Traditional Chinese medicine mouthwash composition for strengthening teeth and refreshing oral cavity and preparation method thereof

Abstract

The invention discloses a traditional Chinese medicine mouthwash composition for strengthening teeth and refreshing oral cavity, the traditional Chinese medicine mouthwash composition comprises traditional Chinese medicine active components and a mouthwash matrix, and the traditional Chinese medicine active components comprise, by weight, 500-900 parts of ginseng, 10-20 parts of prunella vulgaris, 10-20 parts of coptis chinensis and 10-30 parts of turmeric. The invention also discloses a preparation method of the traditional Chinese medicine mouthwash composition. According to the traditional Chinese medicine mouthwash composition disclosed by the invention, the inhibition rate of anaerobic bacteria in the oral cavity can reach 42.1%-55.3%, the proliferation rate of gingival fibroblasts can reach up to 140.5%, the secretion amount of type I collagen of the gingival fibroblasts is increased to 24.4 ng / mL, and the effects of strengthening teeth and refreshing the oral cavity are remarkably realized.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine, and in particular to a traditional Chinese medicine mouthwash composition for strengthening teeth and freshening the mouth, and its preparation method. Background Technology

[0002] Gingival recession refers to the degeneration and shrinkage of the gingival tissue, usually accompanied by the upward movement of the gingival margin, leading to exposure of the tooth roots. Its harm is not limited to oral health but can also affect overall health. One of the most significant consequences of gingival recession is the disruption of the tooth's supporting structure. The gums and alveolar bone interact to provide support for the teeth. When the gums recede, the alveolar bone may also shrink, causing the teeth to lose stability, become loose, and even fall out. Exposed tooth roots make teeth more susceptible to bacterial invasion. The lack of enamel protection on the root surface significantly increases the risk of tooth decay, especially root caries. Furthermore, exposed roots can also make teeth more sensitive to hot and cold stimuli, causing discomfort.

[0003] Gingival fibroblasts (GFs) are the main cell type in gingival connective tissue, playing a crucial role in maintaining gingival tissue homeostasis, repairing damage, and responding to inflammation. Their functions include synthesizing extracellular matrix (such as collagen and glycosaminoglycans), regulating inflammatory responses, and participating in tissue remodeling. Therefore, the activity, secretory function, and responsiveness to environmental changes of gingival fibroblasts are closely related to the occurrence and development of gingival recession.

[0004] Halitosis, medically known as "oral malodor," refers to the foul-smelling gases produced when bacteria in the mouth decompose food residue, dead cells, or other substances. Halitosis has a significant negative impact on an individual's social and professional life. It often leaves a bad impression on others, leading to embarrassment or a lack of confidence in social interactions. Halitosis can also affect an individual's mental health. Long-term halitosis can lead to feelings of inferiority, anxiety, and depression, especially during face-to-face communication, where it can cause embarrassment or shame.

[0005] Halitosis is closely related to the growth of bacteria in the mouth; bacterial activity and metabolic processes are among the main causes of halitosis. The oral cavity is a complex ecosystem containing a large number of bacteria, which are usually harmless under normal circumstances and even help maintain oral health. However, when certain conditions change, the number and types of these bacteria may change abnormally, leading to halitosis. Some anaerobic bacteria in the mouth (bacteria that survive in the absence of oxygen) are a significant source of halitosis. These bacteria are particularly prone to proliferating in the gum pockets, deep in the periodontal tissues, and at the back of the tongue. When they break down food debris and proteins in saliva, the resulting metabolic products often have a strong odor, especially sulfur-containing compounds such as methanethiol and dimethyl sulfide. The odor emitted by these gases may be a major cause of halitosis.

[0006] Mouthwash is an oral care product. It is easy to use and does not require special skills or much time. It is suitable as an auxiliary tool for daily oral care and can maintain oral hygiene more comprehensively. It can also provide targeted solutions for different oral problems, which has a certain degree of flexibility and targeting. However, chlorhexidine (0.12% or 0.2%) is currently a commonly used antibacterial ingredient in mouthwash, but it may cause tooth staining, tartar formation and changes in taste, especially with long-term use. Long-term use of 0.12% chlorhexidine mouthwash (>1 month) resulted in 62% of users experiencing extrinsic tooth staining (Yue Zhaoguo, Liu Qi, Yang Jingwen, Hou Jianxia. A case of peri-implantitis related to toothpaste residue. Chinese Journal of Stomatology. 2023; 58 (3): 277-280. DOI: 10.3760 / cma.j.cn112144-20220824-00458.). Alcohol-containing mouthwashes increased the incidence of dry mouth symptoms by 45% in patients with dry mouth (Xie Yongting, Huang Ruijie, Zou Jing. Research progress on oral manifestations of hypophosphoesterase syndrome [J]. International Journal of Stomatology, 2022, 49(2): 239-243.). Cetylpyridine chloride may also cause similar problems, such as brown staining of teeth and restorations, affecting aesthetics. Alcohol-containing mouthwashes may cause dry mouth, especially for people with dry mouth. Studies suggest that patients with dry mouth should choose alcohol-free formulas to reduce discomfort. In addition, alcohol may cause stinging sensations for some users, affecting the user experience. Furthermore, some mouthwash formulas are expensive, affecting the user experience.

[0007] Existing Chinese herbal mouthwashes mostly focus solely on antibacterial or anti-inflammatory effects. For example, Chinese patent application CN103520057A discloses a mouthwash containing Coptis chinensis, which only emphasizes antibacterial activity and does not address tooth-strengthening effects. CN107184530A discloses a mouthwash containing tea extract, sorbitol, xylitol, citrus peel powder, ginseng extract, and propolis extract, which can quickly and effectively clean the mouth, freshen breath, and effectively inhibit and eliminate oral bacteria, reducing the occurrence of oral diseases; however, it also does not address tooth-strengthening effects. CN117731571A discloses a plant-based mouthwash for freshening breath, containing plant oil, menthol, Prunella vulgaris extract, Chrysanthemum indicum extract, and mulberry leaf extract; its only effect is freshening breath. Summary of the Invention

[0008] The purpose of this invention is to address the above-mentioned technical problems by providing a traditional Chinese medicine mouthwash that can effectively strengthen teeth and freshen the mouth, thereby improving oral health.

[0009] To achieve the above-mentioned objectives, this invention provides a traditional Chinese medicine mouthwash composition for strengthening teeth and freshening oral cavity, and a method for preparing the same. The traditional Chinese medicine mouthwash composition includes active ingredients of traditional Chinese medicine and a mouthwash base. The active ingredients of traditional Chinese medicine are composed of 500-900 parts of ginseng, 10-20 parts of prunella vulgaris, 10-20 parts of coptis chinensis, and 10-30 parts of turmeric by weight.

[0010] Ginseng (Panax ginseng, a plant of the Araliaceae family) Panax ginseng The dried roots and rhizomes of *Camellia sinensis* (CAMey.) greatly replenish vital energy, strengthen the body's foundation, promote the production of qi and blood, and provide an energy basis for gum repair. *Prunella vulgaris* (a plant in the Lamiaceae family) Prunella vulgaris The dried fruit spikes of *L.* clear liver and stomach heat, disperse nodules and reduce swelling, providing local heat-clearing effects for swollen and inflamed gums. (Coptis chinensis, a plant in the Ranunculaceae family) Coptis chinensis The dried rhizome of ginseng (Frankincense) clears heat from the heart and stomach, dries dampness and detoxifies, and directly inhibits anaerobic bacteria in the mouth. The two herbs complement each other in their cooling properties, working together to clear heat and fight bacteria, balancing the warming and tonifying nature of ginseng. Turmeric (from the ginger family, Curcuma longa) Turmeric long The dried rhizome of *L.* invigorates blood circulation and improves local microcirculation, assisting ginseng in promoting tissue repair. *Coptis chinensis* and *Prunella vulgaris* clear excess heat in the oral cavity, balancing the warming and tonifying properties of ginseng and preventing dryness from damaging body fluids. Turmeric invigorates blood circulation and provides a foundation for local microcirculation to support ginseng's "repair-promoting" effect. The four ingredients are balanced in terms of their warming and cooling properties, complementing each other's effects. Ginsenoside Rg1 can upregulate the expression of the COL1A1 gene in gingival fibroblasts, synergistically working with berberine (which inhibits anaerobic bacteria) and curcumin (which improves local blood flow) to form a closed loop of "antibacterial-repair-gingival protection."

[0011] Preliminary experiments have verified that when the amount of ginseng is 500-900 parts by weight, the HGF proliferation rate is ≥128% within this range; when the amount is less than 500 parts, the proliferation rate is <120%; and when the amount is more than 900 parts, the efficacy is not significantly improved, but the raw material cost increases by 25%.

[0012] Preferably, the active ingredients of the traditional Chinese medicine are composed of 900 parts ginseng, 15 parts prunella vulgaris, 15 parts coptis chinensis, and 20 parts turmeric by weight.

[0013] Preferably, the weight ratio of the active ingredient of the traditional Chinese medicine to the mouthwash base is 0.5~1.5:10.

[0014] Preferably, the mouthwash base comprises a humectant, a preservative, a surfactant, a chelating agent, a conditioning agent, and water.

[0015] Preferably, the mouthwash base comprises, by weight, 6.5-19% humectant, 0.21-0.52% preservative, 0.2-0.5% surfactant, 0.01-0.05% chelating agent, and 0.1-0.5% conditioning agent.

[0016] More preferably, the mouthwash base comprises, by its total weight, 6.5-19% humectant, 0.21-0.52% preservative, 0.2-0.5% surfactant, 0.01-0.05% chelating agent, 0.1-0.5% conditioning agent, and the balance being water.

[0017] The total proportion of moisturizer is 6.5% to 19%. When it is less than 6.5%, the oral mucosa dryness score is ≥3 points (out of 5). When it is more than 19%, the mouthwash viscosity is >300mPa·s, which affects the rinsing experience.

[0018] Preferably, the humectant is any one or a combination of sorbitol, erythritol, or xylitol. More preferably, based on the total weight of the mouthwash base, the humectant comprises 5-15% sorbitol, 1-3% erythritol, and 0.5-1% xylitol.

[0019] Preferably, the preservative is potassium sorbate, cetylpyridinium chloride or a combination thereof, more preferably a combination of potassium sorbate and cetylpyridinium chloride, and even more preferably includes 0.2-0.5% potassium sorbate and 0.01-0.02% cetylpyridinium chloride.

[0020] Preferably, the surfactant is polysorbate-20. The surfactant chosen is polysorbate-20, which has an HLB value of 16.7, effectively dissolves the active ingredients of traditional Chinese medicine, and has an oral mucosal irritation score of <1 (out of 5), significantly lower than sodium dodecyl sulfate (irritation score 3.2).

[0021] Preferably, the chelating agent is disodium EDTA.

[0022] Preferably, the conditioning agent is dipotassium glycyrrhizate.

[0023] On the other hand, the present invention also provides a method for preparing the aforementioned herbal mouthwash composition for strengthening teeth and freshening the oral cavity, the method comprising the following steps: (1) Add 75% to 95% v / v ethanol to the mixture of Prunella vulgaris, Coptis chinensis and Curcuma longa, heat and reflux to extract, then filter to remove impurities, and obtain the extract. After concentration by rotary evaporation under reduced pressure at 60°C, vacuum dry to obtain ethanol extracts of Prunella vulgaris, Coptis chinensis and Curcuma longa. The weight-volume ratio of the mixture of Prunella vulgaris, Coptis chinensis and Curcuma longa to ethanol is 1:8 (weight-volume ratio is expressed as "g:mL").

[0024] (2) Add water to ginseng and heat and reflux to extract. The extract is precipitated with ethanol at a concentration of 70% v / v to 80% v / v, then filtered to remove impurities and obtain the extract. After concentration by rotary evaporation under reduced pressure at 60°C, the extract is dried under vacuum to obtain ginseng extract. The weight-to-volume ratio of ginseng to water is 1:10. (3) Mix the ethanol extracts of Prunella vulgaris, Coptis chinensis and Curcuma longa with ginseng extract to obtain the active ingredients of traditional Chinese medicine; (4) Mix the active ingredients of traditional Chinese medicine and the mouthwash base, stir evenly, and obtain the traditional Chinese medicine mouthwash composition.

[0025] Preferably, in step (1), the concentration of ethanol is 75% v / v.

[0026] Preferably, in step (2), the concentration of ethanol is 75% v / v.

[0027] Preferably, the reflux extraction temperature in each step is 100°C.

[0028] Preferably, the reflux extraction time in each step is 1.5-2.5 hours, with 2 hours being the optimal extraction time (highest yield and fewest impurities).

[0029] Preferably, the temperature for vacuum drying is 60°C.

[0030] The herbal mouthwash composition of the present invention, through the synergistic effect of four Chinese herbs, can effectively inhibit the proliferation of anaerobic bacteria in the oral cavity, promote the proliferation of gingival fibroblasts and the secretion of collagen by gingival fibroblasts, thereby achieving the effects of strengthening teeth and keeping the mouth fresh. Detailed Implementation

[0031] The present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0032] Among the various Chinese medicinal materials, ginseng meets the standard on page 9 of Part I of the 2020 edition of the Chinese Pharmacopoeia, with a ginsenoside Rg1+Re content ≥0.3%; Prunella vulgaris meets the standard on page 102 of Part I of the 2020 edition of the Chinese Pharmacopoeia, with a rosmarinic acid content ≥0.2%; Coptis chinensis meets the standard on page 124 of Part I of the 2020 edition of the Chinese Pharmacopoeia, with a berberine content ≥5.5%; and Curcuma longa meets the standard on page 134 of Part I of the 2020 edition of the Chinese Pharmacopoeia, with a curcumin content ≥1.0%.

[0033] In this invention, "parts" refers to parts by weight, and the percentage of components (%) is a percentage by weight.

[0034] Example 1 A traditional Chinese medicine mouthwash composition for strengthening teeth and freshening oral cavity, consisting of active ingredients of traditional Chinese medicine and a mouthwash base.

[0035] The active ingredients of the traditional Chinese medicine are as follows: ginseng 900g, prunella vulgaris 15g, coptis chinensis 15g, and turmeric 20g.

[0036] The mouthwash base components are as follows: humectant (5% sorbitol, 1% erythritol, 0.5% xylitol), preservative (0.2% potassium sorbate, 0.01% cetylpyridinium chloride), surfactant (0.2% polysorbate-20), chelating agent (0.01% disodium EDTA), conditioning agent (0.1% dipotassium glycyrrhizate), and the remainder is made up to 100% with water.

[0037] The specific steps of the preparation method are as follows: (1) Add 8 times the weight volume (g:mL) of 75% v / v ethanol to Prunella vulgaris, Coptis chinensis and Curcuma longa. Extract by reflux extraction, keep the reflux temperature at 100℃ and extract for 2 hours. Then filter to separate and remove impurities to obtain a clear extract. Concentrate by rotary evaporation under reduced pressure at 60℃ and then vacuum dry to obtain ethanol extracts of Prunella vulgaris, Coptis chinensis and Curcuma longa.

[0038] (2) Ginseng was added to softened water at 10 times its weight volume and heated under reflux for extraction at 100°C for 2 hours. The extract was then precipitated with 75% v / v ethanol, filtered to remove impurities, and a clear extract was obtained. The extract was concentrated by rotary evaporation under reduced pressure at 60°C and then vacuum dried to obtain ginseng extract.

[0039] (3) Mix the ethanol extracts of Prunella vulgaris, Coptis chinensis and Curcuma longa with ginseng extract to obtain the active ingredients of traditional Chinese medicine.

[0040] (4) Add the active ingredients of traditional Chinese medicine to the mouthwash base (weight ratio 1:10), stir evenly, and obtain a traditional Chinese medicine mouthwash composition for strengthening teeth and freshening the mouth.

[0041] Example 2 A traditional Chinese medicine mouthwash composition for strengthening teeth and freshening oral cavity, consisting of active ingredients of traditional Chinese medicine and a mouthwash base.

[0042] The active ingredients of the traditional Chinese medicine are as follows: ginseng 500g, prunella vulgaris 10g, coptis chinensis 10g, and turmeric 30g.

[0043] The mouthwash base components are as follows: humectant (10% sorbitol, 2% erythritol, 1% xylitol), preservative (0.3% potassium sorbate, 0.02% cetylpyridinium chloride), surfactant (0.3% polysorbate-20), chelating agent (0.03% disodium EDTA), conditioning agent (0.2% dipotassium glycyrrhizate), and the remainder is made up to 100% with water.

[0044] The specific steps of the preparation method are as follows: (1) Add 8 times the weight volume (g:mL) of 80% v / v ethanol to Prunella vulgaris, Coptis chinensis and Curcuma longa. Extract by reflux extraction, keep the reflux temperature at 100℃ and extract for 2 hours. Then filter to separate and remove impurities to obtain a clear extract. Concentrate by rotary evaporation under reduced pressure at 60℃ and then vacuum dry to obtain ethanol extracts of Prunella vulgaris, Coptis chinensis and Curcuma longa.

[0045] (2) Ginseng was added to softened water at 10 times its weight volume and heated under reflux for extraction at 100°C for 2 hours. The extract was then precipitated with 80% v / v ethanol, filtered to remove impurities, and a clear extract was obtained. The extract was concentrated by rotary evaporation under reduced pressure at 60°C and then vacuum dried to obtain ginseng extract.

[0046] (3) Mix the ethanol extracts of Prunella vulgaris, Coptis chinensis and Curcuma longa with ginseng extract to obtain the active ingredients of traditional Chinese medicine.

[0047] (4) Add the active ingredients of traditional Chinese medicine to the mouthwash base (weight ratio 0.5:10), stir evenly, and obtain a traditional Chinese medicine mouthwash composition for strengthening teeth and freshening the mouth.

[0048] Example 3 A traditional Chinese medicine mouthwash composition for strengthening teeth and freshening oral cavity, consisting of active ingredients of traditional Chinese medicine and a mouthwash base.

[0049] The active ingredients of the traditional Chinese medicine are as follows: ginseng 900g, prunella vulgaris 20g, coptis chinensis 20g, and turmeric 10g.

[0050] The mouthwash base components are as follows: humectant (15% sorbitol, 3% erythritol, 1% xylitol), preservative (0.5% potassium sorbate, 0.02% cetylpyridinium chloride), surfactant (0.5% polysorbate-20), chelating agent (0.05% disodium EDTA), conditioning agent (0.5% dipotassium glycyrrhizate), and the remainder is made up to 100% with water.

[0051] The specific steps of the preparation method are as follows: (1) Add 8 times the weight volume (g:mL) of 95% v / v ethanol to Prunella vulgaris, Coptis chinensis and Curcuma longa. Extract by reflux extraction, keep the reflux temperature at 100℃ and extract for 2 hours. Then filter to separate and remove impurities to obtain a clear extract. Concentrate by rotary evaporation under reduced pressure at 60℃ and then vacuum dry to obtain ethanol extracts of Prunella vulgaris, Coptis chinensis and Curcuma longa.

[0052] (2) Ginseng was added to softened water at 10 times its weight volume and heated under reflux for extraction at 100°C for 2 hours. The extract was then precipitated with 70% v / v ethanol, filtered to remove impurities, and a clear extract was obtained. The extract was concentrated by rotary evaporation under reduced pressure at 60°C and then vacuum dried to obtain ginseng extract.

[0053] (3) Mix the ethanol extracts of Prunella vulgaris, Coptis chinensis and Curcuma longa with ginseng extract to obtain the active ingredients of traditional Chinese medicine.

[0054] (4) Add the active ingredients of traditional Chinese medicine to the mouthwash base (weight ratio 1.5:10), stir evenly, and obtain a traditional Chinese medicine mouthwash composition for strengthening teeth and freshening the mouth.

[0055] Comparative Example 1 A type of Chinese herbal mouthwash, composed of active ingredients of Chinese herbal medicine and a mouthwash base.

[0056] The active ingredients of the traditional Chinese medicine are as follows: 15g of Prunella vulgaris, 15g of Coptis chinensis, and 20g of Curcuma longa.

[0057] The mouthwash base components are as follows: humectant (5% sorbitol, 1% erythritol, 0.5% xylitol), preservative (0.2% potassium sorbate, 0.01% cetylpyridinium chloride), surfactant (0.2% polysorbate-20), chelating agent (0.01% disodium EDTA), conditioning agent (0.1% dipotassium glycyrrhizate), and the remainder is made up to 100% with water.

[0058] The specific steps of the preparation method are as follows: (1) Add 8 times the weight-volume of 75% v / v ethanol to Prunella vulgaris, Coptis chinensis and Curcuma longa, and extract by reflux extraction. The reflux temperature is maintained at 100℃ and the extraction time is 2 hours. Then filter to separate and remove impurities to obtain a clear extract. After concentration by rotary evaporation under reduced pressure at 60℃, vacuum dry to obtain ethanol extracts of Prunella vulgaris, Coptis chinensis and Curcuma longa.

[0059] (2) Add the ethanol extracts of Prunella vulgaris, Coptis chinensis and Curcuma longa to the mouthwash base, stir well, and prepare Chinese medicine mouthwash.

[0060] Comparative Example 2 A type of Chinese herbal mouthwash, composed of active ingredients of Chinese herbal medicine and a mouthwash base.

[0061] The active ingredients of the traditional Chinese medicine are as follows: ginseng 900g, coptis 15g, and turmeric 20g.

[0062] The mouthwash base components are as follows: humectant (5% sorbitol, 1% erythritol, 0.5% xylitol), preservative (0.2% potassium sorbate, 0.01% cetylpyridinium chloride), surfactant (0.2% polysorbate-20), chelating agent (0.01% disodium EDTA), conditioning agent (0.1% dipotassium glycyrrhizate), and the remainder is made up to 100% with water.

[0063] The specific steps of the preparation method are as follows: (1) Add 8 times the weight volume of 75% v / v ethanol to Coptis chinensis and Curcuma longa, and extract by reflux extraction. The reflux temperature is maintained at 100℃ and the extraction time is 2 hours. Then filter and separate to remove impurities and obtain a clear extract. After concentration by rotary evaporation under reduced pressure at 60℃, vacuum dry to obtain ethanol extracts of Coptis chinensis and Curcuma longa.

[0064] (2) Ginseng was added to softened water at 10 times its weight volume and heated under reflux for extraction at 100°C for 2 hours. The extract was then precipitated with 75% v / v ethanol, filtered to remove impurities, and a clear extract was obtained. The extract was concentrated by rotary evaporation under reduced pressure at 60°C and then vacuum dried to obtain ginseng extract.

[0065] (3) Mix the ethanol extracts of Coptis chinensis and Curcuma longa with the ginseng extract to obtain the active ingredients of traditional Chinese medicine.

[0066] (4) Add the active ingredients of traditional Chinese medicine to the mouthwash base, stir evenly, and prepare traditional Chinese medicine mouthwash.

[0067] Comparative Example 3 A type of Chinese herbal mouthwash, composed of active ingredients of Chinese herbal medicine and a mouthwash base.

[0068] The active ingredients of the traditional Chinese medicine are as follows: ginseng 900g, prunella vulgaris 15g, and turmeric 20g.

[0069] The mouthwash base components are as follows: humectant (5% sorbitol, 1% erythritol, 0.5% xylitol), preservative (0.2% potassium sorbate, 0.01% cetylpyridinium chloride), surfactant (0.2% polysorbate-20), chelating agent (0.01% disodium EDTA), conditioning agent (0.1% dipotassium glycyrrhizate), and the remainder is made up to 100% with water.

[0070] The specific steps of the preparation method are as follows: (1) Add 8 times the weight of 75% v / v ethanol to Prunella vulgaris and Curcuma longa, and extract by reflux extraction. The reflux temperature is maintained at 100℃ and the extraction time is 2 hours. Then filter and separate to remove impurities and obtain a clear extract. After concentration by rotary evaporation under reduced pressure at 60℃, vacuum dry to obtain ethanol extracts of Prunella vulgaris and Curcuma longa.

[0071] (2) Ginseng was added to softened water at 10 times its weight volume and heated under reflux for extraction at 100°C for 2 hours. The extract was then precipitated with 75% v / v ethanol, filtered to remove impurities, and a clear extract was obtained. The extract was concentrated by rotary evaporation under reduced pressure at 60°C and then vacuum dried to obtain ginseng extract.

[0072] (3) Mix the ethanol extracts of Prunella vulgaris and Curcuma longa with the ginseng extract to obtain the active ingredients of traditional Chinese medicine.

[0073] (4) Add the active ingredients of traditional Chinese medicine to the mouthwash base, stir evenly, and prepare traditional Chinese medicine mouthwash.

[0074] Comparative Example 4 A type of Chinese herbal mouthwash, composed of active ingredients of Chinese herbal medicine and a mouthwash base.

[0075] The active ingredients of the traditional Chinese medicine are as follows: ginseng 900g, prunella vulgaris 15g, and coptis chinensis 15g.

[0076] The mouthwash base components are as follows: humectant (5% sorbitol, 1% erythritol, 0.5% xylitol), preservative (0.2% potassium sorbate, 0.01% cetylpyridinium chloride), surfactant (0.2% polysorbate-20), chelating agent (0.01% disodium EDTA), conditioning agent (0.1% dipotassium glycyrrhizate), and the remainder is made up to 100% with water.

[0077] The specific steps of the preparation method are as follows: (1) Add 8 times the weight of 75% v / v ethanol to Prunella vulgaris and Coptis chinensis, and extract by reflux extraction. The reflux temperature is maintained at 100℃ and the extraction time is 2 hours. Then filter and separate to remove impurities and obtain a clear extract. After concentration by rotary evaporation under reduced pressure at 60℃, vacuum dry to obtain ethanol extracts of Prunella vulgaris and Coptis chinensis.

[0078] (2) Ginseng was added to softened water at 10 times its weight volume and heated under reflux for extraction at 100°C for 2 hours. The extract was then precipitated with 75% v / v ethanol, filtered to remove impurities, and a clear extract was obtained. The extract was concentrated by rotary evaporation under reduced pressure at 60°C and then vacuum dried to obtain ginseng extract.

[0079] (3) Mix the ethanol extracts of Prunella vulgaris and Coptis chinensis with ginseng extract to obtain the active ingredients of traditional Chinese medicine.

[0080] (4) Add the active ingredients of traditional Chinese medicine to the mouthwash base, stir evenly, and prepare traditional Chinese medicine mouthwash.

[0081] The above examples of mouthwash usage instructions are as follows: Use after brushing your teeth in the morning and evening. Take 15mL each time, rinse for 30 seconds, and then spit it out. No need to rinse with water. Use continuously for 28 days as one care cycle.

[0082] The mouthwash stability of Examples 1-3: After being sealed and stored at room temperature (25℃±2℃) for 6 months, the retention rate of ginsenoside Rg1 was ≥92%, the retention rate of berberine was ≥88%, and the anaerobic bacteria inhibition rate decreased by ≤3%, which met the stability requirements of oral care products.

[0083] Efficacy Experiment The efficacy of the mouthwashes in Examples 1-3 and Comparative Examples 1-4 was tested through experiments.

[0084] 1. Gingival fibroblast (HGF) culture experiment Human gingival fibroblasts (HGF) were cultured in DMEM medium containing 10% fetal bovine serum at 37°C in a 5% CO2 incubator. When the cells reached 80% confluence, they were passaged using trypsin, and cells in the logarithmic growth phase were used for subsequent experiments. The human gingival fibroblast cell line HGF-1 was purchased from Nanjing Kebai Biotechnology Co., Ltd. (catalog number CBP61610).

[0085] The herbal extracts from Examples 1-3 and Comparative Examples 1-4 were diluted to 10 μg / mL with culture medium. A blank control group (without the drug) and a positive control group (containing 10 ng / mL EGF (epidermal growth factor) in culture medium) were established. HGF was seeded in 96-well plates. After 24 hours of cell adhesion, the culture medium was replaced with the drug-containing medium. The treatment lasted 48 hours, and CCK-8 reagent was added to each well. The cells were incubated for 2 hours, and the absorbance at 450 nm was measured using a microplate reader to calculate the cell proliferation rate.

[0086] Gingival fibroblasts (HGF) treated with traditional Chinese medicine extracts were collected and washed twice with pre-chilled PBS. Cells were lysed with RIPA lysis buffer (containing protease inhibitors) and incubated on ice for 30 min, vortexing 2-3 times during incubation. The cells were centrifuged at 12,000 rpm for 15 min at 4°C, and the supernatant was collected to determine the total protein concentration (BCA method).

[0087] Adjust the protein concentration to the same level (1 μg / μL), dilute type I collagen (COL1A1) with carbonate buffer (pH 9.6), add the capture antibody to the working concentration, 100 μL per well of a 96-well microplate, and coat overnight at 4°C. Discard the coating solution, wash three times with PBST (PBS + 0.05% Tween-20), 3 min each time.

[0088] Add 200 μL of 5% skim milk powder or BSA blocking buffer to each well and block at 37°C for 1 hour. After washing, add 100 μL of the sample to be tested to each well. Add biotinylated anti-COL1A1 detection antibody (1:1000 dilution), incubate at 37°C for 1 hour, and wash 3 times with PBST. Add horseradish peroxidase (HRP)-labeled streptavidin (1:5000 v / v dilution), incubate at 37°C for 30 min, and wash 5 times with PBST. Add 100 μL of TMB substrate solution to each well and incubate at room temperature in the dark for 15–30 min (stop when the color development is satisfactory).

[0089] The reaction was terminated by adding 50 μL of 2 M H2SO4, and the absorbance at 450 nm was measured immediately.

[0090] 2. Halitosis Antibacterial Experiment Plaque was collected from periodontal pockets or the dorsum of the tongue of patients with halitosis using a sterile curette and immediately placed in pre-reduced anaerobic transport medium. The scraped plaque sample was vortexed to mix and then inoculated into TSB liquid medium. Anaerobic culture was carried out until the logarithmic growth phase (OD600≈0.5, equivalent to 1×10⁻⁶). 8 (CFU / mL), adjust the bacterial concentration to 1×10⁻⁶. 6 CFU / mL is prepared for use. Add 100 μL of the mouthwash described in Example 1 to each well, followed by 100 μL of bacterial suspension (final bacterial count 5 × 10⁻⁶). 5The mixture (CFU / mL) was thoroughly mixed and then covered with sterile paraffin oil. The 96-well plate was placed in an anaerobic flask and incubated at 37°C for 48 hours. After incubation, the absorbance at 600 nm was measured using a microplate reader to calculate the inhibition rate. 0.12% chlorhexidine mouthwash was used as a positive control. Each experiment was repeated three times, and data are expressed as mean ± standard deviation. One-way ANOVA was performed using SPSS, with p < 0.05 considered statistically significant.

[0091] All experiments passed the Levene test for homogeneity of variance (p>0.05). The significance values ​​are indicated by p: *p<0.05, **p<0.01, ***p<0.001, and NS is p>0.05.

[0092] The experimental results are shown in Tables 1 to 3 below.

[0093] Table 1. Effect of comparative examples of each embodiment on HGF proliferation activity.

[0094] As shown in Table 1, the mouthwashes of Examples 1-3 significantly promoted HGF proliferation activity, while Comparative Examples 1-4 performed poorly. In particular, Comparative Example 3, due to the lack of Coptis chinensis active ingredient, had a lower HGF proliferation rate than the blank control. It is speculated that because Coptis chinensis was missing, the blood-activating effect of curcumin was not counteracted by the "heat-clearing" effect of Coptis chinensis, resulting in a slight increase in local inflammation-related factors (such as IL-6), which inhibited cell proliferation.

[0095] Table 2. Effects of comparative examples on COL1A1 expression in HGFs cells (unit: ng / mL)

[0096] As shown in Table 2, the mouthwashes of Examples 1-3 significantly promoted the expression of COL1A1 in HGF cells, while the comparative examples 1-4 showed poor performance, especially comparative example 3.

[0097] Table 3. Effect of each comparative example on the inhibition rate of oral anaerobic bacteria

[0098] The results above show that the herbal mouthwash composition of the present invention can inhibit oral anaerobic bacteria by 42.1% to 55.3%, increase the proliferation rate of gingival fibroblasts to a maximum of 140.5%, and increase the secretion of type I collagen by gingival fibroblasts to 24.4 ng / mL, significantly achieving the effects of strengthening teeth and freshening the mouth. The herbal mouthwash compositions of Examples 1-3 of the present invention, through the synergistic effect of four herbal ingredients, can effectively inhibit the proliferation of oral anaerobic bacteria and promote the proliferation of gingival fibroblasts and the secretion of collagen by gingival fibroblasts compared with Comparative Examples 1-4, thus achieving the effects of strengthening teeth and keeping the mouth fresh.

[0099] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A traditional Chinese medicine mouthwash composition for fixing teeth and freshening breath, comprising a traditional Chinese medicine active ingredient and a mouthwash base, characterized in that, The active ingredients of the traditional Chinese medicine are composed of 500-900 parts of ginseng, 10-20 parts of prunella vulgaris, 10-20 parts of coptis chinensis, and 10-30 parts of curcuma longa by weight.

2. The traditional Chinese medicine mouthwash composition according to claim 1, characterized in that, The mouthwash base comprises a humectant, a preservative, a surfactant, a chelating agent, a conditioning agent, and water.

3. The traditional Chinese medicine mouthwash composition according to claim 2, characterized in that, The mouthwash base comprises 6.5-19% of a humectant, 0.21-0.52% of a preservative, 0.2-0.5% of a surfactant, 0.01-0.05% of a chelating agent, 0.1-0.5% of a conditioning agent, and the balance of water by weight of the total weight.

4. The traditional Chinese medicine mouthwash composition according to claim 2, characterized in that, The humectant is any one or a combination of sorbitol, erythritol, or xylitol.

5. The traditional Chinese medicine mouthwash composition according to claim 2, characterized in that, The preservative is potassium sorbate, cetylpyridinium chloride, or a combination thereof.

6. The traditional Chinese medicine mouthwash composition according to claim 2, characterized in that, The surfactant is polysorbate-20; the chelating agent is disodium EDTA; and the conditioning agent is dipotassium glycyrrhizinate.

7. The traditional Chinese medicine mouthwash composition according to claim 3, characterized in that, The weight ratio of the active ingredients of the traditional Chinese medicine to the mouthwash base is 0.5-1.5:

10.

8. The method for preparing the traditional Chinese medicine mouthwash composition according to any one of claims 1 to 8, characterized in that The method comprises the following steps: (1) adding 75%-95% v / v ethanol to a mixture of prunella vulgaris, coptis chinensis, and curcuma longa, performing heating reflux extraction, then performing filtration separation to remove impurities, obtaining an extract, vacuum drying after concentration by rotary evaporation under reduced pressure at 60℃, and obtaining an ethanol extract of prunella vulgaris, coptis chinensis, and curcuma longa, wherein the weight-to-volume ratio of the mixture of prunella vulgaris, coptis chinensis, and curcuma longa to ethanol is 1 g:8 mL. (2) adding water to ginseng, performing heating reflux extraction, performing alcohol precipitation on the extract by 70%-80% v / v ethanol, then performing filtration separation to remove impurities, obtaining an extract, vacuum drying after concentration by rotary evaporation under reduced pressure at 60℃, and obtaining a ginseng extract, wherein the weight-to-volume ratio of ginseng to water is 1 g:10 mL; (3) mixing the ethanol extract of prunella vulgaris, coptis chinensis, and curcuma longa with the ginseng extract to obtain active ingredients of traditional Chinese medicine; (4) mixing the active ingredients of traditional Chinese medicine and the mouthwash base, stirring uniformly, and obtaining the traditional Chinese medicine mouthwash composition.

9. The method of claim 8, wherein, The reflux extraction temperature in each step is 100℃.

10. The method of claim 8, wherein, The reflux extraction time is 1.5-2.5 hours.

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