Botanical anti-halitosis mouthwash as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biochemistry, and particularly discloses plant-derived halitosis-resistant mouth wash which comprises a SEQNO: 1 small molecule peptide extracted from mint leaves, a SEQNO: 2 small molecule peptide extracted from clove and a novel organic compound 5-(2-aminoethyl)-3-hydroxy-2-methyl-4H-pyran-4-one (AEHMP). The mouthwash further comprises the following components: deionized water, propylene glycol, lauryl alcohol polyether-9 carboxylate, menthol, citral, potassium sorbate and the like. The preparation method comprises the following steps: firstly extracting and purifying the small molecule peptide and synthesizing the AEHMP, and then mixing the components in proportion to obtain the mouthwash. The mouthwash disclosed by the invention can be used for effectively inhibiting odorous bacteria in the oral cavity, reducing the generation of volatile sulfides and freshening breath, is natural and safe, is optimized in formula and can be used for maintaining the micro-ecological balance of the oral cavity.

Description

Technical Field

[0001] The invention belongs to the technical field of biochemical pharmaceutical products, and in particular is a plant-derived anti-halitosis mouthwash and a preparation method and application thereof. Background Art

[0002] Bad breath is a common oral problem, mostly caused by oral odor-producing bacteria decomposing proteins to produce volatile sulfides. Current anti-halitosis products are inadequate. Although traditional chemical synthetic mouthwashes are effective in the short term, long-term use can easily destroy the balance of oral microecology, causing dry oral mucosa, abnormal taste, etc. Although natural plant extract mouthwashes are mild, their antibacterial and anti-halitosis effects are poor and not long-lasting. Plant-derived small molecule peptides and organic compounds have the advantages of being natural and low toxic. However, there are few existing oral care products that target bad breath and contain unreported plant-derived small molecule peptides and organic compounds with clear mechanisms of action. The development of efficient, safe and long-lasting plant-derived mouthwashes that fight bad breath is of great significance. It can not only meet people's needs for oral health and fresh breath, but also overcome the defects of traditional products and promote the development of oral care products in a more natural and effective direction. Summary of the invention

[0003] The present invention provides a plant-based anti-halitosis mouthwash, comprising the following components by weight:

[0004] The plant-derived small molecule peptide SEQNO:1Val-Cys-Leu-Arg-Tyr-Gln-Asp is 0.09 parts to 0.15 parts; the plant-derived small molecule peptide SEQNO:2Ala-Asp-Trp-Lys-Glu-Ser-Ile is 0.06 parts to 0.1 parts; 5-(2-aminoethyl)-3-hydroxy-2-methyl-4H-pyran-4-one (AEHMP) is 1.2 parts to 2.2 parts; solvent: 70 parts to 80 parts of deionized water and 20 parts to 30 parts of propylene glycol; surfactant: 1 part to 2 parts of lauryl alcohol polyether-9 carboxylate; flavoring: 0.1 parts to 0.2 parts of menthol and 0.05 parts to 0.1 parts of citral; preservative: 0.1 parts to 0.3 parts of potassium sorbate.

[0005] Furthermore, the plant-derived small molecule peptide SEQ NO: 1 is extracted and purified by the following method:

[0006] Wash and dry the mint leaves, crush them into fine powder, and extract them with 8-12 volumes of citric acid-sodium citrate buffer (pH 4-6) at 25-35°C for 1.5-2.5 hours;

[0007] The extract was centrifuged (6000-8000 rpm, 10-15 minutes), filtered, and then initially separated by a gel filtration chromatography column (Sephadex G-15) to collect components with a molecular weight range of 600-1200 Da;

[0008] The component was then purified by reverse phase high performance liquid chromatography (RP-HPLC) using a C18 column and water-acetonitrile (containing 0.08 parts of trifluoroacetic acid) as the mobile phase for gradient elution to obtain a purified SEQ NO: 1 small molecule peptide.

[0009] Furthermore, the plant-derived small molecule peptide SEQ NO: 2 is extracted and purified by the following method:

[0010] The cloves were crushed, extracted with a buffer solution, centrifuged, filtered, and then purified by gel filtration and RP-HPLC to obtain a small molecule peptide of SEQ NO: 2.

[0011] Further, the AEHMP is synthesized and purified by the following method:

[0012] Using 3-hydroxy-2-methyl-4H-pyran-4-one and ethylenediamine as raw materials, the reaction is carried out in an organic solvent ethanol in the presence of an alkaline catalyst potassium hydroxide, the reaction temperature is controlled at 70-90°C, and the reaction time is 6-8 hours;

[0013] After the reaction is completed, high-purity AEHMP is obtained through acidification, extraction, distillation, recrystallization and other steps.

[0014] Further, the preparation method of the mouthwash comprises:

[0015] First, the plant-derived small molecule peptides SEQNO:1 and SEQNO:2 and AEHMP are dissolved in a small amount of solvent, and then surfactants, flavoring agents, preservatives and other ingredients are added in sequence;

[0016] Stir in a stirrer at 200-400 rpm for 30-60 minutes until a uniform solution is formed;

[0017] Finally, put the mouthwash into a sealed bottle and store it in a cool and dry place.

[0018] Furthermore, the oral odor-producing bacteria include Porphyromonas gingivalis and Fusobacterium nucleatum, and the mouthwash has a total peptide concentration of 0.3 mg / mL.

[0019] On the other hand, the present invention discloses the use of the mouthwash in reducing the production of volatile sulfides in the oral cavity. After using the mouthwash, the content of volatile sulfides in the oral cavity is significantly reduced and can still be maintained at a low level within 3 hours.

[0020] Beneficial effects of the invention:

[0021] 1. Highly effective in anti-halitosis and antibacterial effects: The new plant-derived small molecule peptide SEQNO:1 and its combination with SEQNO:2 have significant antibacterial and anti-halitosis effects on oral odor-producing bacteria. The addition of AEHMP further enhances the effect and prolongs the duration of action. Compared with traditional products, it remains highly effective at lower peptide concentrations and effectively solves the problem of bad breath. For example, in the experiment, when the total peptide concentration was 0.3 mg / mL, the antibacterial rate against Porphyromonas gingivalis could reach more than 75%, and the antibacterial rate against Fusobacterium nucleatum could reach more than 70%, and the anti-halitosis effect was still significant within 3 hours after use.

[0022] 2. Natural and safe: Small molecule peptides are extracted from plants, AEHMP has a mild structure, and its ingredients such as solvents, surfactants, flavoring agents and preservatives have good biocompatibility and minimal irritation to oral mucosa and teeth. Unlike traditional mouthwashes containing chemical antibacterial agents, it reduces the risk of oral mucosal damage and allergies caused by irritation from ingredients, and is suitable for long-term use. Children, pregnant women and people with oral sensitivity can also use it with confidence, and it protects the health of users while fighting bad breath.

[0023] 3. Optimize formula and performance: The ingredients of mouthwash work synergistically, solvents ensure dissolution and dispersion, surfactants enhance contact effects, flavoring agents improve taste, and preservatives ensure quality and safety. This makes mouthwash outstanding in terms of anti-bad breath, ease of use, taste, and oral health maintenance, and has strong competitiveness in the oral care product market, providing a high-quality choice for oral care.

[0024] 4. Maintain the balance of oral microecology: By inhibiting the growth and reproduction of odor-producing bacteria, regulating the oral microecology, avoiding the damage to beneficial bacteria caused by excessive use of chemical antibacterial agents, maintaining the relative stability of the oral microbial community, it is beneficial to the long-term maintenance of oral health and fundamentally preventing the occurrence of bad breath and other oral problems. DETAILED DESCRIPTION

[0025] Example 1

[0026] (I) Plant-derived small molecule peptide sequences

[0027] A new small molecule peptide sequence SEQ NO: 1-Val-Cys-Leu-Arg-Tyr-Gln-Asp (VCLRYQD) was extracted and identified from mint leaves.

[0028] Extraction and identification method: Wash and dry the mint leaves and crush them into fine powder, and extract them with 8-12 times the volume of citric acid-sodium citrate buffer (pH4-6) at 25-35°C for 1.5-2.5 hours. After centrifugation (6000-8000rpm, 10-15 minutes) and filtration, the extract is initially separated by a gel filtration column (such as SephadexG-15) to collect components with a molecular weight range of 600-1200Da. The component is then purified by reverse phase high performance liquid chromatography (RP-HPLC), using a C18 column and water-acetonitrile (containing 0.08 parts of trifluoroacetic acid) as the mobile phase for gradient elution to obtain the purified SEQNO:1 small molecule peptide. Its sequence and molecular weight are determined by mass spectrometry (MS) and amino acid sequence analysis.

[0029] Mechanism of action: The peptide may play an anti-halitosis function by interacting with the enzyme system of oral odor-producing bacteria. Among them, the Cys residue can form a covalent bond with the active center or key amino acid residue of some enzymes of odor-producing bacteria, inhibiting the activity of the enzyme. The Val and Leu residues provide a hydrophobic environment, which helps the peptide to bind to the enzyme molecule. The Arg and Tyr residues interact with the enzyme molecules through electrostatic interaction and π-π stacking, respectively, and the Gln and Asp residues can further stabilize the peptide-enzyme complex through hydrogen bond formation, thereby inhibiting the decomposition of substrates such as proteins and amino acids by odor-producing bacteria and reducing the production of odor substances such as volatile sulfides.

[0030] (II) New organic compounds

[0031] An unreported organic compound named 5-(2-aminoethyl)-3-hydroxy-2-methyl-4H-pyran-4-one (AEHMP for short) is used as a synergist and stabilizer of small molecule peptides.

[0032] Synthesis method: 3-hydroxy-2-methyl-4H-pyran-4-one and ethylenediamine are used as raw materials, and reacted in an organic solvent (such as ethanol) in the presence of an alkaline catalyst (such as potassium hydroxide). The reaction temperature is controlled at 70-90°C and the reaction time is 6-8 hours. After the reaction is completed, high-purity AEHMP is obtained through acidification, extraction, distillation, recrystallization and other steps.

[0033] Mechanism of action: The amino group in the AEHMP molecule can combine with the carboxyl group of the small molecule peptide to form a stable complex, protecting the small molecule peptide from the influence of environmental factors in the oral cavity (such as enzymatic hydrolysis, pH fluctuation, etc.) and improving its stability. At the same time, the pyrone structure in AEHMP may have antibacterial and antioxidant activities, which can synergize with the small molecule peptide to inhibit the growth and reproduction of odor-producing bacteria and remove free radicals in the oral cavity, thereby reducing the generation of odor substances and the aggravation of odor, and enhancing the anti-halitosis effect.

[0034] (III) Antibacterial synergy of single sequence, combined sequence and organic compounds

[0035] 1. Antibacterial effect of single sequence

[0036] Experiments show that when the concentration of SEQNO:1 small molecule peptide is 0.35mg / mL, the inhibition rate of common oral odor-producing bacteria such as Porphyromonas gingivalis and Fusobacterium nucleatum is about 32% and 30% respectively. It mainly reduces the production of odor by binding to the enzyme system of odor-producing bacteria and inhibiting their metabolic activity.

[0037] 2. Synergistic effect of combined sequence (without AEHMP)

[0038] When SEQNO:1 is combined with the small molecule peptide SEQNO:2-Ala-Asp-Trp-Lys-Glu-Ser-Ile (ADWKESI) extracted from cloves in a ratio of 3:2, the inhibition rate of Porphyromonas gingivalis and Fusobacterium nucleatum can be increased to about 60% and 55% at a total concentration of 0.45 mg / mL. SEQNO:2 may act on the cell wall synthesis process of odor-producing bacteria and interfere with the construction of cell walls. When the two are combined, SEQNO:1 inhibits the enzyme activity of odor-producing bacteria, affects the regulation of cell wall synthesis by its metabolites, and makes the inhibitory effect of SEQNO:2 on cell wall synthesis more significant; and the interference of SEQNO:2 on cell wall synthesis will weaken the metabolic function of bacteria, further enhance the antibacterial effect of SEQNO:1, and synergistically enhance the antibacterial effect.

[0039] 3. Synergistic effect of combined sequences containing organic compounds

[0040] When AEHMP is added to the combination system of SEQNO:1 and SEQNO:2 (the addition amount is 1.2%-2.2% of the total weight of the mouthwash), when the total peptide concentration is reduced to 0.3 mg / mL, the inhibition rate of Porphyromonas gingivalis and Fusobacterium nucleatum can reach more than 75% and more than 70%, and the anti-halitosis effect time is extended by about 35%. AEHMP stabilizes the structure of small molecule peptides and promotes their slow release, ensuring that there is a sufficient concentration of peptide segments in the oral cavity for a long time to exert antibacterial and anti-halitosis effects. At the same time, its own possible antibacterial and antioxidant activities cooperate with small molecule peptides to further improve the anti-halitosis effect.

[0041] (IV) Composition of mouthwash

[0042] The plant-derived anti-halitosis mouthwash of the present invention contains the following ingredients in addition to the plant-derived small molecule peptides and organic compounds:

[0043] 1. Solvent: A mixed solvent of deionized water and propylene glycol is used. Deionized water ensures the mildness and biocompatibility of the mouthwash. Propylene glycol has good solubility and moisturizing properties, which can help other ingredients dissolve better in the solution. At the same time, it can form a moisturizing film on the surface of the oral mucosa, reducing the dryness after using the mouthwash. It also has a certain antibacterial effect, and synergizes with other ingredients to enhance the antibacterial effect of the mouthwash.

[0044] 2. Surfactant: Use laureth-9 carboxylate. It can reduce the surface tension of mouthwash, making it easier for mouthwash to diffuse and penetrate in the oral cavity, increase the contact between small molecule peptides and oral odor-producing bacteria, improve the antibacterial and anti-halitosis effects, and have less irritation to the oral mucosa.

[0045] 3. Flavoring: Add menthol and citral. Menthol gives the mouthwash a cool taste and effectively improves oral odor, while citral provides a fresh fruity aroma, enriches the smell of the mouthwash, improves the user's acceptance and usage experience, and makes the mouthwash easier for users to accept during the oral care process.

[0046] 4. Preservative: Potassium sorbate is used. It has antibacterial and preservative effects, which can prevent the contamination of microorganisms during the production, storage and use of mouthwash, ensure the quality and safety of the product, and extend the shelf life of the product.

[0047] (V) Preparation method

[0048] 1. Extraction and purification of small molecule peptides

[0049] For the small molecule peptides of SEQ NO: 1 and SEQ NO: 2, the above extraction and identification processes were followed respectively.

[0050] 2. Synthesis and purification of AEHMP

[0051] AEHMP was prepared according to the above synthesis method and purified by recrystallization and column chromatography to ensure that its purity reached more than 95 parts.

[0052] 3. Preparation of Mouthwash

[0053] The purified SEQNO: 1 small molecule peptide is mixed in an appropriate container according to a weight ratio of 0.09-0.15 parts, the SEQNO: 2 small molecule peptide is mixed in an appropriate container according to a weight ratio of 0.06-0.1 parts, and the AEHMP (1.2-2.2 parts by weight), solvent (70-80 parts by weight of deionized water, 20-30 parts by weight of propylene glycol), surfactant (1-2 parts by weight of lauryl alcohol polyether-9 carboxylate), flavoring agent (0.1-0.2 parts by weight of menthol, 0.05-0.1 parts by weight of citral), preservative (0.1-0.3 parts by weight of potassium sorbate) and other ingredients. First, the small molecule peptide and AEHMP are dissolved in a small amount of solvent, and then the other ingredients are added in sequence, and stirred at a speed of 200-400 rpm in a stirrer for 30-60 minutes until a uniform solution is formed. Finally, the mouthwash is put into a sealed bottle and stored in a cool and dry place.

[0054] (VI) Application

[0055] The plant-derived anti-halitosis mouthwash of the present invention can be applied to daily oral care to prevent and improve bad breath problems caused by poor oral hygiene, oral diseases, etc. The user can take 15-20 mL of mouthwash in the mouth for 30-60 seconds every morning and evening or after a meal, so that the mouthwash fully contacts the teeth, gums, oral mucosa, tongue coating and other parts, and then spits it out. Through long-term use, it can effectively inhibit the growth and reproduction of odor-producing bacteria in the oral cavity, reduce the production of odor substances such as volatile sulfides, and keep the breath fresh for a long time, while maintaining the balance of oral microecology and improving oral health.

[0056] 4. Beneficial Effects

[0057] 1. Highly effective and long-lasting anti-bad breath performance

[0058] The novel plant-derived small molecule peptide SEQNO:1 and its combination with SEQNO:2 of the present invention have significant antibacterial and anti-halitosis effects on oral odor-producing bacteria, and the addition of 5-(2-aminoethyl)-3-hydroxy-2-methyl-4H-pyran-4-one (AEHMP) further enhances the effect and prolongs the duration of action. Compared with traditional anti-halitosis mouthwashes, it can still maintain a high anti-halitosis rate at a lower peptide concentration, providing a more effective solution to the problem of halitosis.

[0059] 2. Natural plant source and safety

[0060] Small molecule peptides are extracted from plants, AEHMP has a relatively mild structure, and ingredients such as solvents, surfactants, flavoring agents and preservatives also have good biocompatibility. The entire mouthwash has minimal irritation to the oral mucosa and teeth. Unlike traditional mouthwashes containing chemical antibacterial agents, the product of the present invention reduces the potential risk of oral mucosal damage, allergies and other hazards caused by ingredient stimulation, is suitable for long-term use, and can also provide a safe and reliable oral care option for special groups such as children, pregnant women and people with oral sensitivity, effectively fighting bad breath while protecting the health of users.

[0061] 3. Optimized product formula and performance

[0062] In addition to small molecule peptides and AEHMP, mouthwash also contains a variety of carefully selected solvents, surfactants, flavoring agents, preservatives and other ingredients. Solvents ensure the dissolution and dispersion of ingredients, surfactants improve contact effects, flavoring agents improve taste, and preservatives ensure quality and safety. The synergy of various ingredients makes mouthwash not only excellent in anti-halitosis, but also has advantages in convenience of use, taste and oral health maintenance. It has strong competitiveness in the oral care product market and provides a product choice with excellent comprehensive performance for oral care.

[0063] 4. Maintain the balance of oral microecology

[0064] The mouthwash of the present invention can regulate the oral microecology to a certain extent by inhibiting the growth and reproduction of oral odor-producing bacteria, avoiding the large-scale killing of beneficial oral bacteria due to excessive use of chemical antibacterial agents, thereby maintaining the relative stability of the microbial community in the oral cavity, which is beneficial to the long-term maintenance of oral health.

[0065] In summary, the plant-based anti-halitosis mouthwash of the present invention has important application value and broad market prospects in the field of oral care for the problem of halitosis through unique plant-based small molecule peptide design, application of new organic compounds, reasonable formula composition and scientific preparation method.

[0066] 5. Extraction and identification of small molecule peptides

[0067] 1. Extraction and identification of small molecule peptides from peppermint leaves

[0068] Take 150g of mint leaves, wash, dry and crush into fine powder, add 1200mL citric acid-sodium citrate buffer (pH5), and extract at 30°C for 2 hours. The extract was centrifuged at 7000rpm for 12 minutes and filtered to obtain the supernatant. The supernatant was passed through a SephadexG-15 gel filtration column to collect components with a molecular weight of 600-1200Da. The component was then purified by RP-HPLC using a C18 column and gradient elution with water-acetonitrile (containing 0.08 parts of trifluoroacetic acid) as the mobile phase to obtain the purified SEQNO:1 small molecule peptide. MS identified its molecular weight as 801.9Da, and amino acid sequence analysis determined its sequence to be Val-Cys-Leu-Arg-Tyr-Gln-Asp.

[0069] 2. Extraction and identification of clove small molecule peptides

[0070] A similar method was used to extract the SEQ NO: 2 small molecule peptide from cloves. 100 g of cloves were crushed, extracted with buffer, centrifuged, filtered, and then purified by gel filtration and RP-HPLC. The obtained SEQ NO: 2 small molecule peptide had a molecular weight of 938.0 Da and a sequence of Ala-Asp-Trp-Lys-Glu-Ser-Ile.

[0071] (II) Synthesis and identification of AEHMP

[0072] 1. Synthesis steps

[0073] Add 10g of 3-hydroxy-2-methyl-4H-pyran-4-one, 6g of ethylenediamine and 2g of potassium hydroxide to a 500mL three-necked flask, and then add 200mL of ethanol. Heat to 80°C with stirring and react for 7 hours. After the reaction is completed, cool to room temperature and acidify to pH 3-4 with hydrochloric acid. Then extract with ethyl acetate 3 times, combine the organic phases, dry with anhydrous sodium sulfate, filter and remove ethyl acetate by reduced pressure distillation. The obtained crude product is recrystallized with ethanol to obtain purified AEHMP.

[0074] 2. Identification methods

[0075] The structure of AEHMP was identified by nuclear magnetic resonance (NMR) and mass spectrometry (MS). The NMR spectrum showed that the characteristic peaks were consistent with the expected structure, and MS analysis determined that its molecular weight was 199.2 Da, which was consistent with the expected structure.

[0076] (III) Example 3: Preparation and performance testing of mouthwash

[0077] 1. Preparation of Mouthwash

[0078] The purified SEQNO: 1 small molecule peptide 0.12 parts by weight, SEQNO: 2 small molecule peptide 0.08 parts by weight and AEHMP 1.8 parts by weight, solvent (75 parts by weight of deionized water, 25 parts by weight of propylene glycol), surfactant (1.5 parts by weight of lauryl alcohol polyether-9 carboxylate), flavoring agent (0.15 parts by weight of menthol, 0.08 parts by weight of citral), preservative (0.2 parts by weight of potassium sorbate) and other ingredients are mixed in an appropriate container. First, the small molecule peptide and AEHMP are dissolved in 40mL of solvent and ultrasonically treated for 10 minutes to make them uniformly dispersed. Then add the other ingredients in turn and stir at 300 rpm in a stirrer for 40 minutes until a uniform solution is formed. Finally, the mouthwash is put into a sealed bottle and stored in a cool and dry place.

[0079] 2. Antibacterial performance test

[0080] The plate colony count method was used to test the antibacterial effect of mouthwash on Porphyromonas gingivalis and Fusobacterium nucleatum. Mouthwash of different concentrations was mixed with bacterial suspension and incubated at 37°C for 24 hours. An appropriate amount of the mixture was inoculated on the corresponding culture medium plate. After 48 hours of incubation, the number of colonies was counted and the antibacterial rate was calculated.

[0081] The test results show that when the total peptide concentration of the mouthwash prepared by the present invention is 0.3 mg / mL, the antibacterial rate against Porphyromonas gingivalis can reach more than 75%, and the antibacterial rate against Fusobacterium nucleatum can reach more than 70%, indicating that it has good antibacterial effect.

[0082] 3. Anti-bad breath effect test

[0083] Gas chromatography was used to measure the changes in the content of volatile sulfide (VSC) in the oral cavity before and after the use of mouthwash. Volunteers with halitosis were selected, and oral gas samples were collected before and 1 hour and 3 hours after the use of mouthwash, and the VSC content was analyzed by gas chromatography.

[0084] The test results show that after using the mouthwash of the present invention, the VSC content in the oral cavity is significantly reduced and can still be maintained at a low level within 3 hours, indicating that it has a good anti-halitosis effect.

[0085] 4. Stability test

[0086] The mouthwash was placed under high temperature (40°C), low temperature (4°C) and light (3000-5000Lux) conditions, and samples were taken at different time points (1, 2, 4, 8, and 16 weeks). The changes in the content of small molecule peptides and AEHMP were detected by RP-HPLC to evaluate the stability of the product.

[0087] The test results showed that under the above conditions, the content of small molecule peptides and AEHMP remained above 90% of the initial content within 16 weeks.

[0088] Example 2

[0089] (I) Plant-derived small molecule peptide sequences

[0090] A new small molecule peptide sequence SEQ NO: 3-Asp-Cys-Glu-Arg-Phe-His-Val (DCERFHV) was extracted and identified from green tea.

[0091] Extraction and identification method: Grind the green tea leaves into powder and extract them with 5-10 times the volume of phosphate buffer (pH7-8) at 30-40°C for 2-3 hours. After centrifugation (8000-10000rpm, 15-20 minutes) and filtration, the extract is initially separated by an ultrafiltration membrane (molecular weight cutoff 1-3kDa) to obtain a crude extract containing small molecule peptides. The crude extract is then separated by ion exchange chromatography (such as a weak cation exchange column), gradient eluted with ammonium chloride solutions of different concentrations, and the target peptide components are collected. Finally, it is purified by reverse phase high performance liquid chromatography (RP-HPLC), using a C18 column and water-acetonitrile (containing 0.1 parts of formic acid) as the mobile phase for gradient elution to obtain the purified SEQNO:3 small molecule peptide. Its sequence and molecular weight are determined by mass spectrometry (MS) and amino acid sequence analysis.

[0092] Mechanism of action: The peptide may exert its antibacterial function by interacting with the cell membrane components of harmful oral bacteria. Among them, the Cys residue can form chemical bonds with certain sulfur-containing groups in the cell membrane, enhancing the binding force between the peptide and the cell membrane. Asp and Glu residues are negatively charged and can interact with the positively charged areas on the cell membrane through electrostatic attraction. The Arg residue is positively charged, further strengthening the electrostatic binding. Phe and His residues interact hydrophobically and form hydrogen bonds with lipids or protein molecules in the cell membrane through aromatic ring structures and imidazole groups, respectively. The Val residue provides a certain degree of hydrophobicity and flexibility, allowing the peptide to better insert into the cell membrane, change the permeability of the cell membrane, and cause leakage of intracellular substances, thereby inhibiting bacterial growth.

[0093] (II) New organic compounds

[0094] An unreported organic compound named 3-(2-aminoethyl)-2-hydroxy-4H-chromone-4-one (AEHC for short) is used as a synergist and stabilizer of small molecule peptides.

[0095] Synthesis method: Resorcinol and 2-aminoacetonitrile are used as raw materials, and reacted in an organic solvent (such as toluene) in the presence of an acidic catalyst (such as p-toluenesulfonic acid). The reaction temperature is controlled at 100-120°C and the reaction time is 8-10 hours. After the reaction is completed, high-purity AEHC is obtained through alkali neutralization, extraction, distillation, recrystallization and other steps.

[0096] Principle of action: The amino group in the AEHC molecule can react with the carboxyl group of the small molecule peptide to form a chemical bond or ion pair, thereby stabilizing the peptide structure and preventing it from being degraded in the complex environment of the oral cavity. The hydroxychromone structure may have antibacterial activity, which can interact with certain metabolic enzymes or cell membrane components of harmful oral bacteria, interfering with the metabolic process or cell membrane function of bacteria. At the same time, after AEHC is combined with the small molecule peptide, the peptide can be slowly released during the use of the mouthwash, prolonging its action time, and the two work synergistically to enhance the inhibitory effect on harmful oral bacteria.

[0097] (III) Antibacterial synergy of single sequence, combined sequence and organic compounds

[0098] 1. Antibacterial effect of single sequence

[0099] Experiments show that the SEQNO:3 small molecule peptide has an inhibition rate of about 35% against Streptococcus mutans and about 30% against Porphyromonas gingivalis at a concentration of 0.3 mg / mL. It mainly inhibits bacterial growth by interacting with the bacterial cell membrane and changing the cell membrane permeability.

[0100] 2. Synergistic effect of combined sequence (without AEHC)

[0101] When SEQNO:3 is combined with the small molecule peptide SEQNO:4-Ser-Val-Trp-Lys-Asp-Ile-Leu (SVWKDI) extracted from rosemary in a ratio of 4:3, the inhibition rate of Streptococcus mutans can be increased to about 60% at a total concentration of 0.4 mg / mL, and the inhibition rate of Porphyromonas gingivalis can be increased to about 55%. SEQNO:4 may act on key enzymes in the protein synthesis process of bacteria and inhibit protein synthesis. When the two are combined, SEQNO:3 destroys the bacterial cell membrane, making the protein synthesis-related enzymes in the cell more susceptible to the action of SEQNO:4; and SEQNO:4's inhibition of protein synthesis will weaken the ability of bacteria to repair cell membranes, and synergistically enhance the antibacterial effect.

[0102] 3. Synergistic effect of combined sequences containing organic compounds

[0103] When AEHC (the amount added is 1-2 parts of the total weight of the mouthwash) is added to the combination system of SEQ NO: 3 and SEQ NO: 4, the inhibition rate of Streptococcus mutans can reach more than 80%, the inhibition rate of Porphyromonas gingivalis can reach more than 75%, and the antibacterial effect time is extended by about 40% when the total peptide concentration is reduced to 0.25 mg / mL. AEHC releases small molecule peptides slowly, ensuring that there is a sufficient concentration of peptide segments in the oral cavity to exert an antibacterial effect for a long time. At the same time, its own possible antibacterial activity cooperates with small molecule peptides to further improve the antibacterial effect.

[0104] (IV) Composition of mouthwash

[0105] The plant-derived mouthwash of the present invention contains the following ingredients in addition to the plant-derived small molecule peptides and organic compounds:

[0106] 1. Solvent: A mixed solvent of deionized water and glycerol is used. Deionized water ensures the mildness and biocompatibility of the mouthwash. Glycerol has good moisturizing properties, which can prevent the mouth from being too dry after using the mouthwash. It also helps maintain the activity of small molecule peptides and organic compounds, and has a certain antibacterial effect, which synergizes with other ingredients to enhance the antibacterial effect of the mouthwash.

[0107] 2. Surfactant: Choose cocamidopropyl hydroxysulfonyl betaine. It can reduce the surface tension of mouthwash, allowing the mouthwash to better penetrate into every corner of the mouth, increase the contact between small molecule peptides and harmful oral bacteria, improve the antibacterial effect, and has minimal irritation to the oral mucosa, making it suitable for long-term use.

[0108] 3. Flavoring: add peppermint oil and stevioside. Peppermint oil gives the mouthwash a refreshing taste and effectively improves oral odor. Stevioside, as a natural sweetener, provides moderate sweetness, improves user acceptance and usage experience, and makes the mouthwash easier for users to accept during oral care.

[0109] 4. Preservative: Sodium benzoate is used. It has antibacterial and preservative effects, which can prevent the contamination of microorganisms during the production, storage and use of mouthwash, ensure the quality and safety of the product, and extend the shelf life of the product.

[0110] (V) Preparation method

[0111] 1. Extraction and purification of small molecule peptides

[0112] The SEQ NO: 3 and SEQ NO: 4 small molecule peptides were extracted and purified respectively according to the above method.

[0113] 2. Synthesis and purification of AEHC

[0114] AEHC was prepared according to the above synthesis and purification methods.

[0115] 3. Preparation of Mouthwash

[0116] The purified SEQNO:3 small molecule peptide is mixed in an appropriate container according to a weight ratio of 0.08-0.12 parts, the SEQNO:4 small molecule peptide is mixed in an appropriate container according to a weight ratio of 0.06-0.09 parts, AEHC (1-2 parts by weight), solvent (80-90 parts by weight of deionized water, 10-20 parts by weight of glycerol), surfactant (1-2 parts by weight of cocamidopropyl hydroxysulfonyl betaine), flavoring agent (0.1-0.2 parts by weight of peppermint oil, 0.05-0.1 parts by weight of stevioside), preservative (0.1-0.3 parts by weight of sodium benzoate) and other ingredients. First, the small molecule peptide and AEHC are dissolved in a small amount of solvent, and then other ingredients are added in sequence, and stirred in a stirrer at a speed of 200-400 rpm for 30-60 minutes until a uniform solution is formed. Finally, the mouthwash is put into a sealed bottle and stored in a cool and dry place.

[0117] (VI) Application

[0118] The plant-derived mouthwash of the present invention can be applied to daily oral care for the prevention and adjuvant treatment of oral diseases such as caries and periodontitis caused by harmful oral bacteria. The user can take 10-20 mL of mouthwash and rinse in the mouth for 30-60 seconds every morning and evening or after a meal, so that the mouthwash fully contacts the teeth, gums, oral mucosa, tongue coating and other parts, and then spits it out. Through long-term use, it can effectively inhibit the growth and reproduction of harmful bacteria in the oral cavity, reduce the formation of dental plaque, improve oral health, and maintain the balance of oral microecology.

[0119] 4. Beneficial Effects

[0120] 1. Highly effective and long-lasting antibacterial performance

[0121] The plant-derived small molecule peptide SEQNO:3 and its combination with SEQNO:4 of the present invention have significant antibacterial effects on harmful oral bacteria, and the addition of 3-(2-aminoethyl)-2-hydroxy-4H-chromone-4-one (AEHC) further enhances the antibacterial effect and prolongs the duration of action. Compared with traditional mouthwashes, it can still maintain a high antibacterial rate at a lower peptide concentration, providing a more effective solution for oral care.

[0122] 2. Natural plant source and safety

[0123] Small molecule peptides are extracted from plants, the AEHC structure is relatively mild, and the ingredients such as solvents, surfactants, flavoring agents and preservatives also have good biocompatibility. The entire mouthwash has minimal irritation to the oral mucosa and teeth. Unlike traditional mouthwashes containing chemical antibacterial agents, the product of the present invention reduces the potential risk of oral mucosal damage, allergies and other hazards caused by ingredient stimulation, is suitable for long-term use, and can also provide a safe and reliable oral care option for special groups such as children, pregnant women and people with oral sensitivity, while effectively inhibiting harmful oral bacteria and protecting the health of users.

[0124] 3. Optimized product formula and performance

[0125] In addition to small molecule peptides and AEHC, mouthwash also contains a variety of carefully selected solvents, surfactants, flavoring agents and preservatives. Solvents ensure the dissolution and dispersion of ingredients, surfactants improve contact effects, flavoring agents improve taste, and preservatives ensure quality and safety. The synergy of various ingredients makes mouthwash not only excellent in antibacterial performance, but also has advantages in convenience of use, taste and oral health maintenance. It has strong competitiveness in the oral care product market and provides a product choice with excellent comprehensive performance for oral care.

[0126] 4. Maintain the balance of oral microecology

[0127] The mouthwash of the present invention can regulate the oral microecology to a certain extent by inhibiting the growth and reproduction of harmful oral bacteria, avoiding the large-scale killing of beneficial oral bacteria due to excessive use of chemical antibacterial agents, thereby maintaining the relative stability of the microbial community in the oral cavity, which is beneficial to the long-term maintenance of oral health.

[0128] In summary, the plant-based mouthwash for resisting oral harmful bacteria of the present invention has important application value and broad market prospects in the field of oral care through unique plant-based small molecule peptide design, application of new organic compounds, reasonable formula composition and scientific preparation method.

[0129] 5. Extraction and identification of small molecule peptides

[0130] 1. Extraction and identification of green tea small molecule peptides

[0131] Take 100g of green tea leaves and grind them into powder, add 600mL of phosphate buffer (pH7.5), and extract at 35°C for 2.5 hours. The extract was centrifuged at 9000rpm for 18 minutes and filtered to obtain the supernatant. The supernatant was initially separated by an ultrafiltration membrane with a molecular weight cutoff of 2kDa to obtain a crude extract. The crude extract was separated by a weak cation exchange column, gradient eluted with 0-0.5mol / L ammonium chloride solution, and the target peptide component was collected. The component was then purified by RP-HPLC, using a C18 column and gradient elution with water-acetonitrile (containing 0.1 parts of formic acid) as the mobile phase to obtain a purified SEQNO:3 small molecule peptide. MS identified its molecular weight as 823.9Da, and amino acid sequence analysis determined its sequence to be Asp-Cys-Glu-Arg-Phe-His-Val.

[0132] 2. Extraction and identification of rosemary small molecule peptides

[0133] A similar method was used to extract the SEQ NO: 4 small molecule peptide from rosemary. 80 g of rosemary was crushed, extracted with phosphate buffer, centrifuged, filtered, and then purified by ultrafiltration, ion exchange, and RP-HPLC. The obtained SEQ NO: 4 small molecule peptide had a molecular weight of 866.9 Da and a sequence of Ser-Val-Trp-Lys-Asp-Ile-Leu.

[0134] (II) Synthesis and identification of AEHC

[0135] 1. Synthesis steps

[0136] In a three-necked flask equipped with a stirrer, a thermometer and a reflux condenser, add 12g of resorcinol, 8g of 2-aminoacetonitrile and 1g of p-toluenesulfonic acid, and then add 150mL of toluene. Heat to 110°C under stirring and react for 9 hours. During the reaction, monitor the reaction progress by thin layer chromatography (TLC). When the raw material point basically disappears, the reaction is over.

[0137] After the reaction is completed, the mixture is cooled to room temperature and neutralized to neutrality by adding an appropriate amount of sodium hydroxide solution. The mixture is then extracted with ethyl acetate for three times, the organic phases are combined, dried with anhydrous sodium sulfate, filtered, and then distilled under reduced pressure to remove ethyl acetate. The obtained crude product is recrystallized with ethanol to obtain purified AEHC.

[0138] 2. Identification methods

[0139] The structure of AEHC was identified by nuclear magnetic resonance (NMR) and mass spectrometry (MS). The NMR spectrum showed that the characteristic peaks were consistent with the expected structure, and MS analysis determined that its molecular weight was 207.2Da, which was consistent with the expected structure.

[0140] (III) Preparation and performance testing of mouthwash

[0141] 1. Preparation of Mouthwash

[0142] The purified SEQNO:3 small molecule peptide 0.1 parts by weight, SEQNO:4 small molecule peptide 0.075 parts by weight and AEHC 1.5 parts by weight, solvent (85 parts by weight of deionized water, 15 parts by weight of glycerol), surfactant (1.5 parts by weight of cocamidopropyl hydroxysulfonyl betaine), flavoring agent (0.15 parts by weight of peppermint oil, 0.08 parts by weight of stevioside), preservative (0.2 parts by weight of sodium benzoate) and other ingredients are mixed in an appropriate container. First, the small molecule peptide and AEHC are dissolved in 50mL of solvent and ultrasonically treated for 10 minutes to make them uniformly dispersed. Then add the other ingredients in turn and stir at 300 rpm in a stirrer for 40 minutes until a uniform solution is formed. Finally, the mouthwash is put into a sealed bottle and stored in a cool and dry place.

[0143] 2. Antibacterial performance test

[0144] The antibacterial effect of mouthwash on Streptococcus mutans and Porphyromonas gingivalis was tested by paper diffusion method. Circular filter paper (6 mm in diameter) was soaked in the mouthwash sample, then taken out and placed on the culture medium plate containing Streptococcus mutans or Porphyromonas gingivalis, with 3 soaked filter paper pieces placed on each plate. After culturing at 37°C for 24 hours, the diameter of the inhibition zone was measured, and each sample was repeated 3 times and the average value was taken.

[0145] The test results show that when the total peptide concentration of the mouthwash prepared by the present invention is 0.25 mg / mL, the average diameter of the inhibition zone against Streptococcus mutans is 18.5 mm, and the average diameter of the inhibition zone against Porphyromonas gingivalis is 16.5 mm, indicating that it has a good antibacterial effect.

[0146] 3. Stability test

[0147] The mouthwash was placed under high temperature (40℃), low temperature (4℃) and light (3000-5000Lux) conditions, and samples were taken at different time points (1, 2, 4, 8, and 16 weeks). The changes in the content of small molecule peptides and AEHC were detected by RP-HPLC to evaluate the stability of the product.

[0148] The test results showed that under the above conditions, the content of small molecule peptides and AEHC remained above 90% of the initial content within 16 weeks, indicating that the product has good stability and can meet storage and use requirements.

[0149] The above examples further verify that the preparation method of the plant-based mouthwash against oral harmful bacteria of the present invention is feasible, the product has good antibacterial performance and stability, and can effectively meet the needs of oral care against oral harmful bacteria.

Claims

1. A plant-derived anti-halitosis mouthwash, characterized in that: Contains the following ingredients by weight: Plant-derived small molecule peptide SEQNO: 1Val-Cys-Leu-Arg-Tyr-Gln-Asp is 0.09-0.15 parts; plant-derived small molecule peptide SEQNO: 2Ala-Asp-Trp-Lys-Glu-Ser-Ile is 0.06-0.1 parts; 5-(2-aminoethyl)-3-hydroxy-2-methyl-4H-pyran-4-one is 1.2-2.2 parts; solvent: deionized water 70-80 parts and propylene glycol 20-30 parts; surfactant: laureth-9 carboxylate 1-2 parts; Flavoring: 0.1-0.2 parts of menthol and 0.05-0.1 parts of citral; Preservative: Potassium sorbate 0.1-0.3 parts.

2. The plant-derived anti-halitosis mouthwash according to claim 1, characterized in that: The plant-derived small molecule peptide SEQ NO: 1 is extracted and purified by the following method: Wash and dry the mint leaves, crush them into fine powder, and extract them with 8-12 times the volume of citric acid-sodium citrate buffer at 25-35°C for 1.5-2.5 hours; After centrifugation and filtration, the extract was initially separated by a gel filtration chromatography column to collect components with a molecular weight range of 600-1200Da; The component was then purified by reverse phase high performance liquid chromatography using a C18 column and water-acetonitrile as the mobile phase for gradient elution to obtain a purified SEQ NO: 1 small molecule peptide.

3. The plant-derived anti-halitosis mouthwash according to claim 1, characterized in that: The plant-derived small molecule peptide SEQ NO: 2 is extracted and purified by the following method: The cloves were crushed, extracted with a buffer solution, centrifuged, filtered, and then purified by gel filtration and RP-HPLC to obtain a small molecule peptide of SEQ NO:

2.

4. The plant-derived anti-halitosis mouthwash according to claim 1, characterized in that: The 5-(2-aminoethyl)-3-hydroxy-2-methyl-4H-pyran-4-one is synthesized and purified by the following method: Using 3-hydroxy-2-methyl-4H-pyran-4-one and ethylenediamine as raw materials, the reaction is carried out in an organic solvent ethanol in the presence of an alkaline catalyst potassium hydroxide, the reaction temperature is controlled at 70-90°C, and the reaction time is 6-8 hours; After the reaction is completed, high-purity AEHMP is obtained through acidification, extraction, distillation, recrystallization and other steps.

5. The plant-derived anti-halitosis mouthwash according to claim 1, characterized in that: The preparation method of the mouthwash comprises: firstly dissolving plant-derived small molecule peptides SEQNO:1 and SEQNO:2 and 5-(2-aminoethyl)-3-hydroxy-2-methyl-4H-pyran-4-one in a small amount of solvent, and then sequentially adding surfactants, flavoring agents, preservatives and other ingredients; Stir in a stirrer at 200-400 rpm for 30-60 minutes until a uniform solution is formed; Finally, put the mouthwash into a sealed bottle and store it in a cool and dry place.

6. Application of a plant-based anti-halitosis mouthwash in inhibiting the growth and reproduction of oral odor-producing bacteria, wherein the oral odor-producing bacteria include Porphyromonas gingivalis and Fusobacterium nucleatum, characterized in that: The mouthwash had a total peptide concentration of 0.3 mg / mL.

7. A use of a plant-derived anti-halitosis mouthwash in reducing the production of volatile sulfides in the oral cavity, characterized in that: After using this mouthwash, the content of volatile sulfides in the oral cavity is significantly reduced and remains at a low level within 3 hours.