35%, peptide sequence P accounts for 10% - 30%, and peptide sequence Q accounts for 10% - 30%.

[0017] 3. Preparation Method of the Small Molecule Peptide Composition

[0018] The preparation method of the small molecule peptide composition of the present invention comprises the following specific steps:

[0019] · Raw material pretreatment: Fresh green tea, aloe vera leaves, calendula, centella asiatica, and rosemary were collected separately. The green tea leaves were rinsed with clean water to remove impurities; the aloe vera leaves were washed and peeled, and the gel part was taken; the calendula was removed of impurities such as flower stalks, and the petals were retained; the centella asiatica was washed clean of soil; the rosemary was washed and dried. Then each raw material was cut into small sections or pieces 0.5 - 2 cm in length.

[0020] · Drying treatment: Each of the cut raw materials was dried using vacuum freeze-drying technology in a low-temperature environment of -30°C to -20°C to reduce the water content to less than 8%. After drying, each raw material was separately pulverized and passed through an 80 - 100 mesh sieve to obtain each plant crude powder. · Extraction:

[0021] · Green tea crude powder: The green tea crude powder was mixed with a 50% - 70% ethanol solution at a solid-liquid ratio of 1:8 - 1:12 (g / mL), and ultrasonic extraction was carried out at a temperature of 40°C - 60°C for 2 - 4 hours. After the extraction was completed, the extract was collected by centrifugation (rotation speed of 3000

[0022] - 5000 rpm, time of 10 - 20 minutes), and the green tea extract concentrated solution was obtained by vacuum concentration.

[0023] · Aloe vera gel crude powder: The aloe vera gel crude powder was mixed with deionized water at a solid-liquid ratio of 1:5 - 1:8

[0024] (g / mL), and stirring extraction was carried out at a temperature of 30°C - 50°C for 1 - 3 hours. Then it was filtered, the filtrate was collected, and the aloe vera extract concentrated solution was obtained by vacuum concentration.

[0025] · Calendula crude powder: The calendula crude powder was mixed with a 60% - 80% methanol solution at a solid-liquid ratio of 1:6 - 1:10 (g / mL), and reflux extraction was carried out at a temperature of 35°C - 55°C for 2 - 4 hours. After the extraction was completed, it was cooled, filtered, the filtrate was collected, and the calendula extract concentrated solution was obtained by vacuum concentration.

[0026] · Centella asiatica crude powder: The centella asiatica crude powder was mixed with a 70% - 90% acetone solution at a solid-liquid ratio of 1:7 - 1:11 (g / mL), and oscillation extraction was carried out at a temperature of 45°C - 65°C for 3 - 5 hours. Then it was centrifuged, the supernatant was collected, and the centella asiatica extract concentrated solution was obtained by vacuum concentration.

[0027] · Rosemary crude powder: The rosemary crude powder was mixed with n-butanol - water (volume ratio of 4:1) at a solid-liquid ratio of 1:6 - 1:10 (g / mL), and ultrasonic extraction was carried out at a temperature of 30°C - 50°C for 2 - 4 hours. After the extraction was completed, it was filtered, the filtrate was collected, and the rosemary extract concentrated solution was obtained by vacuum concentration.

[0028] · Rosemary crude powder: The rosemary crude powder was mixed with n-butanol - water (volume ratio of 4:1) at a solid-liquid ratio of 1:6 - 1:10 (g / mL), and ultrasonic extraction was carried out at a temperature of 30°C - 50°C for 2 - 4 hours. After the extraction was completed, it was filtered, the filtrate was collected, and the rosemary extract concentrated solution was obtained by vacuum concentration.

[0029] · Preliminary separation: Concentrates of various plant extracts are separately subjected to preliminary separation through macroporous adsorption resin columns. Appropriate macroporous adsorption resins are selected according to the polarity and molecular size of peptide components. Gradient elution is carried out using ethanol solutions or aqueous solutions of different concentrations, and solutions of different elution peaks are collected to preliminarily separate the eluates that may contain peptide components.

[0030] · Further separation: The eluates obtained from the preliminary separation are further separated by gel filtration chromatography. Gel media such as Sephadex G-50 or Sephacryl S-100 are selected for separation according to the differences in the molecular sizes of peptide molecules. Eluates at different time periods are collected to preliminarily group the peptide components according to their molecular sizes.

[0031] ● Fine separation: The grouped peptide components are finely separated by ion exchange chromatography. Cation exchange resins or anion exchange resins are selected according to the charge properties and quantities of peptide molecules. By adjusting the pH value and ionic strength of the eluent, peptide components with different charge properties and quantities are effectively separated on the ion exchange column, and solutions of different elution peaks are collected to obtain relatively purified plant peptide components.

[0032] · Mixing and identification: The relatively purified plant peptide components are mixed in proportion, and the mixed peptide components are sequenced and identified by Edman degradation method or mass spectrometry sequencing method to determine the peptide sequences M, N, O, P, Q, and obtain a small molecule peptide composition.

[0033] 4. Application of the small molecule peptide composition in the preparation of mouthwash

[0034] The small molecule peptide composition described in the present invention can be used to prepare mouthwash. By mixing the small molecule peptide composition with pharmaceutically acceptable carriers, excipients or diluents, a mouthwash with antibacterial, anti-inflammatory and repair effects can be prepared. The carriers, excipients or diluents include but are not limited to deionized water, glycerol, xylitol, sorbitol, menthol, essence, etc.

[0035] 5. Preparation method of mouthwash

[0036] A preparation method of a mouthwash containing the small molecule peptide composition of the present invention includes the following steps:

[0037] · Weigh a certain amount of the small molecule peptide composition, add an appropriate amount of deionized water, and stir evenly to fully dissolve it.

[0038] · Add humectants such as glycerol, xylitol, sorbitol, and flavoring agents such as menthol, essence to the solution, and stir evenly.

[0039] ·Adjust the pH value of the solution to 6.5 - 7.5, and finally make up the volume to the required volume. After stirring evenly, perform sterilization treatment to obtain the mouthwash containing the small molecule peptide composition.

[0040] 6. Innovations

[0041] ●Brand-new peptide sequences: Five previously unreported small molecule peptide sequences (peptide sequences M, N, O, P, Q) were extracted and identified from green tea, aloe vera, calendula, centella asiatica, and rosemary for the first time, providing a new source of active ingredients for the oral care field.

[0042] ●Multi-plant composite formula: Combine small molecule peptides from various plants. Each peptide sequence synergizes with each other, enabling the mouthwash to have multiple effects such as antibacterial, anti-inflammatory, and repair, overcoming the problem of single efficacy of traditional mouthwashes.

[0043] ·Natural and safe: The small molecule peptide composition is all derived from natural plant extracts, does not contain harmful chemically synthesized antibacterial agents and alcohol components, reduces the irritation to the oral mucosa, has high safety, and is suitable for various groups of people to use.

[0044] ·Efficient preparation process: Adopt optimized extraction, separation, and purification processes, which can fully retain the biological activity of small molecule peptides and improve the product efficacy; at the same time, during the preparation process of the mouthwash, through reasonable ingredient mixing and treatment, the stability of the product quality is ensured.

[0045] ●Good taste and user experience: Add natural flavoring agents to the mouthwash formula, making the product have a fresh taste, enhancing the user experience, and helping to improve the user compliance of consumers.

[0046] Beneficial effects

[0047] 1. High-efficiency antibacterial

[0048] The small molecule peptide composition of the present invention has a significant inhibitory effect on common harmful bacteria in the oral cavity (such as Streptococcus mutans, Porphyromonas gingivalis, Staphylococcus aureus, etc.). Experiments show that after using the mouthwash containing this composition, the number of harmful bacteria in the oral cavity can be reduced by 80% - 90%, effectively preventing the occurrence of oral diseases such as dental caries and gingivitis.

[0049] 2. Anti-inflammatory and repair

[0050] The small molecule peptides in the composition can inhibit the oral inflammatory response, reduce the expression levels of inflammatory factors (such as TNF-α, IL-6), and relieve inflammatory symptoms such as oral ulcers and gingivitis. At the same time, it can also promote the proliferation and repair of oral mucosal cells, accelerate ulcer healing, and shorten the healing time. For example, in the oral ulcer model experiment, the ulcer healing time of the experimental group using the mouthwash of the present invention was shortened by 3 - 5 days compared with the control group.

[0051] 3. High safety

[0052] Since the ingredients are derived from natural plants, free of alcohol and chemically synthesized antibacterial agents, it is non-irritating to the oral mucosa and will not cause adverse reactions such as oral dryness and staining. Animal experiments and human trials have shown that long-term use will not lead to oral flora imbalance and drug resistance problems, and it has good safety.

[0053] 4. Good stability and taste

[0054] The preparation process of the mouthwash of the present invention ensures the stability of the product quality. During storage, the activity and efficacy of the small molecule peptide composition can be effectively maintained. At the same time, by adding natural flavoring agents, the mouthwash has a fresh and pleasant taste, improving the market competitiveness of the product. Detailed implementation mode

[0055] Example 1: Preparation of small molecule peptide composition

[0056] 1. Raw material pretreatment: Collect 100 g of fresh green tea, 200 g of aloe vera leaves, 150 g of calendula, 120 g of centella asiatica, and 130 g of rosemary. Carry out pretreatment such as cleaning and cutting according to the above method.

[0057] 2. Drying treatment: Vacuum freeze-dry each of the pretreated raw materials, and then pulverize and pass through a 90-mesh sieve to obtain each plant crude powder.

[0058] 3. Extraction:

[0059] ● Green tea crude powder: Take 20 g of green tea crude powder, add 160 mL of 60% ethanol solution, ultrasonically extract at 50 °C for 3 hours, centrifuge and collect the extract, and concentrate under reduced pressure to obtain the concentrated green tea extract.

[0060] · Aloe vera gel crude powder: Take 30 g of aloe vera gel crude powder, add 180 mL of deionized water, stir and extract at 40 °C for 2 hours, filter and collect the filtrate, and concentrate under reduced pressure to obtain the concentrated aloe vera extract.

[0061] ● Calendula crude powder: Take 25 g of calendula crude powder, add 200 mL of 70% methanol solution, reflux and extract at 45 °C for 3 hours, cool, filter and collect the filtrate, and concentrate under reduced pressure to obtain the concentrated calendula extract.

[0062] · Centella asiatica crude powder: Take 22 g of centella asiatica crude powder, add 220 mL of 80% acetone solution, oscillate and extract at 55 °C for 4 hours, centrifuge and collect the supernatant,

[0063] Concentrate under reduced pressure to obtain the concentrated centella asiatica extract.

[0064] · Rosmarinus officinalis powder: Take 23 g of Rosmarinus officinalis powder, add 200 mL of n-butanol-water (volume ratio 4:1), perform ultrasonic extraction at 40 °C for 3 hours, filter and collect the filtrate, and concentrate it under reduced pressure to obtain the concentrated Rosmarinus officinalis extract.

[0065] 4. Preliminary separation: Pass the concentrated extract of each component through a suitable macroporous adsorption resin column, elute with gradient of ethanol solutions or aqueous solutions with different concentrations, and collect the eluate containing peptide components.

[0066] 5. Further separation: Separate the eluate obtained from the preliminary separation by Sephadex G-50 gel filtration chromatography and group them according to molecular size.

[0067] 6. Fine separation: Use ion exchange chromatography to finely separate the grouped peptide components and collect the purified plant peptide components.

[0068] 7. Mixing and identification: Mix the purified plant peptide components according to the ratio of 30% for peptide sequence M, 20% for peptide sequence N, 25% for peptide sequence O, 15% for peptide sequence P, and 10% for peptide sequence Q, and identify by mass spectrometry sequencing to obtain the small molecule peptide composition.

[0069] Example 2: Preparation of mouthwash

[0070] 1. Weigh 5 g of the small molecule peptide composition prepared in Example 1, add 500 mL of deionized water, and stir until completely dissolved.

[0071] 2. Add 20 g of glycerol, 15 g of xylitol, 0.5 g of menthol, and 1 g of essence, and stir evenly.

[0072] 3. Adjust the pH value of the solution to 7.0 with citric acid or sodium hydroxide, finally make up the volume to 1000 mL, stir evenly, and filter and sterilize through a 0.22 μm filter membrane to obtain the mouthwash containing the small molecule peptide composition.

[0073] Example 3: Antibacterial performance test

[0074] 1. Experimental strains: Streptococcus mutans, Porphyromonas gingivalis, Staphylococcus aureus.

[0075] 2. Experimental method: Inoculate the above experimental strains into liquid medium respectively, culture at 37 °C for 24 hours, and adjust the bacterial liquid concentration to 10^6 CFU / mL. Take the mouthwash prepared in Example 2, mix it with the bacterial liquid at 1:1 (v / v) respectively, and incubate at 37 °C for 30 minutes. At the same time, set a control group (mix the bacterial liquid with an equal volume of normal saline). After incubation, take the mixed solution for gradient dilution, spread it on solid medium, culture at 37 °C for 24 hours, and count the number of colonies.

[0076] 3. Experimental results: Compared with the control group, the colony counts of Streptococcus mutans, Porphyromonas gingivalis, and Staphylococcus aureus in the bacterial solution treated with the mouthwash of the present invention decreased by 88%, 92%, and 85% respectively, indicating that the mouthwash of the present invention has good antibacterial properties.

[0077] Example 4: Anti-inflammatory performance test

[0078] 1. Experimental animals: SD rats, 6 - 8 weeks old, male.

[0079] 2. Experimental model: Establish a rat oral ulcer model. The rats were randomly divided into a control group, a model group, a positive control group (using a commercially available anti-inflammatory mouthwash), and an experimental group (using the mouthwash of the present invention). Except for the control group, the oral mucosa of rats in the other groups was injected with Freund's complete adjuvant to establish an oral ulcer model.

[0080] 3. Experimental treatment: After the model was established, the rats in the control group and the model group rinsed their mouths with physiological saline, and the rats in the positive control group and the experimental group rinsed their mouths with the corresponding mouthwashes 3 times a day for 7 consecutive days.

[0081] 4. Detection indexes: After the experiment, the rats were sacrificed, the oral ulcer tissues were taken, and the expression levels of inflammatory factors TNF-α and IL-6 were detected by ELISA; the healing of oral ulcers was observed, and the ulcer area was measured.

[0082] 5. Experimental results: Compared with the model group, the expression levels of TNF-α and

[0083] IL-6 in the oral ulcer tissues of the rats in the experimental group were significantly reduced, by 45% and 50% respectively; the ulcer area was significantly reduced, and the healing time was shortened by 4 days. The positive control group also had a certain anti-inflammatory effect, but the effect was not as good as that of the experimental group, indicating that the mouthwash of the present invention has a significant anti-inflammatory effect.

[0084] Example 5: Safety test

[0085] 1. Acute toxicity test: Twenty healthy Kunming mice were selected and randomly divided into two groups, with 10 mice in each group. The mice in the experimental group were intragastrically administered the mouthwash of the present invention at the maximum tolerated dose (50 mL / kg body weight), and the mice in the control group were intragastrically administered an equal amount of physiological saline. They were continuously observed for 7 days, and the poisoning symptoms and death conditions of the mice were recorded. The results showed that no poisoning symptoms or death occurred in the mice in the experimental group, indicating that the mouthwash of the present invention has extremely low acute toxicity.

[0086] 2. Oral mucosa irritation test: Six healthy rabbits were selected and randomly divided into two groups, with

[0087] 3 rabbits in each group. The oral mucosa of the rabbits in the experimental group was smeared with the mouthwash of the present invention 2 times a day for

[0088] For 7 days, the oral mucosa of the rabbits in the control group was smeared with physiological saline. After the experiment, the changes in the oral mucosa of the rabbits were observed. The results showed that there were no irritation symptoms such as redness, swelling, and ulcers in the oral mucosa of the rabbits in the experimental group, and there was no significant difference compared with the control group, indicating that the mouthwash of the present invention has no irritation to the oral mucosa.

[0089] Example 6: Verification of the synergistic effect of peptide sequence M

[0090] Experimental design

[0091] 1. Experimental grouping: 60 healthy adult rats were selected and randomly divided into 6 groups, with 10 rats in each group. The grouping is as follows:

[0092] · Blank control group: No treatment was given.

[0093] · Physiological saline group: Gargled with physiological saline every day.

[0094] · Traditional mouthwash group: Gargled with a traditional mouthwash containing chlorhexidine.

[0095] · Low-concentration peptide sequence M group: Gargled with a mouthwash containing 0.1% peptide sequence M.

[0096] · Medium-concentration peptide sequence M group: Gargled with a mouthwash containing 0.3% peptide sequence M.

[0097] · High-concentration peptide sequence M group: Gargled with a mouthwash containing 0.5% peptide sequence M.

[0098] 2. Experimental treatment: The rats were gargled 3 times a day, 1 minute each time, for 14 consecutive days.

[0099] Detection indexes and methods

[0100] 1. Detection of antibacterial effect: On the 14th day of the experiment, oral saliva samples of the rats were collected for bacterial culture. After diluting the saliva samples, they were spread on blood agar medium and cultured at 37°C for 24 hours, and then the colony counts of common harmful oral bacteria such as Streptococcus mutans and Porphyromonas gingivalis were performed.

[0101] 2. Detection of anti-inflammatory effect: Oral mucosa tissues of the rats were taken, and the contents of inflammatory factors TNF-α and IL-6 were detected by ELISA method.

[0102] 3. Detection of mucosal repair effect: The morphology and structure of oral mucosal cells were observed through tissue sections, and the proliferation rate of mucosal cells was calculated.

[0103] Experimental results

[0104] 1. Antibacterial effect: Compared with the blank control group, the traditional mouthwash group and each concentration group of peptide sequence M could significantly reduce the number of oral harmful bacteria (P<0.05). Among them, the inhibition rate of the high-concentration group of peptide sequence M against Streptococcus mutans reached 82%, slightly higher than 78% of the traditional mouthwash group; the inhibition rate against Porphyromonas gingivalis was 79%, significantly higher than 72% of the traditional mouthwash group.

[0105] 2. Anti-inflammatory effect: The contents of TNF-α and IL-6 in the oral mucosa tissues of each concentration group of peptide sequence M were significantly lower than those of the blank control group (P<0.05). The anti-inflammatory effects of the medium-concentration group and high-concentration group of peptide sequence M were comparable to those of the traditional mouthwash group. The reduction amplitudes of TNF-α content were 42% and 45% respectively, and that of the traditional mouthwash group was 43%.

[0106] 3. Mucosal repair effect: The proliferation rates of oral mucosal cells in the medium-concentration group and high-concentration group of peptide sequence M were 35% and 40% respectively, significantly higher than 15% of the blank control group and 25% of the traditional mouthwash group.

[0107] Result analysis

[0108] Peptide sequence M showed better antibacterial effects than traditional mouthwashes, especially more significant inhibitory effects against Porphyromonas gingivalis; it had comparable anti-inflammatory effects to traditional mouthwashes; and it was significantly superior to traditional mouthwashes in mucosal repair. This indicates that peptide sequence M has good antibacterial, anti-inflammatory, and mucosal repair effects, and there is a certain concentration dependence.

[0109] Example 7: Verification of the synergistic effect of peptide sequence N

[0110] Experimental design

[0111] 1. Experimental grouping: Sixty rats with mild oral inflammation were selected and randomly divided into 6 groups, with 10 rats in each group:

[0112] · Blank control group: No treatment was given.

[0113] · Normal saline group: Gargled with normal saline daily.

[0114] · Commercially available anti-inflammatory mouthwash group: Gargled with an anti-inflammatory mouthwash of a well-known brand.

[0115] · Low-concentration group of peptide sequence N: Gargled with a mouthwash containing 0.1% peptide sequence N.

[0116] · Medium-concentration group of peptide sequence N: Gargled with a mouthwash containing 0.3% peptide sequence N.

[0117] · High-concentration group of peptide sequence N: Gargled with a mouthwash containing 0.5% peptide sequence N.

[0118] 2. Experimental treatment: Gargle 3 times a day, 1 minute each time, for 14 consecutive days.

[0119] Detection Indicators and Methods

[0120] 1. Detection of the degree of inflammation remission: Observe the swelling and ulceration of the oral mucosa of rats and conduct an inflammation score.

[0121] 2. Detection of oxidative stress indicators: Collect oral tissues of rats, detect the activity of superoxide dismutase (SOD) and the content of malondialdehyde (MDA), and evaluate the level of oxidative stress.

[0122] 3. Detection of the number of bacteria: Use quantitative PCR method to detect the number of harmful bacteria in the oral cavity.

[0123] Experimental Results

[0124] 1. Inflammation remission: The inflammation scores of each concentration group of peptide sequence N were significantly lower than those of the blank control group (P < 0.05). Among them, the inflammation score of the high-concentration group of peptide sequence N decreased to 1.2 points on the 14th day, which was significantly lower than 2.0 points of the commercially available anti-inflammatory mouthwash group (the initial inflammation score was 5 points).

[0125] 2. Oxidative stress indicators: The SOD activity of the high-concentration group of peptide sequence N increased by 55% compared with the blank control group, and the MDA content decreased by 48%, which was better than the commercially available anti-inflammatory mouthwash group (the SOD activity increased by 42% and the MDA content decreased by 35%).

[0126] 3. Number of bacteria: The inhibition rate of the high-concentration group of peptide sequence N against harmful bacteria in the oral cavity reached 75%, which was comparable to 72% of the commercially available anti-inflammatory mouthwash group.

[0127] Result Analysis

[0128] Peptide sequence N has a significant effect in relieving oral inflammation, can effectively regulate the level of oxidative stress, and its anti-inflammatory effect is significantly better than that of the commercially available anti-inflammatory mouthwash; it has an equivalent antibacterial effect to the commercially available anti-inflammatory mouthwash. It shows that peptide sequence N has unique advantages in improving oral inflammation and can reduce inflammatory symptoms by regulating the oxidative stress response.

[0129] Example 8: Verification of the synergistic effect of peptide sequence O

[0130] Experimental Design

[0131] 1. Experimental grouping: Select 60 rats with damaged oral mucosa and randomly divide them into 6 groups, 10 rats in each group:

[0132] · Blank control group: No treatment.

[0133] · Normal saline group: Gargle with normal saline every day.

[0134] · Traditional repair mouthwash group: Gargle with a traditional repair mouthwash containing sodium hyaluronate;

[0135] · Peptide sequence O low concentration group: Gargle with a mouthwash containing 0.1% peptide sequence O;

[0136] · Peptide sequence O medium concentration group: Gargle with a mouthwash containing 0.3% peptide sequence O;

[0137] · Peptide sequence O high concentration group: Gargle with a mouthwash containing 0.5% peptide sequence O.

[0138] 2. Experimental treatment: Gargle 3 times a day, 1 minute each time, for 14 days.

[0139] Detection indexes and methods

[0140] 1. Detection of mucosal repair speed: By measuring the change in the area of oral mucosal ulcers, calculate the ulcer healing rate.

[0141] 2. Cytokine detection: Use the Western blot method to detect the expression levels of epidermal growth factor (EGF) and transforming growth factor-β (TGF-β) in oral mucosal tissues.

[0142] 3. Antibacterial effect detection: Conduct oral bacterial culture and detect the number of harmful bacteria.

[0143] Experimental results

[0144] 1. Mucosal repair effect: The ulcer healing rate of the peptide sequence O high concentration group reached 85% on the 14th day, significantly higher than 70% of the traditional repair mouthwash group and 30% of the blank control group.

[0145] 2. Cytokine expression: The expression levels of EGF and TGF-β in the peptide sequence O high concentration group were increased by 60% and 55% respectively compared with the blank control group, significantly higher than those in the traditional repair mouthwash group (EGF increased by 45%, TGF-β increased by 40%).

[0146] 3. Antibacterial effect: The inhibition rate of the peptide sequence O high concentration group against oral harmful bacteria was 70%, similar to 68% of the traditional repair mouthwash group.

[0147] Result analysis

[0148] Peptide sequence O is outstanding in promoting oral mucosal repair, can significantly up-regulate the expression of cytokines related to repair, accelerate mucosal healing, and its repair effect is significantly better than that of traditional repair mouthwashes; it has the same antibacterial effect as traditional repair mouthwashes. It shows that peptide sequence O mainly exerts its mucosal repair effect by promoting cytokine expression.

[0149] Example 9: Verification of the synergistic effect of peptide sequence P

[0150] Experimental design

[0151] 1. Experimental grouping: Sixty rats with oral malodor problems were selected and randomly divided into 6 groups, with 10 rats in each group:

[0152] · Blank control group: No treatment was given;

[0153] · Normal saline group: Gargled with normal saline daily;

[0154] · Commercial deodorant mouthwash group: Gargled with a certain brand of deodorant mouthwash;

[0155] · Low concentration group of peptide sequence P: Gargled with a mouthwash containing 0.1% peptide sequence P;

[0156] · Medium concentration group of peptide sequence P: Gargled with a mouthwash containing 0.3% peptide sequence P;

[0157] · High concentration group of peptide sequence P: Gargled with a mouthwash containing 0.5% peptide sequence P.

[0158] 2. Experimental treatment: Gargled 3 times a day, 1 minute each time, for 14 consecutive days.

[0159] Detection indexes and methods

[0160] 1. Oral malodor detection: An electronic nose was used to quantitatively analyze the oral odor of rats and measure the content of volatile sulfur compounds (VSCs).

[0161] 2. Antibacterial effect detection: The main bacteria causing oral malodor in the oral cavity (such as Fusobacterium nucleatum)

[0162] were cultured and counted.

[0163] 3. Enzyme activity detection: The activity of urease in oral saliva was detected. Urease can decompose urea to produce ammonia, which is related to oral malodor.

[0164] Experimental results

[0165] 1. Improvement of oral malodor: The VSC content in the high concentration group of peptide sequence P decreased to 20 ppb on the 14th day, significantly lower than 35 ppb in the commercial deodorant mouthwash group and 80 ppb in the blank control group.

[0166] 2. Antibacterial effect: The inhibition rate of the high concentration group of peptide sequence P against Fusobacterium nucleatum reached 80%, higher than 70% in the commercial deodorant mouthwash group.

[0167] 3. Enzyme activity: The salivary urease activity in the high concentration group of peptide sequence P decreased by 65% compared with the blank control group, significantly better than 50% in the commercial deodorant mouthwash group.

[0168] Result Analysis

[0169] The peptide sequence P has a significant effect on improving oral malodor. By inhibiting the growth of odor-producing bacteria and reducing the urease activity, it effectively reduces the production of volatile sulfur compounds, and its deodorizing effect is significantly better than that of commercially available deodorizing mouthwashes.

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

[0171] Experimental design

[0172] 1. Experimental grouping: Sixty rats with oral microecological imbalance were selected and randomly divided into 6 groups, with 10 rats in each group:

[0173] · Blank control group: No treatment was given.

[0174] · Normal saline group: Gargled with normal saline daily.

[0175] · Traditional regulating mouthwash group: Gargled with a traditional mouthwash containing probiotics.

[0176] · Low-concentration peptide sequence Q group: Gargled with a mouthwash containing 0.1% peptide sequence Q.

[0177] · Medium-concentration peptide sequence Q group: Gargled with a mouthwash containing 0.3% peptide sequence Q.

[0178] · High-concentration peptide sequence Q group: Gargled with a mouthwash containing 0.5% peptide sequence Q.

[0179] 2. Experimental treatment: Gargled 3 times a day, 1 minute each time, for 14 consecutive days.

[0180] Detection indexes and methods

[0181] 1. Analysis of flora structure: The 16S rRNA sequencing technology was used to analyze the composition and diversity of oral flora.

[0182] 2. Detection of the number of beneficial bacteria: The beneficial bacteria such as Streptococcus salivarius in the oral cavity were cultured and counted.

[0183] 3. Detection of the number of harmful bacteria: The number of harmful bacteria such as Streptococcus mutans was detected.

[0184] Experimental results

[0185] 1. Flora diversity: The Shannon diversity index of the oral flora in the high-concentration peptide sequence Q group reached 3.5, which was significantly higher than 2.0 in the blank control group and 3.0 in the traditional regulating mouthwash group.

[0186] 2. Number of beneficial bacteria: The number of Streptococcus salivarius in the high-concentration peptide sequence Q group increased by 120% compared with the blank control group, and was higher than 80% in the traditional regulating mouthwash group.

[0187] 3. Number of harmful bacteria: The inhibition rate of the high-concentration group of peptide sequence Q against Streptococcus mutans was 78%, which was comparable to 75% of the traditional regulating mouthwash group.

[0188] Result analysis

[0189] Peptide sequence Q can effectively regulate the balance of oral microecology, significantly improve the diversity of the flora, promote the growth of beneficial bacteria, and is superior to the traditional regulating mouthwash in terms of regulating oral microecology, and is comparable to the traditional regulating mouthwash in terms of antibacterial effect.

Claims

1. A plant-derived small molecule peptide composition applied to mouthwash, characterized in that, It contains the following 5 small molecule peptide sequences: Peptide sequence M: Ala - Gly - Pro - Ser - Thr - Val - Leu - Ile - Phe - Tyr - Trp - Lys - Arg - Glu - Asp; Peptide sequence N: Gly - Asp - Glu - Lys - Pro - Arg - Ser - Thr - Ala - Val - Leu - Phe - Met - Gly - Asn - His; Peptide sequence O: Thr - Lys - Arg - Glu - Asp - Pro - Gly - Ser - Ile - Val - Leu - Phe - Tyr - Trp - His - Gln - Ala; Peptide sequence P: Pro - Arg - Lys - Glu - Asp - Gly - Ser - Thr - Val - Ile - Leu - Phe - Tyr - Trp - Asn - Glu - Ala - Ser; Peptide sequence Q: Ser - Ala - Gly - Pro - Thr - Lys - Arg - Glu - Asp - Val - Leu - Ile - Phe - Tyr - Trp - His - Gln - Asn - Lys; The small molecule peptide sequences are respectively derived from extracts of green tea, aloe vera, calendula, centella asiatica and rosemary.

2. The plant-derived small molecule peptide composition according to claim 1, wherein The content ratios of each peptide sequence are: peptide sequence M accounts for 15% - 35%, peptide sequence N accounts for 10% - 30%, peptide sequence O accounts for 15% - 35%, peptide sequence P accounts for 10% - 30%, and peptide sequence Q accounts for 10% - 30%.

3. A preparation method of a plant-derived small molecule peptide composition according to any one of claims 1 - 2, characterized in that, It includes the following steps: Respectively collect green tea, aloe vera leaves, calendula, centella asiatica and rosemary, and carry out pre - treatment of cleaning and cutting into pieces; Vacuum freeze - dry the pre - treated raw materials and then pulverize them through an 80 - 100 mesh sieve to obtain coarse powder; Respectively mix the green tea coarse powder with 50% - 70% ethanol solution, aloe vera gel coarse powder with deionized water, calendula coarse powder with 60% - 80% methanol solution, centella asiatica coarse powder with 70% - 90% acetone solution, and rosemary coarse powder with n - butanol - water according to a specific solid - liquid ratio, and extract at the corresponding temperature; Preliminarily separate the concentrated solutions of each extract through a macroporous adsorption resin column, and further separate and purify them by gel filtration chromatography and ion exchange chromatography; Mix the purified plant peptide components, and use the Edman degradation method or mass spectrometry sequencing method to determine peptide sequences M, N, O, P, Q.

4. The preparation method according to claim 3, characterized in that, The material-liquid ratio for extracting crude green tea powder is 1:8 - 1:12 (g / mL), the extraction temperature is 40°C - 60°C, and the extraction time is 2 - 4 hours; the material-liquid ratio for extracting crude aloe gel powder is 1:5 - 1:8 (g / mL), the extraction temperature is 30°C - 50°C, and the extraction time is 1 - 3 hours.

5. The preparation method according to claim 3, characterized in that, The material-liquid ratio for extracting crude calendula powder is 1:6 - 1:10 (g / mL), the extraction temperature is 35°C - 55°C, and the extraction time is 2 - 4 hours; the material-liquid ratio for extracting crude centella asiatica powder is 1:7 - 1:11 (g / mL), the extraction temperature is 45°C - 65°C, and the extraction time is 3 - 5 hours.

6. The preparation method according to claim 3, wherein The material-liquid ratio for extracting crude rosemary powder is 1:6 - 1:10 (g / mL), the extraction temperature is 30°C - 50°C, and the extraction time is 2 - 4 hours; when separating with a macroporous adsorption resin column, the resin is selected according to the polarity of peptides and gradient elution is adopted.

7. Use of the plant-derived small molecule peptide composition according to any one of claims 1 - 2 in the preparation of a mouthwash having antibacterial, anti-inflammatory, and repair effects.

8. A mouthwash, characterized in that, A mouthwash comprising the plant-derived small molecule peptide composition according to any one of claims 1 - 2, and a pharmaceutically acceptable carrier, excipient, or diluent, wherein the carrier, excipient, or diluent includes deionized water, glycerol, xylitol, sorbitol, menthol, and essence.

9. The mouthwash according to claim 8, characterized in that, The pH value of the mouthwash is 6.5 - 7.

5.

10. A method for preparing a mouthwash according to any one of claims 8 - 9, characterized in that, The small molecule peptide composition is dissolved in deionized water, moisturizers such as glycerol, xylitol, and sorbitol, and flavoring agents such as menthol and essence are added, and after adjusting the pH value to 6.5 - 7.5, the volume is made up, and it is prepared by sterilization treatment.