阅读说明：本技术 一种口腔菌群多态性调节剂 (Oral flora polymorphism regulator ) 是由 王亚静 高秀梅 赵鑫 刘二伟 商利娜 王雁雯 高迪 于 2021-01-22 设计创作，主要内容包括：本发明涉及一种口腔菌群多态性调节剂,具体涉及一种维护口腔生态多样性、调节菌群结构的组合物及其用途,属于药物制剂、口腔保健制品技术领域。一种口腔菌群多态性调节剂,其活性成分由黄芩提取物和杜仲叶提取物组成,黄芩提取物和杜仲叶提取物折合生药的质量比为1:16～16:1。本发明活性分为为天然植物提取物,药味数量少,安全无毒最佳工艺简单易行,产品具有优异的维护口腔生态多样性、调节菌群结构、调节口腔菌群平衡、防治口臭、牙周炎及口腔溃疡的效果。(The invention relates to an oral flora polymorphism regulator, in particular to a composition for maintaining oral ecological diversity and regulating flora structure and application thereof, belonging to the technical field of pharmaceutical preparations and oral health products. An oral flora polymorphism regulator comprises active ingredients of a scutellaria baicalensis extract and an eucommia ulmoides leaf extract, wherein the mass ratio of the scutellaria baicalensis extract to the eucommia ulmoides leaf extract to a crude drug is 1: 16-16: 1. The invention has the advantages of small quantity of medicinal ingredients, safety, no toxicity, simple and feasible optimal process, and excellent effects of maintaining the ecological diversity of the oral cavity, adjusting the flora structure, adjusting the oral flora balance and preventing and treating halitosis, periodontitis and oral ulcer.)

1. An oral flora polymorphism modulator, characterized in that: the active ingredients of the composition comprise a scutellaria baicalensis extract and an eucommia ulmoides leaf extract, wherein the mass ratio of the scutellaria baicalensis extract to the eucommia ulmoides leaf extract to the crude drug is 1: 16-16: 1.

2. The oral flora polymorphism regulator according to claim 1, wherein: the mass ratio of the scutellaria baicalensis extract to the eucommia ulmoides leaf extract to the crude drug is 1: 2-2: 1.

3. The oral flora polymorphism modulator according to claim 1 or 2, wherein: the extraction method of the scutellaria baicalensis extract is selected from one or more of reflux water extraction, reflux alcohol extraction, warm-soaking water extraction or warm-soaking alcohol extraction; the extraction method of the eucommia ulmoides leaf extract is one or more selected from reflux water extraction, reflux ethanol extraction, warm-soaking water extraction and warm-soaking ethanol extraction.

4. The oral flora polymorphism regulator according to claim 3, wherein the oral flora polymorphism regulator is characterized in that: the extraction method of the scutellaria baicalensis extract comprises any one of the following steps:

(1) refluxing and water extracting: adding 10-20 times of water into Scutellariae radix coarse powder, reflux extracting for 1-3 times for 1-3 hr, filtering, and concentrating the filtrate to obtain Scutellariae radix extract;

(2) refluxing and alcohol extracting: extracting Scutellariae radix coarse powder with 10-20 times of 30-70% (V/V) ethanol under reflux for 1-3 hr, filtering, and concentrating the filtrate to obtain Scutellariae radix extract;

(3) warm soaking and water extraction: adding 10-20 times of purified water into Scutellariae radix coarse powder, extracting at 50-70 deg.C for 1-3 hr, filtering, and concentrating the filtrate to obtain Scutellariae radix extract;

(4) warm-dipping and alcohol extraction: extracting Scutellariae radix coarse powder with 10-20 times of 30-70% (V/V) ethanol at 50-70 deg.C for 1-3 hr, filtering, and concentrating the filtrate to obtain Scutellariae radix extract;

the method for extracting the eucommia ulmoides leaves comprises the following steps:

(1) refluxing and water extracting: taking folium Eucommiae, adding 15-25 times of purified water, reflux extracting for 1-3 times, each time for 1-3 hr, filtering, and concentrating the filtrate to obtain folium Eucommiae extract;

(2) refluxing and alcohol extracting: taking folium Eucommiae, adding 15-25 times of 30-50% (V/V) ethanol, reflux extracting for 1-3 times (1-3 hr each time), filtering, and concentrating the filtrate to obtain folium Eucommiae extract;

(3) warm soaking and water extraction: soaking in 15-25 times of water at 60 deg.C for 1 time, reflux extracting for 1-3 times (each time for 1-3 hr), filtering, and concentrating the filtrate to obtain folium Eucommiae extract;

(4) warm-dipping and alcohol extraction: taking folium Eucommiae, adding 15-25 times of 30-50% (V/V) ethanol, extracting at 60 deg.C for 1-3 hr, filtering, and concentrating the filtrate to obtain folium Eucommiae extract.

5. The oral flora polymorphism regulator according to claim 4, wherein the oral flora polymorphism regulator is: the extraction method of the scutellaria baicalensis extract comprises any one of the following steps:

(1) refluxing and water extracting: taking Scutellariae radix coarse powder, adding 15 times of water, reflux extracting for 1 time for 2 hr, filtering, and concentrating the filtrate to obtain Scutellariae radix extract;

(2) refluxing and alcohol extracting: taking Scutellariae radix coarse powder, adding 15 times of 50% (V/V) ethanol, reflux extracting for 2 hr, filtering, and concentrating the filtrate to obtain Scutellariae radix extract;

(3) warm soaking and water extraction: taking Scutellariae radix coarse powder, adding 15 times of purified water, extracting at 60 deg.C for 2 hr, filtering, and concentrating the filtrate to obtain Scutellariae radix extract;

(4) warm-dipping and alcohol extraction: taking Scutellariae radix coarse powder, adding 15 times of 40% (V/V) ethanol, extracting at 60 deg.C for 2 hr, filtering, and concentrating the filtrate to obtain Scutellariae radix extract;

the method for extracting the eucommia ulmoides leaves comprises the following steps:

(1) refluxing and water extracting: taking folium Eucommiae, adding 20 times of purified water, reflux extracting for 2 hr, filtering, and concentrating the filtrate to obtain folium Eucommiae extract;

(2) refluxing and alcohol extracting: taking folium Eucommiae, adding 20 times of 30% (V/V) ethanol, reflux extracting for 2 hr, filtering, and concentrating the filtrate to obtain folium Eucommiae extract;

(3) warm soaking and water extraction: soaking in 20 times of water at 60 deg.C for 1 time, extracting for 2 hr, filtering, and concentrating the filtrate to obtain folium Eucommiae extract;

(4) warm-dipping and alcohol extraction: taking folium Eucommiae, adding 20 times of 30% (V/V) ethanol, extracting at 60 deg.C for 2 hr, filtering, and concentrating the filtrate to obtain folium Eucommiae extract.

6. The oral flora polymorphism regulator according to claim 5, wherein the oral flora polymorphism regulator is characterized in that: the extraction method of the scutellaria baicalensis is reflux water extraction, the extraction process is 15 times of water, reflux extraction is carried out for 1 time, the extraction time is 2 hours, the filtration is carried out until the clarification is achieved, the filtrate is decompressed and concentrated into flowable clear paste, and the scutellaria baicalensis extract is obtained through filtration;

the extraction method of the eucommia ulmoides leaves comprises warm-soaking water extraction, wherein the extraction process comprises 20 times of water, warm-soaking water extraction is carried out for 1 time at 60 ℃, the extraction time is 2 hours, the filtration is carried out until the clarification is achieved, and the filtrate is decompressed and concentrated into flowable clear paste, so that the eucommia ulmoides leaf extract is obtained.

7. The oral flora polymorphism regulator according to claim 3, wherein the oral flora polymorphism regulator is characterized in that: the active ingredient accounts for 0.5-15% of the total mass of the oral flora polymorphism regulator (wherein the concentration unit of the liquid preparation is W/V, and the concentration unit of the solid and semisolid preparation is W/W) in terms of the mass of the crude drug.

8. The oral flora polymorphism modulator according to claim 7, wherein the polymorphism modulator is characterized in that: the active ingredient accounts for 1-6% of the total mass of the oral flora polymorphism regulator (wherein the liquid preparation is W/V, and the solid and semisolid preparations are W/W) in terms of the mass of the crude drug.

9. The oral flora polymorphism modulator according to any one of claims 1 to 8, wherein: the Scutellariae radix extract and folium Eucommiae extract are extractive solution, extract, dry extract or powder; the oral flora polymorphism regulator is in a liquid preparation, a semisolid preparation or a solid preparation, wherein the liquid preparation is a gargle, a gargle or a spray; the semisolid preparation is cream, lotion, ointment, gel, or soft capsule; the solid preparation is in the form of powder, granule, tablet, capsule, dripping pill, or pellicle.

10. Use of a modulator of polymorphism in oral flora according to claims 1 to 9, characterized in that: comprises the steps of maintaining the ecological diversity of the oral cavity, adjusting the flora structure, adjusting the balance of the oral flora, and preventing and treating halitosis, periodontitis (gingivitis) and oral ulcer.

Technical Field

The invention relates to an oral flora polymorphism regulator, in particular to a composition for maintaining oral ecological diversity and regulating flora structure and application thereof, belonging to the technical field of pharmaceutical preparations, oral health products and daily chemical products.

Background

The oral cavity is a channel which is externally connected with a respiratory tract and a digestive tract, and provides proper temperature, humidity and nutrient source for the permanent planting of microorganisms, and the diversity of the microorganisms in the oral cavity is favorable for maintaining the physiological function of the oral cavity and resisting the invasion of external adverse factors to organisms. Once the microflora is dysregulated, oral diseases such as caries, periodontitis (gingivitis), and oral ulcer can result. The research shows that: the human oral microorganisms consist of more than 700 kinds of bacteria, and under normal conditions, normal flora or inherent flora in the oral cavity can not cause diseases, and actually most of the bacteria in the oral cavity are beneficial bacteria and play an important role in maintaining the physiological functions of the oral cavity. These microorganisms generally exhibit beneficial effects on the body when the body maintains a state of mutual equilibrium with normal flora; when the organism is influenced by self or external factors to destroy the balance state, such as long-term use of antibacterial substances and the like, probiotics which originally have an inhibiting effect on conditional pathogenic bacteria are reduced, and some frequently living conditional pathogenic bacteria which originally exist in low quantity are relatively increased when the oral environment is changed and even become dominant bacteria, so that the harmful effect of the pathogenic bacteria can be displayed, and oral diseases and oral problems such as oral ulcer, decayed tooth, periodontitis (gingivitis), oral peculiar smell and the like can be caused. The diversity of oral microorganisms and the relative balance of the flora are therefore critical to maintaining oral health.

With the increasing awareness of oral hygiene of people, oral care with mouth wash and the like is the most convenient and feasible method at present, so that not only can food residues and residual dirt in the oral cavity be cleaned, but also bacteria in the oral cavity can be reduced and fresh breath can be obtained, and the oral care is favored by more and more people. If the gargle ingredients are not selective in bacteriostasis, beneficial bacteria in the oral cavity can be killed while pathogenic bacteria are inhibited, the diversity of oral microorganisms and the relative balance of flora are influenced, adverse effects are brought to the oral health, and even the oral health is caused or the problems are aggravated.

Research shows that most of the currently marketed oral gargles pay attention to the killing effect on pathogenic bacteria, influence on probiotics in the oral cavity is ignored, and long-term repeated application can cause disorder of oral flora. In addition, western medicines such as mouth washes and sprays which are widely used in the market and clinically have strong antibacterial and bactericidal effects, large side effects, easy generation of drug resistance and poor taste. Exogenous probiotic supplement also has certain risks, potential safety problems are not ignored, and abuse of the probiotic supplement is reported to possibly cause adverse effects such as infection, horizontal transfer drug resistance, gastrointestinal ischemia, metabolic disorder, anaphylactic reaction, immune dysfunction and the like. Therefore, mobilizing the endogenous flora regulation capacity of the oral cavity is still the safest intervention method at present.

Disclosure of Invention

The technical problem to be solved by the invention is as follows: an oral flora polymorphism regulator is provided.

In order to achieve the purpose, the invention adopts the following technical scheme:

an oral flora polymorphism regulator comprises active ingredients of a scutellaria baicalensis extract and an eucommia ulmoides leaf extract, wherein the mass ratio of the scutellaria baicalensis extract to the eucommia ulmoides leaf extract to a crude drug is 1: 16-16: 1.

Natural plants are treasury of active substance components, and have various components and different functions. The invention inspects the action of the oral cavity-related bacteria of plants such as wild chrysanthemum and the like and plants such as eucommia leaves and the like. The invention investigates the bacteriostatic activity influence of 7 natural plant extracts such as dahongpao tea, wild chrysanthemum, magnolia officinalis, scutellaria baicalensis, folium cortex eucommiae, clove, dandelion and the like on oral pathogenic bacteria, and the result shows that the inhibition effect of the scutellaria baicalensis on the oral pathogenic bacteria candida albicans is strongest; the influence of 5 natural plants such as astragalus, bighead atractylodes rhizome, medlar, folium cortex eucommiae and liquorice on oral probiotic streptococcus salivarius is examined by adopting a trace broth dilution method, and the result shows that the folium cortex eucommiae shows an obvious bacteria promoting trend compared with other plants. Therefore, the scutellaria baicalensis and the eucommia ulmoides leaves are determined to be used as natural plants which are helpful for regulating the balance of oral flora for follow-up research. The raw materials used in the invention are extracted from natural plants, do not contain any chemical additive, and are safe and nontoxic to human bodies.

Preferably, the mass ratio of the scutellaria baicalensis extract to the eucommia ulmoides leaf extract to the crude drug is 1: 2-2: 1.

The extraction method of the scutellaria baicalensis extract is selected from one or more of reflux water extraction, reflux alcohol extraction, warm-soaking water extraction or warm-soaking alcohol extraction; the extraction method of the eucommia ulmoides leaf extract is one or more selected from reflux water extraction, reflux ethanol extraction, warm-soaking water extraction and warm-soaking ethanol extraction.

The extraction method of the scutellaria baicalensis extract comprises any one of the following steps:

(1) refluxing and water extracting: adding 10-20 times of water into Scutellariae radix coarse powder, reflux extracting for 1-3 times for 1-3 hr, filtering, and concentrating the filtrate to obtain Scutellariae radix extract;

(2) refluxing and alcohol extracting: taking Scutellariae radix coarse powder, adding 10-20 times of 30-70% (V/V) ethanol, reflux extracting for 1-3 hr, standing overnight for removing impurities, filtering, and concentrating the filtrate to obtain Scutellariae radix extract;

(3) warm soaking and water extraction: adding 10-20 times of purified water into Scutellariae radix coarse powder, extracting at 50-70 deg.C for 1-3 hr, standing overnight for removing impurities, filtering, and concentrating the filtrate to obtain Scutellariae radix extract;

(4) warm-dipping and alcohol extraction: extracting Scutellariae radix coarse powder with 10-20 times of 30-70% (V/V) ethanol at 50-70 deg.C for 1-3 hr, filtering, and concentrating the filtrate to obtain Scutellariae radix extract;

the method for extracting the eucommia ulmoides leaves comprises the following steps:

(1) refluxing and water extracting: taking folium Eucommiae, adding 15-25 times of purified water, reflux extracting for 1-3 times, each time for 1-3 hr, filtering, and concentrating the filtrate to obtain folium Eucommiae extract;

(2) refluxing and alcohol extracting: taking folium Eucommiae, adding 15-25 times of 30-50% (V/V) ethanol, reflux extracting for 1-3 times (1-3 hr each time), filtering, and concentrating the filtrate to obtain folium Eucommiae extract;

(3) warm soaking and water extraction: soaking in 15-25 times of water at 60 deg.C for 1 time, reflux extracting for 1-3 times (each time for 1-3 hr), filtering, and concentrating the filtrate to obtain folium Eucommiae extract;

(4) warm-dipping and alcohol extraction: taking folium Eucommiae, adding 15-25 times of 30-50% (V/V) ethanol, extracting at 60 deg.C for 1-3 hr, filtering, and concentrating the filtrate to obtain folium Eucommiae extract.

Preferably, the extraction method of the scutellaria baicalensis extract is any one of the following methods:

(1) refluxing and water extracting: taking Scutellariae radix coarse powder, adding 15 times of water, reflux extracting for 1 time for 2 hr, filtering, and concentrating the filtrate to obtain Scutellariae radix extract;

(2) refluxing and alcohol extracting: taking Scutellariae radix coarse powder, adding 15 times of 50% (V/V) ethanol, reflux extracting for 2 hr, filtering, and concentrating the filtrate to obtain Scutellariae radix extract;

(3) warm soaking and water extraction: taking Scutellariae radix coarse powder, adding 15 times of purified water, extracting at 60 deg.C for 2 hr, filtering, and concentrating the filtrate to obtain Scutellariae radix extract;

(4) warm-dipping and alcohol extraction: taking Scutellariae radix coarse powder, adding 15 times of 40% (V/V) ethanol, extracting at 60 deg.C for 2 hr, filtering, and concentrating the filtrate to obtain Scutellariae radix extract;

preferably, the method of the eucommia ulmoides leaf extract is any one of the following methods:

(1) refluxing and water extracting: taking folium Eucommiae, adding 20 times of purified water, reflux extracting for 2 hr, filtering, and concentrating the filtrate to obtain folium Eucommiae extract;

(2) refluxing and alcohol extracting: taking folium Eucommiae, adding 20 times of 40% (V/V) ethanol, reflux extracting for 2 hr, filtering, and concentrating the filtrate to obtain folium Eucommiae extract;

(3) warm soaking and water extraction: soaking folium Eucommiae in 20 times of water at 60 deg.C for 1 time for 2 hr, filtering, and concentrating the filtrate to obtain folium Eucommiae extract;

(4) warm-dipping and alcohol extraction: taking folium Eucommiae, adding 20 times of 30% (V/V) ethanol, extracting at 60 deg.C for 2 hr, filtering, and concentrating the filtrate to obtain folium Eucommiae extract.

Most preferably, the extraction method of the scutellaria baicalensis is reflux water extraction, the extraction process is 15 times of water, reflux extraction is carried out for 1 time, the extraction time is 2 hours, the filtration is carried out until the filtrate is clear, and the filtrate is decompressed and concentrated to obtain clear paste with crude drug mass concentration of about 1g/mL at 60 ℃ to obtain the scutellaria baicalensis extract; most preferably, the extraction method of the eucommia ulmoides leaves is warm-soaking water extraction, the extraction process comprises 20 times of water, warm-soaking water extraction is carried out for 1 time at 60 ℃, the extraction time is 2 hours, filtration is carried out until clarification is achieved, and filtrate is subjected to vacuum concentration at 60 ℃ until clear paste with the crude drug mass concentration of about 1g/mL is obtained, so that the eucommia ulmoides leaf extract is obtained.

The invention obtains 4 kinds of scutellaria baicalensis extracting solutions with different extraction modes by refluxing water extraction, refluxing alcohol extraction, warm-soaking water extraction and warm-soaking alcohol extraction, and establishes HPLC fingerprint spectrums thereof. Candida albicans is used as an indicator bacterium, and a trace broth dilution method is adopted to carry out an in-vitro bacteriostasis experiment on the extracting solution, so that the bacteriostasis rate is determined. And correlating the fingerprint spectrum and the bacteriostatic efficacy information of the scutellaria baicalensis by adopting a grey correlation analysis method, sequencing the contribution degrees of bacteriostatic components of the scutellaria baicalensis, and determining the material basis of the scutellaria baicalensis for inhibiting the candida albicans. According to the result of the strong and weak contribution degree in the 'spectrum-effect' relationship of the scutellaria baicalensis, the scutellaria baicalensis extracting solution is weighted by adopting an AHP-Critic mixed weighting method, and the optimal extracting process of the scutellaria baicalensis is determined by combining an orthogonal test. The Weissella cibaria is taken as experimental probiotics, the probiotic effect of 4 different extraction modes of the eucommia leaves of warm immersion water extraction, warm immersion alcohol extraction, reflux water extraction and reflux alcohol extraction on the Weissella cibaria is examined, and the optimal extraction process of the eucommia leaves is determined by taking the growth rate of promoting the growth of bacteria of the eucommia leaves as an index.

The experimental result shows that the inhibition rate of 4 different extracting solutions to candida albicans is 2.43% -98.10%, wherein the inhibition rate of the scutellaria baicalensis extracted by refluxing and water is 98.10%, the inhibition effect is the best, and the inhibition rate of the scutellaria baicalensis extracted by the other three extracting modes is lower than 40%. The HPLC fingerprint has 26 bacteriostatic quality marker peaks, wherein 17 of the peaks have promoting effect on baikal skullcap root bacteriostasis, and baicalein has the strongest activity and the correlation degree of 0.7689, so that the baicalein is the main active component of baikal skullcap root for playing the role of bacteriostasis. Carrying out comprehensive scoring on the orthogonal experiment result by combining a weight coefficient obtained by calculation of the AHP-Critic mixed weighting method and the contribution degree of the scutellaria baicalensis bacteriostatic component to obtain the optimal scutellaria baicalensis extraction process: 15 times of water, reflux extracting for 1 time for 2h each time. The folium cortex eucommiae promotes the best probiotic effect in a warm-soaking water extraction mode, the determined process is 20 times of water, the warm-soaking water extraction is carried out for 2 hours at the temperature of 60 ℃, and the extraction times are 1 time.

The mass of the active ingredient in terms of crude drug is 0.5-15% of the total oral flora polymorphism regulator (wherein the concentration unit of the liquid preparation is W/V, and the concentration unit of the solid or semisolid preparation is W/W), and preferably the mass of the active ingredient in terms of crude drug is 1.0-6% of the total oral flora polymorphism regulator (wherein the concentration unit of the liquid preparation is W/V, and the concentration unit of the solid or semisolid preparation is W/W).

The influence of the scutellaria baicalensis and eucommia ulmoides leaf extract in the optimal extraction process on the growth of oral related bacteria is inspected by adopting a trace broth dilution method, and the proportion of the extract which plays the optimal effect of regulating the balance of oral flora is further determined. Experimental results show that when the mass ratio of the scutellaria baicalensis extract to the eucommia ulmoides leaf extract to the crude drug is 1: 2-2: 1, and the sum of the mass of the scutellaria baicalensis extract and the eucommia ulmoides leaf extract accounts for 1-6% of the whole oral flora polymorphism regulator (wherein the liquid preparation is W/V, and the solid preparation and the semisolid preparation are W/W), the natural active substance composition of the scutellaria baicalensis extract and the eucommia ulmoides leaf extract has the strongest inhibition effect on candida albicans (pathogenic bacteria related to oral ulcer), fusobacterium nucleatum (pathogenic bacteria related to halitosis and periodontal (gingivitis) inflammation) and porphyromonas gingivalis (pathogenic bacteria related to periodontal (gingivitis)) and has the promotion effect on Weissella chinensis (oral ulcer, halitosis, probiotics related to periodontitis) and streptococcus salivarius (oral ulcer and halitosis).

The scutellaria baicalensis extract and the eucommia ulmoides leaf extract can be extracted liquid, extract, dry paste or powder; the oral flora polymorphism regulator is in a liquid preparation, a semisolid preparation or a solid preparation, wherein the liquid preparation is a gargle, a gargle or a spray; the semisolid preparation is cream, lotion, ointment, gel, or soft capsule; the solid preparation is in the form of powder, granule, tablet, capsule, dripping pill, or pellicle.

The use of an oral flora polymorphism regulator includes but is not limited to maintaining oral ecological diversity, regulating flora structure, regulating oral flora balance, preventing and treating halitosis, periodontitis (gingivitis) and oral ulcer.

The invention can take the natural active substance composition of the scutellaria and the eucommia leaves as the main active ingredient, and water and other auxiliary materials are added according to a certain proportion to prepare oral preparations or local administration preparations, such as oral care products, such as gargle, gargle tablets, toothpaste, and the like.

In order to compare the composition of the invention with the oral care products sold on the market, the composition of the invention is prepared into a simple and convenient liquid gargle and is named as conditioning liquid of the invention, and the specific preparation method is as follows: the yellow and Du natural active substance composition is used as a main active ingredient, preservative, essence, surfactant, sweetener, antioxidant, etc. with the prescription amount are added, the pH value is adjusted, and purified water is added for constant volume, thus obtaining the product. In order to investigate whether other auxiliary materials possibly influence oral bacteria, a group of natural active substance compositions without adding the huangdu is prepared, and the gargle with the same auxiliary materials is marked as a blank auxiliary material.

In-vitro experiments show that the conditioning liquid has a remarkable inhibiting effect on candida albicans which is a main pathogenic bacterium of oral ulcer, and basically has no influence on weissella civora and streptococcus salivarius which are probiotics related to oral ulcer; the conditioning liquid has extremely strong inhibiting effect on main pathogenic bacteria fusobacterium nucleatum related to halitosis and periodontitis (gingivitis), and basically has no influence on probiotics streptococcus salivarius related to halitosis and Weissella sinus related to periodontitis (gingivitis); blank auxiliary materials have weak inhibiting effect on pathogenic bacteria related to oral ulcer, periodontitis (gingivitis) and halitosis, have obvious inhibiting effect on probiotics, do not have the potential of regulating the balance of oral flora, namely, the main effective substance for regulating the balance of the oral flora is the active substance composition of the Dudu Huang; the rest of the commercially available mouth wash has obvious inhibition effect on oral pathogenic bacteria and probiotics, so that the number of the pathogenic bacteria and the probiotics are reduced after the mouth wash acts on the oral cavity, and the effect of regulating the oral flora cannot be achieved. Therefore, experimental results show that the active substance composition of the extracts of the scutellaria baicalensis and the eucommia ulmoides leaves, which is a main functional substance, can inhibit pathogenic bacteria and reduce the influence on probiotics, so that the ratio of the probiotics is increased, and the balance of oral flora is adjusted.

From the perspective of the cell structure of oral flora, the invention explores the influence mechanism of the conditioning solution and blank auxiliary materials and commercially available mouth wash on the cell wall, cell membrane and cell content of the oral flora, and further discloses the characteristics of the influence of the natural active substance composition on oral related bacteria. The experimental result shows that compared with the blank control group, the alkaline phosphatase activity, the potassium ion and the protein content of the extracellular supernatant of the oral ulcer of the experimental group are obviously increased (p is less than 0.01) in comparison with the blank control group by taking the PBS suspension only added with water as the blank control group, taking the PBS suspension added with the conditioning fluid of the invention as the experimental group, taking the PBS suspension added with the blank auxiliary material as the negative control group, taking the PBS suspension added with commercial mouthwash cetylpyridinium chloride mouthwash (batch number: K1906282, Hangzhou national pharmaceutical industry Limited) and the PBS suspension added with Korea shellfish tooth mouthwash (batch number: 0035TH1113) as the positive control group, comparing the difference of the alkaline phosphatase activity, the potassium ion and the protein content in the extracellular supernatant of the oral ulcer of the experimental group and other groups at each time point after acting on the bacteria, the conditioning liquid is shown to play a role in bacteriostasis by acting on cell walls, cell membranes and proteins of pathogenic bacteria of dental ulcer, halitosis and periodontitis (gingivitis); at each time point after the conditioning liquid acts on the bacteria, the alkaline phosphatase activity, the potassium ion content and the protein content in the supernatant are obviously increased (p is less than 0.01) compared with those of a negative control group, which shows that the main effective substance which destroys cell walls, cell membranes and proteins of pathogenic bacteria of oral ulcer, halitosis and periodontitis (gingivitis) in the conditioning liquid so as to play a role in bacteriostasis is a natural active substance composition of the yellow Du; compared with the commercial mouthwash, the conditioning fluid has stronger effect of destroying cell walls, cell membranes and proteins of pathogenic bacteria of dental ulcer, halitosis and periodontitis (gingivitis).

According to the invention, the change of flora in oral cavity of volunteers before and after the conditioning solution is used is compared by using 16S rDNA V4 regional amplicon and ITS sequencing technology, and the influence of the conditioning solution on oral health and oral pathogenic bacteria and probiotics is further analyzed, so that the action characteristics of the natural active substance composition of the huangdu on preventing and treating oral problems are disclosed, and a basis is provided for the application of the natural active substance composition of the huangdu. The result of the alpha diversity analysis of the oral flora shows that the microbial diversity index of the oral cavity of a volunteer has no obvious difference (P is more than 0.05) compared with the non-dry pre-healthy group before and after the conditioning liquid is applied, and the conditioning liquid has mild effect and certain protection effect on normal physiological functions of the oral cavity; based on the results of species-level relative abundance analysis, the relative abundance values of pathogenic bacteria, namely Porphyromonas gingivalis, Fusobacterium nucleatum, Prevotella intermedia, Fossella and Primazala melanogenes, related to periodontitis (gingivitis) are reduced to different degrees after the conditioning fluid is applied; the relative abundance value of the most main pathogenic bacterium, namely the Moraxella, of the oral odor is reduced; except that the Weissella civora relative abundance value is slightly reduced, the relative abundance values of other oral probiotics are all increased; and for the oral health subjects, after the conditioning fluid is used, the relative abundance value of the candida albicans in the oral cavity is still maintained within a normal range; for the subjects with oral ulcer, the relative abundance value of the candida albicans in the oral cavity is in a higher level before intervention, and after the conditioning fluid is used, the relative abundance value of the candida albicans is remarkably reduced, which shows that the conditioning fluid has almost no influence on the normal oral cavity, and has obvious improvement effect on the abnormal oral cavity state of pathogenic bacteria, and prompts that: the conditioning liquid can be used as a daily oral care product to maintain the balance of oral flora and prevent and treat oral diseases. The conditioning liquid has the inhibiting effect on main pathogenic bacteria of periodontitis (gingivitis), oral odor and oral ulcer, and can also up-regulate the proportion of probiotics related to the three oral diseases. The experimental results of the subjects are basically consistent with the results of the prior in vitro effects, which indicates that: the conditioning liquid has certain prevention and treatment effects on periodontitis (gingivitis), oral odor and oral ulcer by adjusting the balance of oral flora.

The invention has the following advantages: the active ingredients of the invention are natural plant extracts, the medicine is small in quantity, safe, nontoxic and optimal, the process is simple and feasible, and the product has excellent effects of maintaining the ecological diversity of the oral cavity, adjusting the flora structure, adjusting the flora balance of the oral cavity, and preventing and treating halitosis, periodontitis (gingivitis) and oral ulcer, and is used for preparing oral care products such as gargle, gargle tablets, toothpaste and the like, and the mouth feel is fresh, sweet and comfortable.

Drawings

The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.

FIG. 1 shows the results of comparison of the bacteriostatic activity of seven natural plants on C.albicans

Fig. 2 shows the mixed HPLC fingerprints of each internal standard and the reflux aqueous scutellaria baicalensis liquid: 1. linarin; 2. rutin; 3. paeonol; 4. chlorogenic acid; 5. hesperidin; 6. ferulic acid

FIG. 3 shows the 3D chromatogram of the fingerprint of Scutellaria baicalensis Georgi at 202nm

FIG. 4 shows the 3D chromatogram of fingerprint of Scutellaria baicalensis Georgi at 278nm

Fig. 5 shows HPLC fingerprints of the scutellaria baicalensis mixed reference substance and the test substance: the mixed reference substance is filtered through a 0.45 mu m filter membrane and then is injected for measurement

Fig. 6 shows HPLC fingerprints of the scutellaria baicalensis mixed reference substance and the test substance: the mixed solution of the sample and the internal standard substance is filtered by a 0.45 mu m filter membrane and then is injected for determination

FIG. 7 shows the components and their degree of contribution to the promotion (left) and attenuation (right) of inhibition of C.albicans by Scutellaria baicalensis

FIG. 8 shows the time-inhibition curves of different concentrations of aqueous extracts of Scutellaria baicalensis to C.albicans

FIG. 9 shows the time-growth rate curves of different concentrations of aqueous extracts of folium Eucommiae against Streptococcus salivarius

FIG. 10 is a graph showing the time-growth rate curves of different concentrations of aqueous extracts of folium Eucommiae against Weissella sinus

FIG. 11 shows the time-growth rate curves of aqueous extracts of Scutellariae radix at concentrations of 7.81 and 15.63mg/mL against Streptococcus salivarius

FIG. 12 shows the time-growth rate curves of 7.81 and 15.63mg/mL concentrations of aqueous extracts of Scutellariae radix against Weissella civora

FIG. 13 shows the growth kinetics curves of the respective mouthwashes against Candida albicans

FIG. 14 shows growth kinetics curves of each mouthwash against Fusobacterium nucleatum

FIG. 15 shows the growth kinetics curves of various mouthwashes against Weissella cibaria

FIG. 16 shows the growth kinetics curves of the respective mouthwashes against Streptococcus salivarius

Figure 17 shows the results of the determination of the change in alkaline phosphatase activity in the extracellular supernatant of the bacterial suspension after the action of each mouthwash on the pathogenic candida albicans.

Figure 18 shows the results of the determination of the change in alkaline phosphatase activity in the extracellular supernatant of the bacterial suspension after the action of each mouthwash on the pathogenic clostridium nucleatum.

Figure 19 shows the results of the determination of the change in alkaline phosphatase activity of each rinse on the extracellular supernatant of the pathogenic porphyromonas gingivalis suspension.

Figure 20 shows the results of the determination of the change in potassium ion content in the extracellular supernatant of the bacterial suspension after the action of each mouthwash on the pathogenic candida albicans.

FIG. 21 shows the results of measuring the change in the potassium ion content of the extracellular supernatant of the bacterial suspension after each mouthwash was exposed to the pathogenic bacterium Fusobacterium nucleatum.

FIG. 22 shows the results of the determination of the change in the potassium ion content of the extracellular supernatant of the bacterial suspension after the action of each mouthwash on the pathogenic bacterium, Porphyromonas gingivalis.

Figure 23 shows the results of the determination of the change in protein content in the extracellular supernatant of the bacterial suspension after the action of each mouthwash on the pathogenic candida albicans.

FIG. 24 shows the results of measuring the change in protein content in the extracellular supernatant of the bacterial suspension after each mouthwash was exposed to the pathogenic Clostridium nucleatum.

FIG. 25 shows the results of the determination of the change in protein content in the extracellular supernatant of the bacterial suspension after the action of each mouthwash on the pathogenic bacterium, Porphyromonas gingivalis.

FIG. 26 shows a graph of the relative abundance of oral probiotics before and after use of the conditioner of the present invention (n-13)

Fig. 27 shows the relative abundance of oral pathogens before and after use of the conditioning fluid of the present invention, with bacteria (n ═ 13) at the left, and candida albicans (n ═ 5) at the right, note: q: before using the conditioning solution, H: after the conditioning liquid is used

Detailed Description

Example 1: screening of natural plant extracts

1. Apparatus and materials

1.1 instruments

HCB-900V vertical laminar flow clean bench (Qingdao Haier Special appliances Co., Ltd.); SW-CJ-JF clean bench (Suzhou purification plant); HPX-9052MBE electric heating constant temperature incubator (Shanghai Bochen industry Co., Ltd.); BSD-TX345 desktop shaker (Shanghai Boxun industries, Inc. medical facilities); an infinite M200PRO microplate reader (TECAN, USA); EYELA N-1210B rotary evaporator (Shanghai Elang instruments, Inc.); EYELA-1000S circulating water type vacuum pump (Tokyo physical and chemical instruments Co., Ltd.); FA124 electronic balance (tianjin yinuo scientific instruments ltd); LS-B50L vertical pressure steam sterilization pot (Shanghai medical nuclear instrument factory).

1.2 materials

Streptococcus salivarius (Streptococcus salivarius, s.salivarius, ATCC13419) was purchased from the guangdong province collection of microorganisms; candida albicans (c. albicans, ATCC1231) was purchased from north na biotechnology limited; BHI broth (LA0360), nutrient agar powder (A8190), and modified Martin agar medium (1108S304) were purchased from Beijing Solaibao Biotech Ltd; modified Martin Medium (M4101B) was purchased from Shandong Tuopu bioengineering, Inc.; folium Eucommiae is provided by Jiangxi Puzheng pharmaceutical Co Ltd; the wild chrysanthemum flower, the dandelion and the scutellaria baicalensis decoction pieces are all provided by republic of agriculture and drug industry of Hebei Anguo; radix astragali, rhizoma Atractylodis Macrocephalae, fructus Lycii, radix Glycyrrhizae, Dahongpao, cortex Magnoliae officinalis, and flos Caryophylli were purchased from Tongrentang pharmacia, Tianjin.

2 method of experiment

2.1 screening of extracts of Natural plants

Preparation of extracts of different natural plants

Weighing 10g of each natural plant, adding purified water twenty times the amount of the natural plant, carrying out reflux extraction for 2 times, each time for 1h, carrying out suction filtration on an extracting solution while the extracting solution is hot, standing overnight, centrifuging, removing precipitate, carrying out rotary evaporation and concentration at 60 ℃ to 10mL to prepare a natural plant extracting solution with the crude drug concentration of 1g/mL, cooling, and placing in a refrigerator at 4 ℃ for later use.

2.1.1 screening of antibacterial activity of seven natural plant extracts to oral pathogenic bacteria C.albicans-scutellaria baicalensis

The antibacterial activity of the natural plants to the oral pathogenic bacteria is determined by adopting an improved Oxford cup method. Under aseptic conditions, 15mL of a fused improved martin agar solid culture medium is taken to be plated (a first layer), 9 Oxford cups are vertically placed after solidification, 10mL of fused improved martin agar solid culture medium (a second layer) containing 50 muL of diluted C.albicans bacterial liquid is added into a culture dish after the fixation, the Oxford cups are removed after the second layer is solidified, and 50 muL of extracting solutions of wild chrysanthemum, dandelion, scarlet robo, scutellaria baicalensis, folium cortex eucommiae, mangnolia officinalis and clove are respectively added into holes; the negative control is 50 mu L of sterile water, the positive control is 20 mu L of diluted econazole nitrate spray, the culture dish is placed in a constant temperature incubator at 30 ℃ for culture for 36h, the diameter of the inhibition zone is determined by a cross method, and the experiment is carried out in parallel for 3 times, and the average value is taken to compare the antibacterial activity.

Evaluation indexes are as follows: the diameter of the inhibition zone is more than or equal to 20mm, and the inhibition zone is extremely sensitive; the diameter of the bacteriostatic circle is less than 20mm and is more than or equal to 15mm, and the bacteriostatic eye is highly sensitive; the diameter of the bacteriostatic circle is more than or equal to 10mm and less than 15mm, and the bacteriostatic circle is moderately sensitive; no inhibition zone or diameter of inhibition zone less than 10mm, low sensitivity or drug resistance.

2.1.2 screening of oral Probiotics S.Salivarius by invigorating plants- -folium Eucommiae

And (3) inspecting the bacterium promoting capability of the tonifying plants on the probiotic streptococcus salivarius by adopting a trace broth dilution method, and screening out the plants with the strongest bacterium promoting effect. Diluting the 5 kinds of tonic plant extractive solutions by 2 times. Setting an experimental group, a negative control group, a control group and a blank control group in a 96-well plate, wherein the experimental group comprises 100 mu L of diluted S.salivarius bacterial liquid and 100 mu L of tonifying plant extracting solution with different concentrations; the negative control group comprises 100 μ L of LBHI liquid culture medium and 100 μ L of tonifying plant extractive solutions with different concentrations; the control group is diluted S.salivarius bacterial liquid 100 μ L and LBHI 100 μ L liquid culture medium; the blank control was 200. mu.LBHI liquid medium. And (3) placing the 96-well plate in an enzyme labeling instrument to set parameters for measuring the absorbance of each well, recording data, and carrying out parallel measurement for 3 times.

3. Results of the experiment

3.1 screening of antibacterial activity of seven natural plant extracts on dental ulcer pathogenic bacteria C.albicans- -results Scutellaria baicalensis

The bacteriostatic activity of seven natural plants on c.albicans was examined, and the results of the zone of inhibition diameter are shown in table 1 and fig. 1. The result shows that the scutellaria baicalensis in the seven natural plants has the strongest inhibition effect on the C.albicans under the same extraction mode and the same experimental conditions, the diameter of the inhibition zone can reach 18.76 +/-0.09 mm, and the scutellaria baicalensis belongs to high-sensitivity natural plants; the cloves, the magnolia officinalis and the eucommia leaves belong to middle-allergy natural plants, and the dandelion and the wild chrysanthemum are low-allergy natural plants; the diameters of the inhibition zones of the seven natural plants to C.albicans are ranked as follows: scutellaria root, cloves, magnolia bark, eucommia bark, dandelion and wild chrysanthemum flower.

Table 1 results of inhibition zones and drug sensitivity of seven natural plants to c

Note: p <0.05, P <0.01 for each group compared to negative controls.

3.2 screening of oral Probiotics S.Salivarius by invigorating plants- -results eucommia ulmoides leaves

The results of investigating the bacteriogenic activity of seven tonifying plants on probiotic s.salivarius are shown in table 2. The result shows that compared with the rest 4 tonifying natural plants, the eucommia ulmoides leaves show obvious growth tendency of promoting probiotics, the growth rate can reach 104.90%, and the medicine concentration of the eucommia ulmoides leaves is 7.81mg (crude drug amount)/mL; therefore, the scutellaria baicalensis and the eucommia ulmoides leaves are determined to be used as natural plants which are helpful for regulating the balance of oral flora for follow-up research.

Table 2 effect of seven natural plants on s.salivarius bacteriogenic activity

Example 2 extraction Process of extract and matching Studies

1. Apparatus and materials

1.1 instruments

HCB-900V vertical laminar flow clean bench (Qingdao Haier Special appliances Co., Ltd.); SW-CJ-JF clean bench (Suzhou purification plant); HPX-9052MBE electric heating constant temperature incubator (Shanghai Bochen industry Co., Ltd.); BSD-TX345 desktop shaker (Shanghai Boxun industries, Inc. medical facilities); an infinite M200PRO microplate reader (TECAN, USA); FA124 electronic balance (tianjin yinuo scientific instruments ltd); LS-B50L vertical pressure steam sterilization pot (Shanghai medical nuclear instrument factory). Agilent1260 high performance liquid chromatograph, Agilent 20RBAX Eclipse Plus column (250 mm. times.4.6 mm, 5 μm), YP1001 electronic balance (Shanghai Kappan instruments Co., Ltd.), AX205 one in ten thousand electronic balance (METTLER TOLEDO, Switzerland), Mili-Q ultra-pure water processor (Milipore, USA), EYELA N-1100 rotary evaporator (Tokyo chemical and physical instruments Co., Ltd.); EYELA A-1000S circulating water type vacuum pump (Tokyo physical and chemical instruments Co., Ltd.); HH.S21-6 electric heating constant temperature water bath (Jinan Ji island analytical instruments Co., Ltd.).

1.2 materials

Streptococcus salivarius (Streptococcus salivarius, s.salivarius, ATCC13419) was purchased from the guangdong province collection of microorganisms; porphyromonas gingivalis (p. gingivalis, ATCC33277), Fusobacterium nucleatum (f. nucleatum, ATCC10953), Candida albicans (Candida albicans, c. albicans, ATCC1231) were purchased from north nana biotechnology limited; weissella cibaria (weissella. cibaria, w. cibaria, CPCC101289) was purchased from the chinese pharmaceutical microbial strain collection management center; columbia blood plates (batch: 352241) were purchased from North Na Biotechnology Ltd; BHI broth (LA0360), MRS broth (M8540), nutrient agar powder (A8190), modified Martin agar medium (1108S304), hemin solution (H8132), and vitamin K3(V8170) all provided by Beijing Solelaibao Biotechnology, Inc.; modified Martin Medium (M4101B) was supplied by Shandong Tuo general bioengineering, Inc.; anaerobic gas-generating bags, anaerobic culture bags (trigonal gas chemical co., ltd. japan); yeast Extract Yeast powder LP0021 (OXOID); folium Eucommiae is provided by Jiangxi Puzheng pharmaceutical Co Ltd; scutellariae radix tablet (batch No. 190102, producing area: Shanxi) is provided by Dai Ling pharmaceutical Co., Ltd in North An river; baicalin control (P20A9F59353), baicalein control (C20MBY31962), wogonin control (W30M10284622), and hesperidin control (P06D9F77001) are all provided by Shanghai-sourced leaf Biotech Co., Ltd; wogonoside reference (823B022) was provided by Beijing Sorleibao technologies, Inc.; methanol (chromatographic grade) was supplied by kang scientific technologies, ltd, Tianjin; acetonitrile (chromatographic grade) was supplied by Fisher, usa; deionized water (MIILLIPORE ultra pure water system).

2. Experimental methods

Determination of optimal extraction process of natural plant extract

2.1 study of the extraction Process of the Chinese medicine Scutellaria baicalensis Georgi

Early-stage preliminary experiments find that scutellaria baicalensis liquid medicine prepared by 4 modes of refluxing water extraction, refluxing 60% ethanol extraction, warm-soaking water extraction and warm-soaking 60% ethanol extraction has obvious difference on the inhibition zone of C.albicans, therefore, 4 different extraction modes are specially set in the experiment, so that the chemical components and the drug effect (inhibition rate) of the scutellaria baicalensis liquid medicine have obvious difference, and the material basis of the scutellaria baicalensis for inhibiting the C.albicans is searched from the viewpoint of the inhibition difference.

2.1.1 preparation of extracts by different extraction methods

Precisely weighing 4 parts of scutellaria baicalensis coarse powder, respectively adding 10g of scutellaria baicalensis coarse powder into 200mL (water or 60% ethanol) of a corresponding solvent, extracting for 2h in a mode of refluxing water extraction, refluxing 60% ethanol extraction, warm-soaking water extraction and warm-soaking 60% ethanol extraction, cooling an extracting solution, complementing weight loss with the corresponding solvent, centrifuging, removing medicine residues, filtering with a 0.45 mu m filter membrane, and keeping the temperature at 4 ℃ for later use, wherein the prepared solution is marked as a test solution.

2.1.2 establishment of fingerprint analysis method

2.1.2.1 chromatographic conditions

A chromatographic column: agilent ZORBAX Eclipse Plus column (250 mm. times.4.6 mm, 5 μm); mobile phase: acetonitrile (a) -0.1% phosphoric acid water (B); gradient elution (0-25 min, 85-75% B, 25-40 min, 75-70% B, 40-45 min, 70-60% B, 45-60 min, 60-57% B, 60-65 min, 57-30% B, 65-70 min, 30-85% B, 70-75 min, 85% B); detection wavelength: 202 nm; column temperature: 30 ℃; flow rate: 1 mL/min; sample introduction amount: 10 μ L.

2.1.2.2 internal standard substance and selection of wavelength

Selection of internal standard: precisely absorbing 6 parts of scutellaria baicalensis water extract, wherein each part is 1mL, respectively adding 1mL of linarin, rutin, paeonol, chlorogenic acid, hesperidin and ferulic acid reference solution, uniformly mixing, filtering by a 0.45-micrometer microporous filter membrane, injecting into a high performance liquid chromatograph under the chromatographic condition of '2.1.2.1' in the embodiment, and screening out an optimal internal standard substance by taking the peak appearance time and the separation degree as indexes.

Selection of wavelength: absorbing a proper amount of scutellaria baicalensis water extract, filtering the scutellaria baicalensis water extract by a 0.45-micron microporous filter membrane, injecting the scutellaria baicalensis water extract into a high performance liquid chromatograph according to the chromatographic condition under the section '2.1.2.1' of the embodiment, setting full-wavelength scanning (190 plus 400nm and step length of 2nm), and determining the wavelength of the scutellaria baicalensis fingerprint according to the response of all peaks of the fingerprint.

2.1.2.3 preparation of solution

Hesperidin internal standard substance solution: 1.47mg of hesperidin is precisely weighed, methanol is added to the hesperidin to a constant volume of 25mL, an internal standard substance solution with the mass concentration of 58.8 mu g/mL is prepared, and the internal standard substance solution is reserved at 4 ℃.

Mixing the reference solution: accurately weighing appropriate amount of baicalin, wogonoside, baicalein, and wogonin, dissolving in methanol, and making into reference stock solution with mass concentration of 0.516, 1.000, 0.506, and 0.515 mg/mL. Respectively sucking 1mL of each reference substance solution, placing in a 10mL volumetric flask, adding methanol to a constant volume to a scale, ultrasonically mixing, filtering with a 0.45 μm filter membrane, and taking the subsequent filtrate as the mixed reference substance solution.

Mixed solution of test sample and internal standard substance: 1mL of the sample solution under the item "2.1.1" is placed in a 5mL volumetric flask, the volume is determined to 5mL by the corresponding solvent, 1mL of the diluted sample solution is mixed with 1mL of the internal standard solution, and the mixture is injected into a high performance liquid chromatograph for fingerprint determination under the chromatographic conditions under the item "2.1.2.1" in the embodiment after passing through a 0.45 μm microporous filter membrane.

2.1.2.4 repeatability

The same mixed solution of the sample and the internal standard substance was sampled for 6 times continuously under the chromatographic conditions of "2.1.2.1" in this example, the peak appearance time of each chromatographic peak and the ratio of the peak area of each chromatographic peak to the peak area of the internal standard substance (relative peak area ratio) were measured, and the relative standard deviation RSD (%) was calculated to examine the reproducibility of the method.

2.1.2.5 precision

Taking 6 parts of mixed solution of the test sample and the internal standard substance in the same batch, injecting according to the chromatographic conditions under the item '2.1.2.1' in the embodiment, measuring the peak appearance time of each chromatographic peak and the ratio (relative peak area ratio) of the peak area of each chromatographic peak to the peak area of the internal standard substance, calculating the relative standard deviation RSD (%), and inspecting the precision of the instrument.

2.1.2.6 stability

The same mixed solution of the sample and the internal standard substance was taken, sample injection was performed for 0, 2, 4, 8, 12, 16 and 24 hours respectively under the chromatographic conditions of item "2.1.2.1" in this example, the peak appearance time of each chromatographic peak, the ratio of the peak area of each chromatographic peak to the peak area of the internal standard substance (relative peak area ratio) were measured, the relative standard deviation RSD (%) was calculated, and the reproducibility of the method was examined.

2.1.3 study on C.albicans antibacterial Activity

In vitro bacteriostasis experiment of scutellaria baicalensis extract on c.albicans was determined by a broth dilution method. Taking a sterile 96-well plate, respectively adding 100 μ L of Scutellariae radix extract with concentration of 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98mg (crude drug amount)/mL into A1-A11, respectively, and respectively adding 100 μ L into A1-A11 well to dilute to 10 μ L⁴Multiplying the C.albicans bacterial liquid to make the final concentration of each hole be 500, 250, 125, 62.5, 31.25, 15.63, 7.81 and 3.911.95, 0.98, 0.49mg (crude drug)/mL as experimental group; sequentially adding 100 μ L of Scutellariae radix extractive solution with concentration of 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98mg (crude drug amount)/mL into B1-B11 wells, and respectively adding 100 μ L of improved Martin liquid culture medium as negative control group; 100 μ L of sterile water and 100 μ L of diluted solution are added into A12 hole⁴The multiplied bacterial liquid is used as a control group; b12 was added 100. mu.L of sterile water and 100. mu.L of modified Martin broth as a blank control. The 96-well plate was placed in a microplate reader at 600nm to measure absorbance (OD), and the experiment was performed 3 times in parallel.

The formula for calculating the bacteriostasis rate is as follows:

2.1.4 establishment of Gray correlation analysis

2.1.4.1 determination of reference and comparison sequences

This example studies the influence of various components (i.e. peak areas of various components) of the scutellaria baicalensis extracting solution on the bacteriostatic ratio of candida albicans under different extraction processes. Therefore, the bacteriostatic rate is taken as a reference sequence (mother sequence) and is marked as X₀(k) (ii) a k is 1,2,3, 4; the peak values of 27 components were used as comparison sequences (subsequences) and X_i(k)；i＝1、2、3···27；k＝1、2、3、4。

2.1.4.2 dimensionless processing

In order to ensure the accuracy of the experimental result and facilitate the comparison of each index, before calculating the gray correlation, the data needs to be subjected to non-dimensionalization processing, the experimental study adopts an averaging method to process, i.e. each sequence element is divided by the average value of the corresponding sequence, and the calculation formula is as follows: x_i’(k)＝X_i(k)/X_iI is 0, 1,2,3 · 27; k is 1,2,3, 4. Wherein represents the average value, X, of sample data of sequence i_i(k) Representing the original data, X_i' (k) represents data after the dimensionless quantization process. Resulting in an averaged sequence { Xi' (k) }.

2.1.4.3 creating a difference series

I.e., calculating the absolute difference between the reference and comparative sequences.

The difference array calculation formula is as follows:

ΔX_i(k)＝|X_i'(k)-X₀'(k)| (3)

2.1.4.4 determining the correlation coefficient

The grey correlation coefficient is calculated by the formula:

in the formula | X₀(k)－X_i(k) I represents the sequence X₀And sequence X_iThe absolute value of the difference at point k; expressing the second-level minimum value of the absolute value of the difference, namely further obtaining the minimum value of the difference of all the sequences on the basis of the minimum value of the difference of each sequence; the difference value is a second-order maximum value of the absolute value of the difference value, namely, the maximum value of the difference value of all the sequences is further obtained on the basis of the maximum value of the difference value of each sequence. ρ is a resolution coefficient, a value interval is (0, 1), and ρ is 0.5 in the experiment.

2.1.4.5 calculating Grey relevance

Correlation coefficient xi_i(k) Denotes the reference sequence X₀And comparison of sequence X_iDegree of association at k (k ═ 1,2,3, 4). Further, the average value of the correlation coefficients in a certain sequence i (i 1,2, 3.. 27) can be used to represent the reference sequence X₀With the comparison sequence X_iThe overall degree of correlation between the two sequences is the gray degree of correlation between the two sequences.

Degree of gray correlation gamma_0iThe calculation formula of (a) is as follows:

(n is the number of samples in any sequence i, in this experiment n-4) (5)2.1.4.6 correlation polarity analysis

Sequence X_i' (k) and X₀The correlation polarity between' (k) is calculated by the following equation:

if sgn (σ)_i)＝sgn(σ₀) Then Y is_iAnd Y₀Positive association, i.e. Y_iFor Y₀Plays a role in enhancing; if sgn (σ)_i)＝-sgn(σ₀) Then Y is_iAnd Y₀Negative correlation, meaning Y to Y₀The weakening effect is realized, i is 1,2 and 3 … … 27. Here sgn (x) is a sign function, i.e.:

2.1.5 determination of optimal extraction Process

2.1.5.1 establishment of weights

And determining the weight by adopting an AHP-Critic mixed weighting method. According to the 'spectrum-effect' relationship result of the scutellaria root for inhibiting the candida albicans, all indexes and the bacteriostasis rate are quantified as weight indexes, namely 11 indexes are divided into 6 levels, and the priority of all indexes is determined: the bacteriostatic ratio > component 24 (baicalein) > component 27 (oroxylin a) > component 19 ═ component 2 ═ component 15 ═ component 16 ═ component 11 (baicalin) ═ component 18 ═ component 25 (wogonin) > component 20 (wogonoside), a preferential judgment matrix for comparison was constructed, and relative scores given to each index were given to determine the AHP weight.

The Critic method is a weighting method for objectively reflecting the index weight, and is based on the contrast strength and the conflict among indexes, wherein the contrast strength is reflected in the form of standard deviation, and the conflict is reflected in the form of correlation among indexes. In the research, a Critic method is adopted to determine the weight among all indexes, orthogonal test data are subjected to standardization processing through linear interpolation [ index components are (measured value-minimum value)/(maximum value-minimum value) ], data are processed according to SPSS 17.0 software to obtain a correlation coefficient matrix, and then the contrast strength (si), the conflict (delta i), the comprehensive weight (ci) and the weight (omega i) of all indexes are calculated.

According to the spectrum-effect relation characteristic of the scutellaria for inhibiting the candida albicans, the AHP method obtains a weight coefficient based on subjective evaluation, and basically embodies the primary and secondary sequence of each index of the scutellaria for inhibiting bacteria; on the basis, objective weight coefficients of all indexes are obtained by adopting a Critic method, so that the influence of variability of all sample data and correlation among the indexes on weighting is considered, and the deviation of subjective weighting is avoided. In order to make the evaluation weight pay attention to objectivity and not lose subjectivity, the evaluation weight and the subjectivity are combined together so that the evaluation result is more objective and real.

The integrated weight calculation formula is as follows:

2.1.5.2 orthogonal experimental design

Arranging L according to the property of the medicine and the actual production requirement, taking the extraction time (A), the extraction times (B) and the solvent times (C) as the investigation factors₉(3⁴) Orthogonal experiments, factors and levels are shown in table 3.

TABLE 3 Scutellaria baicalensis extraction Process orthogonal test factors and levels

2.2 study of the extraction Process of Chinese medicine folium Eucommiae

In the early period of the laboratory, factors such as solvent multiple, temperature and time of folium cortex eucommiae under a reflux water extraction mode are examined, the optimal process of extracting the folium cortex eucommiae at 60 ℃ for 2 hours by using 20 times of water is found, but systematic research on the extraction mode is not carried out, and pre-experiments show that the folium cortex eucommiae has a remarkable bacterium promoting effect on W.cibaria, and the bacterium promoting effect is closely related to the extraction mode of the folium eucommiae.

2.2.1 preparation of extracts by different extraction methods

Precisely weighing 4 parts of eucommia ulmoides leaf medicinal material, adding 10g of eucommia ulmoides leaf medicinal material into corresponding solvent 200mL (water or 40% ethanol) respectively, extracting for 2h in a mode of refluxing water extraction, refluxing 40% ethanol extraction, warm-soaking water extraction and warm-soaking 40% ethanol, centrifuging and filtering to be clear, concentrating the filtrate at 60 ℃ under reduced pressure, and carrying out rotary evaporation to 10mL (crude drug amount is 1g/mL) for later use at 4 ℃.

2.2.2 Studies on the prohibitionism Activity of W.cibaria

And (3) observing the influence of the eucommia ulmoides leaf extracting solution on the growth condition of the W.cibaria by adopting a trace broth dilution method. The experiment was set up with an experimental group, a negative control group, a positive control group and a blank control group. Taking a sterile 96-well plate, respectively adding 100 mu L of folium Eucommiae extract with concentration of 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95 and 0.98mg (crude drug amount)/mL into A1-A11, respectively, and respectively adding 100 mu L into A1-A11 to dilute to 10⁴Multiplying the W.cibaria bacterial liquid to make the final concentration of each hole be 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 and 0.49mg (crude drug amount)/mL, thus obtaining an experimental group; sequentially adding 100 mu L of eucommia ulmoides leaf extract with the concentration of 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95 and 0.98mg (crude drug amount)/mL into B1-B11 holes, and respectively adding 100 mu L of MRS liquid culture medium as a negative control; 100 μ L of sterile water and 100 μ L of diluted solution are added into A12 hole⁴The multiplied bacterial liquid is used as a positive control; b12 was added 100. mu.L of sterile water and 100. mu. of LMRS broth as a blank. The 96-well plate was placed in a 600nm microplate reader to determine the OD value, and the experiment was performed 3 times in parallel.

The growth rate calculation formula is as follows:

2.2.3 determination of optimal extraction Process

According to the experimental method under the item "2.2.2" in this example, the optimum extraction process of eucommia ulmoides leaves was determined using the growth rate of eucommia ulmoides leaves to w.cibaria as an index.

Second, research on the mixture ratio of extracts

And (3) inspecting the influence of the extract of the scutellaria baicalensis and the eucommia ulmoides leaves on the growth condition of oral flora by adopting a trace broth dilution method. The experiment was set up with an experimental group, a negative control group, a positive control group and a blank control group. Taking a sterile 96-well plate, sequentially adding 100 mu L of 2-time diluted extracts of scutellaria baicalensis and eucommia ulmoides leaves into A1-A11 respectively, and then adding 100 mu L of diluted bacterial suspension into A1-A11 respectively to obtain an experimental group; sequentially adding 2 times diluted extracts of Scutellariae radix and folium Eucommiae into B1-B11 wells, and adding 100 μ L corresponding liquid culture medium as negative control; adding 100 mu L of corresponding liquid culture medium and 100 mu L of diluted bacterial suspension into a hole A12 to be used as a control group; b12 was added 200. mu.L of the corresponding liquid medium as a blank control. Determination of OD in 0, 2, 4, 6, 8, 10, 12, 24, 36, 48h with Fusobacterium nucleatum₆₀₀The OD of the rest bacteria is measured every 2h₆₀₀The experiment was performed 3 times in parallel, and a time-growth curve was plotted with time as abscissa and growth rate as ordinate.

Growth rate calculation formula:

the formula for calculating the bacteriostasis rate is as follows:

3. results of the experiment

First, research on extraction process of extract

3.1 study of extraction Process of Chinese medicine Scutellariae radix

3.1.1 establishment of fingerprint analysis method

3.1.1.1 selection of internal standards and wavelength

Selection of internal standard: the fingerprint chromatogram of internal standard substance containing linarin, rutin, paeonol, chlorogenic acid, hesperidin and ferulic acid is shown in figure 2 (the mixed HPLC fingerprint chromatogram of each internal standard substance and reflux water-extracted scutellaria baicalensis liquid in figure 2 is 1, linarin, 2, rutin, 3, paeonol, 4, chlorogenic acid, 5, hesperidin, 6 and ferulic acid), the retention time and the separation degree are shown in table 4, and the result shows that the separation degree of chromatographic peaks of 4 internal standard substances of linarin and rutin is not good; the accuracy of the experiment may be affected by the fact that the peak position of chlorogenic acid is close to the front (5.957min) and the peak position of paeonol is close to the back (47.717 min); the peak position of ferulic acid is about 16.175min, but the surrounding peaks are denser; therefore, the peak emergence time and the separation degree are comprehensively considered, the hesperidin with better separation degree and peak emergence time is selected as an internal standard substance, the retention time is 22.600min, and the separation degree is 12.24.

TABLE 4 results of retention time and separation of each internal standard fingerprint

Selection of wavelength: 190-400nm full-wavelength scanning is carried out in the experiment, the 3D map at 202nm is shown in figure 3, and the 3D map at 278nm is shown in figure 4, and the result shows that 27 marking peaks in the scutellaria baicalensis at 202nm have strong ultraviolet absorption, and a part of peaks at 278nm have low ultraviolet absorption; and the ultraviolet absorption band with the wavelength of more than 200nm comprises an R band, a K band, a B band and an E band, and can detect unsaturated compounds containing hetero atoms, conjugated unsaturated compounds, aromatic rings, aromatic heterocyclic compounds and the like, namely the wavelength can comprehensively express most substances in the scutellaria baicalensis extracting solution. Therefore, the wavelength of the fingerprint spectrum of the scutellaria baicalensis in this section is determined to be 202 nm.

3.1.1.2 fingerprint spectrum determination result

According to the chromatographic conditions under the item "2.1.2.1" in this example, the mixed reference substance is filtered through a 0.45 μm filter membrane and then subjected to sample measurement, and the chromatogram is shown in FIG. 5; the mixed solution of the sample and the internal standard substance is filtered through a 0.45 μm filter membrane, and then is subjected to sample injection and determination, and the atlas is shown in figure 6 (in the figure, A is a reference substance, B is the sample, a is hesperidin, b is baicalin, c is wogonoside, d is baicalein, and e is wogonoside). The established fingerprint spectrum has good separation degree, is simple and easy to implement, and can be used for investigating the spectrum-effect relationship of the scutellaria baicalensis medicinal liquid.

3.1.1.3 repeatability

The result of a repeatability experiment, with the peak No. 7 as a reference peak, the RSD of the relative retention time and the relative peak area of the 26 characteristic peaks is calculated to be respectively less than 0.1% and less than 3.3%, which shows that the method has good repeatability.

3.1.1.4 precision

The result of the precision experiment, with peak 7 as the reference peak, the RSD of the relative retention time and the relative peak area of the 26 characteristic peaks calculated were < 0.3% and < 3.4%, respectively, indicating that the instrument has good precision.

3.1.1.5 stability

The result of the stability experiment, with peak 7 as the reference peak, the RSD of the relative retention time and the relative peak area of the 26 characteristic peaks calculated are respectively < 0.4% and < 4.6%, indicating that the test article has good stability within 24 h.

3.1.2 fingerprint and its bacteriostatic activity on C.albicans

The data of the scutellaria fingerprint spectrum are expressed by adopting the ratio of the peak area of each mark peak to the peak area of an internal standard substance, the bacteriostasis effect is expressed by the bacteriostasis rate when the concentration of the scutellaria is 7.81mg (crude drug amount)/mL (experiments prove that the minimum bacteriostasis concentration of the scutellaria for inhibiting C.albicans is 7.81mg (crude drug amount)/mL, and the bacteriostasis rates of the extracting solutions of 4 modes have obvious difference under the concentration), the data are shown in table 4, and the results show that the bacteriostasis rate of the backflow water extraction is 98.10%, the bacteriostasis effect is the best, and the bacteriostasis rates of the other three modes are all lower than 40%.

TABLE 4 Experimental data sheet for peak ratio and bacteriostasis rate of fingerprint spectrum of radix Scutellariae

3.1.3 results analysis of Gray correlation

The correlation degree and the correlation polarity between 27 marker peaks and the bacteriostatic rate in the extract of scutellaria baicalensis can be obtained by analyzing the spectrum-effect relationship of scutellaria baicalensis according to the method under the item "2.1.4" in this example, which is shown in table 5. The positive correlation shows that the component has the effect of promoting the growth of the C.albicans inhibited by the scutellaria baicalensis, and the negative correlation shows that the component has the effect of weakening the growth of the C.albicans inhibited by the scutellaria baicalensis. The results show that except that the components 1, 3, 8, 10, 12, 14, 21, 22 and 26 weaken bacteriostasis, the other components promote bacteriostasis, and the influence degree of each component on bacteriostasis rate is shown in figure 7; among the components promoting bacteriostasis, the peak 24 (baicalein) with the strongest correlation degree is 0.7689; among the ingredients that impair bacteriostasis, the peak 22 with the strongest correlation degree is 0.6247, i.e., the peak 24 (baicalein) is the main active ingredient of scutellaria baicalensis that promotes its inhibition of c.albicans growth, while the ingredient represented by the peak 22 is the main ingredient that impairs its inhibition of c.albicans growth.

TABLE 5 correlation degree and correlation polarity between bacteriostatic mass marker peak and bacteriostatic rate in Scutellariae radix extractive solution

Note: each peak associated polarity with X₀The same correlation polarity (-1) is positive correlation with X₀But is inversely correlated.

3.1.4 determination of optimal extraction Process

3.1.4.1 determination of index weight

Determining a weight coefficient by an AHP method: according to the 'spectrum-effect' relationship result of the scutellaria root for inhibiting the candida albicans, 11 indexes are divided into 6 levels, and the priority order of each index is determined: the results of table 6 show that the weight coefficients obtained by hierarchical analysis of 11 indexes are 0.3195, 0.2159, 0.1413, 0.0717, 0.0632, 0.0537, 0.0366, 0.0311, 0.0274, 0.0233, and 0.0163, and the consistency scale factor (CR) 0.0571 < 0.10, that is, the index priority comparison determination matrix has satisfactory consistency, and the obtained weight coefficients are effective.

TABLE 6 determination priority matrix for paired comparison of indices

Critic method determines the weight coefficients: according to the experimental method under the '2.1.5.1' item in this embodiment, the orthogonal 9 sets of data are normalized by linear interpolation, and the contrast strength (si), the conflict (δ i), the integrated weight (ci) and the weight (ω i) of each index are calculated, and the correlation calculation results are shown in table 7.

TABLE 7 related calculation data

Determining the weight by an AHP-Critic mixed weighting method: according to the spectrum-effect relationship of Scutellaria baicalensis for inhibiting Candida albicans, according to the experimental method under the section 2.1.5.1 in this example, AHP analytic hierarchy process and Critic process are combined to calculate comprehensive weightSo that the evaluation result is more objective and real. The AHP, Critic, and AHP-Critic method weights for each index component are shown in Table 8.

TABLE 8 AHP, Critic, AHP-Critic method weight coefficients

3.1.4.2 analysis of orthogonal test results

The weight coefficient obtained by calculation of the AHP-Critic mixed weighting method is used for carrying out comprehensive scoring on the orthogonal 9 groups of experimental results, the visual analysis result of the orthogonal test is shown in a table 9, and the variance analysis is shown in a table 10. By visual analysis, the pairs of factorsThe extraction process of the scutellaria baicalensis has the following main and secondary effects: c (solvent multiple) > A (extraction time) > B (extraction times), and the analysis of variance shows that the factor of C (solvent multiple) has obvious influence on the test result (p)<0.05). The combination of higher levels of all factors is taken to obtain the optimal process A₂B₁C₂I.e. 15 times the amount of water, is extracted under reflux for 2h each time 1 time.

TABLE 9 results of the composite scores of the orthogonal tests

TABLE 10 analysis of variance results

3.1.4.3 validation test

Taking the same batch of Scutellariae radix, and determining according to the above A₂B₁C₂The extraction process of (1) is carried out for 3 times of verification tests, the relative peak area of each characteristic peak is determined according to the chromatographic condition under the item '2.1.2.1' in the embodiment, the inhibition rate of scutellaria baicalensis for inhibiting C.albicans is determined according to the method under the item '2.1.3' in the embodiment, the comprehensive scores are calculated, the scores are respectively 83.23, 82.09 and 81.43, the RSD is 1.1%, and the verification test result shows that the extraction process is stable and feasible.

3.2 study of extraction Process of Chinese medicine folium Eucommiae

3.2.1 determination of optimal extraction Process

The best extraction method of the eucommia leaves is determined according to the method under the item of '2.2.2' in the embodiment, the results are shown in table 11, and the results show that the eucommia leaves subjected to warm-immersion water extraction have a remarkable bacterium promoting effect, the growth rate can reach 131.98%, and the concentration of the liquid medicine is 7.81mg (crude drug amount)/mL, so that the best extraction method of the eucommia leaves is warm-immersion water extraction, the extraction process is 20 times of water, and the warm-immersion water extraction is carried out for 2 hours at 60 ℃.

TABLE 114 statistics of the optimum growth rate of folium Eucommiae to W.cibaria in different extraction methods

Second, research on the mixture ratio of extracts

The scutellaria baicalensis extract is obtained by extracting with an optimal process: i.e. 15 times the amount of water, is extracted under reflux for 1 time, 2h each time.

The eucommia ulmoides leaf extract is extracted by an optimal process to obtain: 20 times of water is soaked in warm water at 60 ℃ for 2 hours.

1. Effect of Scutellaria baicalensis Georgi extract on growth of Candida albicans oral pathogenic bacteria

The MIC (minimum inhibitory concentration) of scutellaria baicalensis to c.albicans, the time-inhibitory rate curve and the MIC results are shown in fig. 8. The results show that the inhibition effect on C.albicans is generated when the concentration of the scutellaria baicalensis liquid medicine is more than or equal to 3.91mg (crude drug amount)/mL, and the inhibition rate on C.albicans is higher than 80% when the concentration of the scutellaria baicalensis liquid medicine is more than or equal to 7.81mg (crude drug amount)/mL.

2. Effect of folium Eucommiae extract on growth of oral probiotic Streptococcus salivarius and Weissella cibaria

The influence of eucommia ulmoides leaves on the growth of the probiotic streptococcus salivarius and weissella civorans is determined, and the time-growth kinetic curves are shown in fig. 9 and fig. 10. The result shows that when the concentration of the eucommia ulmoides leaf extracting solution is less than 62.5mg (crude drug amount)/mL, the influence on Weissella sinus can be small, and the growth rate can reach more than 80%; when the concentration of the folium cortex eucommiae extracting solution is 7.81-15.63 mg (crude drug amount)/mL, compared with a control group, the growth rate of weissella civora is over 100 percent, the growth rate shows an obvious bacteria promoting trend and is obviously higher than that of weissella civora under the action of the folium cortex eucommiae extracting solution with other concentration; when the concentration of the eucommia ulmoides leaf extracting solution is less than 31.25mg (crude drug amount)/mL, the influence on streptococcus salivarius is small, and the growth rate can reach more than 80%; when the concentration of the eucommia ulmoides leaf extracting solution is 7.81-15.63 mg (crude drug amount)/mL, compared with a control group, the growth rate of streptococcus salivarius is over 100 percent, and the remarkable bacterium promoting trend is shown.

3. Effect of Scutellaria baicalensis extract on growth of oral probiotic Streptococcus salivarius and Weissella cibaria

The influence of scutellaria baicalensis with concentration of 7.81-15.63 mg (crude drug amount)/mL on the growth of the probiotic streptococcus salivarius and weissella civorans is measured, and a time-growth kinetic curve is shown in fig. 11 and fig. 12. The result shows that when the concentration of the scutellaria baicalensis extracting solution is 7.81-15.63 mg (crude drug amount)/mL, the growth rate of Weissella cibaria is over 100 percent compared with that of a control group, a remarkable bacteria promoting trend is shown, the growth rate of streptococcus salivarius is about 90 percent, and the scutellaria baicalensis in the concentration range has no influence on the streptococcus salivarius basically.

When the concentration of the scutellaria baicalensis liquid medicine is 3.91-500 mg (crude drug amount)/mL, the scutellaria baicalensis liquid medicine has an inhibition effect on C.albicans; when the concentration of the eucommia ulmoides leaf extracting solution is less than 31.25mg (crude drug amount)/mL, the influence on streptococcus salivarius is small, and the growth rate can reach more than 80%; when the concentration of the eucommia ulmoides leaf extract is less than 62.5mg (crude drug amount)/mL, the influence on Weissella cibaria is small, the growth rate can reach more than 80%, namely the mass ratio of the scutellaria baicalensis to the eucommia ulmoides leaf extract to the crude drug is 1: 16-16: 1. When the concentration of the scutellaria baicalensis is 7.81-15.63 mg (crude drug amount)/mL, the scutellaria baicalensis extract has a remarkable inhibiting effect on pathogenic bacteria candida albicans, and basically has no influence on probiotics; when the concentration of the eucommia ulmoides leaves is 7.81-15.63 mg (crude drug amount)/mL, the eucommia ulmoides leaves have obvious bacterium promoting effect on probiotic streptococcus salivarius and weissella sinus, namely the mass ratio of the scutellaria baicalensis to the eucommia ulmoides leaf extract is 1: 2-2: 1, and the effect of adjusting the balance of oral flora is best.

Example 3 preparation of active ingredients according to the invention

Taking 15g of radix Scutellariae coarse powder, adding 15 times of purified water, reflux extracting for 2h, centrifuging, filtering to clarify, concentrating the filtrate at 60 deg.C under reduced pressure to obtain fluid extract with crude drug mass concentration of about 1g/mL

Taking 15g of eucommia ulmoides leaves, adding 20 times of purified water, extracting for 2h at 60 ℃, centrifuging and filtering until the mixture is clear, and concentrating the filtrate at 60 ℃ under reduced pressure until the mass concentration of crude drugs is about 1 g/mL.

Example 4 preparation of the active ingredients of the invention

Taking 15g of scutellaria baicalensis coarse powder, adding 10 times of purified water, performing reflux extraction for 1.5h, performing reduced pressure filtration to clarify, and performing reduced pressure concentration on the filtrate at 60 ℃ to obtain clear paste with crude drug mass concentration of about 0.8 g/mL;

taking 15g of eucommia ulmoides leaves, adding 15 times of purified water, extracting for 1.5h at the temperature of 60 ℃, filtering under reduced pressure until the mixture is clear, and concentrating the filtrate at the temperature of 60 ℃ under reduced pressure until the mass concentration of crude drugs is about 0.8g/mL of clear paste.

Example 5 preparation of active ingredients according to the invention

Taking 15g of scutellaria baicalensis coarse powder, adding 15 times of purified water, performing reflux extraction for 2.5h, performing centrifugal filtration to clarify, and concentrating the filtrate at 60 ℃ under reduced pressure to obtain clear paste with crude drug mass concentration of about 1.2 g/mL;

taking 15g of eucommia ulmoides leaves, adding 25 times of purified water, extracting for 2.5h at the temperature of 60 ℃, centrifuging and filtering until the mixture is clear, and concentrating the filtrate at the temperature of 60 ℃ under reduced pressure until the mass concentration of crude drugs is about 1.2 g/mL.

Example 6 preparation of active ingredients according to the invention

Taking 15g of scutellaria baicalensis coarse powder, adding 15 times of purified water, extracting for 2h at 60 ℃, filtering to be clear, and concentrating the filtrate at 60 ℃ under reduced pressure to obtain clear paste with crude drug mass concentration of about 1 g/mL;

taking 15g of eucommia ulmoides leaves, adding 20 times of purified water, carrying out reflux extraction for 2h, filtering to clarify, and concentrating the filtrate at 60 ℃ under reduced pressure to obtain clear paste with crude drug mass concentration of about 1 g/mL.

Example 7 preparation of active ingredients according to the invention

Taking 15g of scutellaria baicalensis coarse powder, adding 15 times of purified water, extracting for 2h at 60 ℃, filtering to be clear, and concentrating the filtrate at 60 ℃ under reduced pressure to obtain clear paste with crude drug mass concentration of about 1 g/mL;

taking 15g of eucommia ulmoides leaves, adding 20 times of purified water, carrying out reflux extraction for 2h, filtering to clarify, and concentrating the filtrate at 60 ℃ under reduced pressure to obtain clear paste with crude drug mass concentration of about 1 g/mL.

Example 8 preparation of active ingredients according to the invention

Taking 15g of scutellaria baicalensis coarse powder, adding 15 times of purified water, performing reflux extraction for 2 hours, filtering to clarify, and concentrating the filtrate at 60 ℃ under reduced pressure to obtain clear paste with crude drug mass concentration of about 1 g/mL;

taking 15g of eucommia ulmoides leaves, adding 20 times of purified water, carrying out reflux extraction for 2h, filtering to clarify, and concentrating the filtrate at 60 ℃ under reduced pressure to obtain clear paste with crude drug mass concentration of about 1 g/mL.

Example 9 preparation of active ingredients according to the invention

Taking 15g of radix scutellariae coarse powder, adding 15 times of 60% (V/V) ethanol, performing reflux extraction for 2h, filtering to clarify, and concentrating the filtrate at 60 ℃ under reduced pressure to obtain a clear paste with crude drug mass concentration of about 1 g/mL;

taking 15g of folium Eucommiae, adding 20 times of 40% (V/V) ethanol, extracting at 60 deg.C for 2h, filtering to clarify, and concentrating the filtrate at 60 deg.C under reduced pressure to obtain fluid extract with crude drug mass concentration of about 1 g/mL.

EXAMPLE 10 preparation of the active ingredients of the invention

Taking 10g of radix scutellariae coarse powder, adding 10 times of 70% (V/V) ethanol, performing reflux extraction for 1.5h, filtering to clarify, and concentrating the filtrate at 60 ℃ under reduced pressure to obtain a fluid extract with crude drug mass concentration of about 0.9 g/mL;

taking 15g of folium Eucommiae, adding 15 times of 30% (V/V) ethanol, extracting at 60 deg.C for 1.5h, filtering to clarify, and concentrating the filtrate at 60 deg.C under reduced pressure to obtain fluid extract with crude drug mass concentration of about 0.9 g/mL.

Example 11 preparation of the active ingredients of the invention

Taking 10g of radix scutellariae coarse powder, adding 20 times of 50% (V/V) ethanol, performing reflux extraction for 3h, filtering to clarify, and concentrating the filtrate at 60 ℃ under reduced pressure to obtain a clear paste with crude drug mass concentration of about 1.1 g/mL;

taking 15g of folium Eucommiae, adding 25 times of 35% (V/V) ethanol, extracting at 60 deg.C for 3h, filtering to clarify, and concentrating the filtrate at 60 deg.C under reduced pressure to obtain fluid extract with crude drug mass concentration of about 1.1 g/mL.

EXAMPLE 12 preparation of the active ingredients of the invention

Taking 15g of radix scutellariae coarse powder, adding 15 times of 60% (V/V) ethanol, extracting for 2h at 60 ℃, filtering to be clear, and concentrating the filtrate at 60 ℃ under reduced pressure to obtain clear paste with crude drug mass concentration of about 1 g/mL;

taking 15g of eucommia ulmoides leaves, adding 20 times of 40% (V/V) ethanol, performing reflux extraction for 2h, filtering to clarify, and concentrating the filtrate at 60 ℃ under reduced pressure to obtain a clear paste with the crude drug mass concentration of about 1 g/mL.

Example 13 preparation of the active ingredients of the invention

Taking 15g of radix scutellariae coarse powder, adding 10 times of 70% (V/V) ethanol, extracting at 60 ℃ for 1.5h, filtering to be clear, and concentrating the filtrate at 60 ℃ under reduced pressure to obtain clear paste with crude drug mass concentration of about 1 g/mL;

taking 15g of folium Eucommiae, adding 15 times of 50% (V/V) ethanol, reflux extracting for 1.5h, filtering to clarify, and concentrating the filtrate at 60 deg.C under reduced pressure to obtain fluid extract with crude drug mass concentration of about 1 g/mL.

EXAMPLE 14 preparation of the active ingredients of the invention

Taking 15g of radix scutellariae coarse powder, adding 20 times of 60% (V/V) ethanol, extracting for 3h at 60 ℃, filtering to be clear, and concentrating the filtrate at 60 ℃ under reduced pressure to obtain clear paste with crude drug mass concentration of about 1.1 g/mL;

taking 15g of eucommia ulmoides leaves, adding 25 times of 40% (V/V) ethanol, performing reflux extraction for 3h, filtering to clarify, and concentrating the filtrate at 60 ℃ under reduced pressure to obtain a clear paste with the crude drug mass concentration of about 1.1 g/mL.

Example 15 preparation of active ingredients according to the invention

Taking 15g of scutellaria baicalensis coarse powder, adding 15 times of purified water, performing reflux extraction for 2 hours, filtering to clarify, and concentrating the filtrate at 60 ℃ under reduced pressure to obtain clear paste with crude drug mass concentration of about 1 g/mL;

taking 15g of folium Eucommiae, adding 20 times of 40% (V/V) ethanol, extracting at 60 deg.C for 2h, filtering to clarify, and concentrating the filtrate at 60 deg.C under reduced pressure to obtain fluid extract with crude drug mass concentration of about 1 g/mL.

EXAMPLE 16 preparation of the active ingredients of the invention

Taking 15g of scutellaria baicalensis coarse powder, adding 15 times of purified water, performing reflux extraction for 2 hours, filtering to clarify, and concentrating the filtrate at 60 ℃ under reduced pressure to obtain clear paste with crude drug mass concentration of about 1 g/mL;

taking 15g of eucommia ulmoides leaves, adding 20 times of 40% (V/V) ethanol, performing reflux extraction for 2h, filtering to clarify, and concentrating the filtrate at 60 ℃ under reduced pressure to obtain a clear paste with the crude drug mass concentration of about 1 g/mL.

Example 17 preparation of active ingredients of the invention

Taking 15g of scutellaria baicalensis coarse powder, adding 15 times of purified water, extracting for 2h at 60 ℃, filtering to be clear, and concentrating the filtrate at 60 ℃ under reduced pressure to obtain clear paste with crude drug mass concentration of about 1 g/mL;

taking 15g of folium Eucommiae, adding 20 times of 40% (V/V) ethanol, extracting at 60 deg.C for 2h, filtering to clarify, and concentrating the filtrate at 60 deg.C under reduced pressure to obtain fluid extract with crude drug mass concentration of about 1 g/mL.

EXAMPLE 18 preparation of the active ingredients of the invention

Taking 15g of radix scutellariae coarse powder, adding 15 times of 60% (V/V) ethanol, extracting for 2h at 60 ℃, filtering to be clear, and concentrating the filtrate at 60 ℃ under reduced pressure to obtain clear paste with crude drug mass concentration of about 1 g/mL;

taking 15g of eucommia ulmoides leaves, adding 20 times of 40% (V/V) ethanol, performing reflux extraction for 2h, filtering to clarify, and concentrating the filtrate at 60 ℃ under reduced pressure to obtain a clear paste with the crude drug mass concentration of about 1 g/mL.

EXAMPLE 19 preparation of the active ingredients of the invention

Taking 15g of radix scutellariae coarse powder, adding 15 times of 30% (V/V) ethanol, performing reflux extraction for 2h, filtering to clarify, and concentrating the filtrate at 60 ℃ under reduced pressure to obtain a clear paste with crude drug mass concentration of about 1 g/mL;

taking 15g of eucommia ulmoides leaves, adding 20 times of purified water, extracting for 2h at 60 ℃, standing overnight to remove impurities, filtering to be clear, and concentrating the filtrate at 60 ℃ under reduced pressure to obtain clear paste with crude drug mass concentration of about 1 g/mL.

EXAMPLE 20 preparation of the active ingredients of the invention

Taking 15g of radix scutellariae coarse powder, adding 15 times of 60% (V/V) ethanol, extracting for 2h at 60 ℃, filtering to be clear, and concentrating the filtrate at 60 ℃ under reduced pressure to obtain clear paste with crude drug mass concentration of about 1 g/mL;

taking 15g of eucommia ulmoides leaves, adding 20 times of purified water, carrying out reflux extraction for 2h, filtering to clarify, and concentrating the filtrate at 60 ℃ under reduced pressure to obtain clear paste with crude drug mass concentration of about 1 g/mL.

Example 21 preparation of liquid mouthwash (1.6 g of active extract per 100 mL)

Taking 600mL of purified water, sequentially adding 9.0g of PEG-60 hydrogenated castor oil, 9.0g of xylitol, 1.0g of citric acid, 4.5g of sodium citrate, 2.0g of 2.0gVC and 2.0g of lysozyme, and dissolving to obtain a solution A;

adding 0.7g of mint essence into 20mL of 95% (V/V) edible ethanol for dissolving, dissolving in 200g of glycerol, and adding the solution A into the solution A under stirring to obtain a solution B;

and (3) respectively adding 8mL of the scutellaria baicalensis extract (equivalent to 8g of crude scutellaria baicalensis medicines) and 8mL of the eucommia ulmoides leaf extract (equivalent to 8g of crude eucommia ulmoides leaf medicines) prepared in the example 3 into the solution B, fully and uniformly stirring, adjusting the pH to 5.5 by using a 5% (W/V) sodium citrate aqueous solution, fixing the volume to 1000mL by using purified water, filtering to clarify, and subpackaging under a clean condition to obtain the traditional Chinese medicine composition. And (3) taking a proper amount (10-20 mL of the common dosage) to rinse the mouth as required each time, and spitting out after rinsing.

Example 22 preparation of liquid mouthwash (3.6 g of active extract per 100 mL)

Taking 600mL of purified water, sequentially adding 15.0g of PEG-40 hydrogenated castor oil, 8.5g of xylitol, 1.2g of citric acid, 4.5g of sodium citrate, 2.0gVC and 3.0g of lysozyme, and dissolving to obtain a solution A;

taking 0.5g of mint essence and 0.4g of green tea essence, adding 15mL of 95% (V/V) edible ethanol for dissolving, dissolving in 220g of glycerol, and adding the solution A into the solution B under stirring to obtain a solution B;

and (3) respectively adding 10mL of the scutellaria baicalensis extract (equivalent to 12g of crude scutellaria baicalensis) prepared in example 5 and 20mL of the eucommia ulmoides leaf extract (equivalent to 24g of crude eucommia ulmoides leaf) into the solution B, fully and uniformly stirring, adjusting the pH to 6.4 by using 6% (W/V) sodium citrate aqueous solution, fixing the volume to 1000mL by using purified water, filtering to clarify, and subpackaging under a clean condition to obtain the traditional Chinese medicine composition. And (3) taking a proper amount (10-20 mL of the common dosage) to rinse the mouth as required each time, and spitting out after rinsing.

Example 23 preparation of liquid mouthwash (1.2 g of active extract per 100 mL)

Taking 650mL of purified water, sequentially adding 9.0g of sodium lauroyl sarcosinate, 9.5g of xylitol, 1.2g of citric acid, 4.8g of sodium citrate, 2.3gVC and 3.4g of lysozyme, and dissolving to obtain a solution A;

taking 0.6g of mint essence, adding 10mL of 95% (V/V) edible ethanol, dissolving and dissolving in 200g of glycerol, and adding the solution A into the solution A under stirring to obtain a solution B;

and (3) respectively adding 10mL of the scutellaria baicalensis extract (equivalent to 8g of crude scutellaria baicalensis medicines) and 5mL of the eucommia ulmoides leaf extract (equivalent to 4g of crude eucommia ulmoides leaf medicines) prepared in the embodiment 4 into the solution B, fully and uniformly stirring, adjusting the pH to 4.7 by using 6% (W/V) sodium citrate aqueous solution, fixing the volume to 1000mL by using purified water, filtering to clarify, and subpackaging under a clean condition to obtain the traditional Chinese medicine composition. And (3) taking a proper amount (10-20 mL of the common dosage) to rinse the mouth as required each time, and spitting out after rinsing.

Example 24 preparation of solid mouthwash tablets (5.2 g per 100g active extract-containing composition)

1. Preparation of spray-dried powder of active ingredient

Spray drying the scutellaria baicalensis extract obtained in the example 3 at the air inlet temperature of 130 ℃ and the liquid inlet speed of 10ml/min to obtain scutellaria baicalensis spray-dried powder, and sieving the powder with a 80-mesh sieve for later use;

the eucommia ulmoides extract obtained in the example 3 is taken and spray-dried under the conditions that the air inlet temperature is 150 ℃ and the liquid inlet speed is 10ml/min, and the obtained eucommia ulmoides spray-dried powder is sieved by a sieve of 80 meshes for later use.

2. Preparation of solid mouthwash tablets

Mixing 0.7g Scutellariae radix spray-dried powder (equivalent to Scutellariae radix crude drug 3.9g) and 0.2g Eucommiae cortex spray-dried powder (equivalent to Eucommiae cortex leaf crude drug 1.3g), sequentially adding 0.1g sodium dodecyl sulfate, 2.0g herba Menthae essence, 2.0g xylitol, 0.02g saccharin sodium, 22.5g citric acid, 46.9g lactose, 22.5g NaHCO₃And 3.0g of PEG6000 are sieved and mixed until the color is uniform, the powder is directly tabletted, and the weight of the tablet is 1g, so that 100 solid mouthwash tablets are obtained. Taking 1-3 tablets (usually 1 tablet), adding appropriate amount of water (usually about 10-20mL), and dissolving to obtain liquid collutory.

Example 25 preparation of solid mouthwash tablets (3.4 g per 100g active extract-containing composition)

1. Preparing active ingredient vacuum drying powder

The extracts of the scutellaria baicalensis and the eucommia ulmoides leaves obtained in the example 5 are respectively dried under reduced pressure at the temperature of 60 ℃, crushed and sieved by a 80-mesh sieve to respectively obtain scutellaria baicalensis extract powder and eucommia ulmoides leaf extract powder for later use.

2. Preparation of solid mouthwash tablets

Taking 0.5g of radix Scutellariae, and subtractingPressing dry powder (2.8 g as crude drug of radix Scutellariae), 0.1g folium Eucommiae vacuum dry powder (0.6 g as crude drug of folium Eucommiae), mixing with 25g citric acid, 0.1g sodium dodecyl sulfate, 2.0g herba Menthae essence, 2.0g xylitol, and 0.02g saccharin sodium, making soft mass with 70% ethanol, sieving with 20 mesh sieve, and granulating to obtain granule 1; another 25g of NaHCO was taken₃Mixing 92.5g of lactose, preparing a soft material by 70% ethanol, sieving with a 20-mesh sieve, and granulating to obtain granules 2; taking the granules 1 and 2, adding 3.0g of PEG6000, mixing uniformly, pressing into triangular tablets by using a special-shaped die, wherein the tablet weight is 1.5g, and obtaining 100 solid mouthwash tablets.

EXAMPLE 26 preparation of solid mouthwash tablets (5.9 g per 100g active extract-containing composition)

1. Preparation of active ingredient lyophilized powder

The scutellaria baicalensis and eucommia ulmoides extracts obtained in the example 3 are respectively pre-frozen at the temperature of minus 40 ℃, then placed in a freeze dryer, freeze drying is carried out at the temperature of minus 45 ℃ and the vacuum degree of 0.06kPa in a cold trap, and the scutellaria baicalensis freeze-dried powder and the eucommia ulmoides freeze-dried powder which are respectively obtained are sieved by a sieve of 80 meshes for later use.

2. Preparation of solid mouthwash tablets

Mixing 0.7g of lyophilized Scutellariae radix powder (4.7 g of crude drug equivalent to Scutellariae radix), 0.35g of lyophilized Eucommiae cortex powder (1.2 g of crude drug equivalent to folium Eucommiae), 26.5g of citric acid, 0.1g of sodium laurylsulfate, 2.0g of herba Menthae essence, 3.0g of xylitol, and 0.02g of saccharin sodium, making soft mass with 75% ethanol, sieving with 20 mesh sieve, and granulating to obtain granule 1; another 25g of NaHCO was taken₃Mixing with 39.3g of maltodextrin, preparing a soft material by 70% of ethanol, sieving with a 20-mesh sieve, and granulating to obtain granules 2; taking granule 1 and granule 2, adding 3.0g PEG6000, mixing, and tabletting with punch with diameter of 12 mm to obtain solid gargle tablet with weight of 0.8g, and making into 125 tablets.

Example 27 preparation of other oral Care products (12 g of active extract per 100g of active extract)

Dissolving 0.12g saccharin sodium, 0.24g lysozyme, 4.0mL Scutellariae radix extract (prepared by example 18 in a proportion of 4g crude drug to Scutellariae radix), and 8.0mL folium Eucommiae extract (prepared by example 17 in a proportion of 8g crude drug to folium Eucommiae) in about 20mL purified water to obtain solution A;

taking 28g of glycerol and dispersing 0.36g of titanium dioxide to obtain a mixed solution B;

dispersing 0.75g of cellulose gum and 0.35g of xanthan gum by 6g of polyethylene glycol-400 to obtain a mixed solution C;

sequentially adding the solution A, the mixed solution B and the mixed solution C into a homogenizer respectively, and stirring for 30min to prepare a mixed solution D;

adding 20g of silicon dioxide into the mixed solution D, homogenizing and stirring for 20min again, adding 1.5g of sodium dodecyl sulfate, adding water to adjust the total weight to 100g, continuing stirring for 15min, adding 1.5g of mint essence, stirring for 5min, vacuumizing for 15min, taking out the paste, pumping into a filling machine, and filling to obtain the traditional Chinese medicine.

Example 28 preparation of other oral Care products (active extract contained per 100g, equivalent to crude drug amount 15.6g)

Dissolving 0.12g saccharin sodium, 0.26g lysozyme, 8.0mL Scutellariae radix extract (prepared by the method of example 20, reduced to 8g crude drug of Scutellariae radix), and 8.0mL folium Eucommiae extract (prepared by the method of example 19, reduced to 8g crude drug of folium Eucommiae) in about 20mL purified water to obtain solution A;

taking 24g of glycerol and dispersing 0.36g of titanium dioxide to obtain a mixed solution B;

dispersing 0.72g of cellulose gum and 0.36g of xanthan gum by 6g of polyethylene glycol-400 to obtain a mixed solution C;

sequentially adding the solution A, the mixed solution B and the mixed solution C into a homogenizer respectively, and stirring for 30min to prepare a mixed solution D;

adding 25g of silicon dioxide into the mixed solution D, adding water to adjust the total weight to be 100g, homogenizing and stirring for 20min again, adding 1.5g of sodium dodecyl sulfate, continuing stirring for 15min, adding 1.2g of mint essence, stirring for 5min, vacuumizing for 15min, taking out the paste, pumping into a filling machine, and filling to obtain the compound.

Example 29 use and application forms of the Natural active substance compositions

1. Apparatus and materials

1.1 instruments

HCB-900V vertical laminar flow clean bench (Qingdao Haier Special appliances Co., Ltd.); SW-CJ-JF clean bench (Suzhou purification plant); HPX-9052MBE electric heating constant temperature incubator (Shanghai Bochen industry Co., Ltd.); BSD-TX345 desktop shaker (Shanghai Boxun industries, Inc. medical facilities); an infinite M200PRO microplate reader (TECAN, USA); EYELA N-1210B rotary evaporator (Shanghai Elang instruments, Inc.); EYELA-1000S circulating water type vacuum pump (Tokyo physical and chemical instruments Co., Ltd.); FA124 electronic balance (tianjin yinuo scientific instruments ltd); LS-B50L vertical pressure steam sterilization pot (Shanghai medical nuclear instrument factory).

1.2 materials

Streptococcus salivarius (Streptococcus salivarius, s.salivarius, ATCC13419) was purchased from the guangdong province collection of microorganisms; porphyromonas gingivalis (p. gingivalis, ATCC33277), Fusobacterium nucleatum (f. nucleatum, ATCC10953), Candida albicans (Candida albicans, c. albicans, ATCC1231) were purchased from north nana biotechnology limited; weissella cibaria (weissella. cibaria, w. cibaria, CPCC101289) was purchased from the chinese pharmaceutical microbial strain collection management center; columbia blood plates (batch: 352241) were purchased from North Na Biotechnology Ltd; BHI broth (LA0360), MRS broth (M8540), nutrient agar powder (A8190), modified Martin agar medium (1108S304), hemin solution (H8132), and vitamin K3(V8170) all provided by Beijing Solelaibao Biotechnology, Inc.; modified Martin Medium (M4101B) was supplied by Shandong Tuo general bioengineering, Inc.; anaerobic gas-generating bags, anaerobic culture bags (trigonal gas chemical co., ltd. japan); yeast Extract Yeast powder LP0021 (OXOID); folium Eucommiae is provided by Jiangxi Puzheng pharmaceutical Co Ltd; the wild chrysanthemum flower, the dandelion and the scutellaria baicalensis decoction pieces are all provided by republic of agriculture and drug industry of Hebei Anguo; radix astragali, rhizoma Atractylodis Macrocephalae, fructus Lycii, radix Glycyrrhizae, radix Campylotropis Hirtella, cortex Magnoliae officinalis, and flos Caryophylli from Hovenia drugstore in Tianjin; lysozyme (pharmaceutical grade, 20190126) was supplied by wanbang industries ltd, south china; the alkaline phosphatase kit A059-2, the potassium ion quantitative kit (C001-2-1) and the total protein quantitative kit (A045-4) are all built by Nanjing to the bioengineering institute; a blank adjuvant group, huangdu oral conditioning liquid (prepared according to the method of example 21); yixin Piraconium chloride gargle (batch: K1906282) was purchased from Hangzhou Minsheng pharmaceutical Co., Ltd; gaolujie beige mouthwash (0035TH 1113); yeast Extract Yeast powder LP0021 from OXOID; compound chlorhexidine gargle (20190616, Shenzhen Nanyue pharmaceutical Co., Ltd.); jiajieshi mouthwash for treating gingival red swelling and bleeding (00735395UB, Guangzhou Baojie Co., Ltd.); shuke (M0150337, Guangzhou Wei Meizi practice Co., Ltd.); europe B (92345395UA, Guangzhou Baojie, Inc.); binah's mouthwash (741-18L053, McKerley technologies, Shenzhen); happy tooth bright white clear mouthwash (20190411, prosperous potential reaching International trade (Shanghai) Limited); haoyijian sodium bicarbonate fresh and whitening mouthwash (2019090601, Guangdong Kangwang Rihua Co., Ltd.); a lisford mouthwash (C110L, qiangsheng (china) ltd); lion king fine tooth gingival cleaning care (190603CA, lion king daily chemical (Qingdao) ltd); blackman dual mint mouthwash (20190831HH4121, good chemical (zhongshan) ltd); OKINA mouthwash (20190401, OKINA corporation, Japan).

2. Experimental methods

2.1 in vitro Effect of the Conditioning liquid of the invention on oral flora

Effects on pathogenic bacteria c, albicans, f, nuclearum and probiotic bacteria w, cibaria, s

The influence of the conditioning solution (prepared by the method of example 21), blank auxiliary materials and commercially available mouthwash on oral flora was determined, and 100 μ L of the bacterial suspension was added to a 96-well plate, and then 100 μ L of the conditioning solution of the present invention and the commercially available mouthwash preparation were added to form an experimental group; simultaneously setting a control group (without adding drugs), a negative control (without adding bacteria) and a blank control (culture medium), filling up less than 200 mu L of final volume in the hole by using a corresponding liquid culture medium, measuring OD600 by using the fusobacterium nucleatum at 0, 2, 4, 6, 8, 10, 12, 24, 36 and 48h, measuring the OD600 by using the other bacteria at intervals of 2h, carrying out parallel measurement for 3 times in the experiment, and drawing a time-growth curve by using time as a horizontal coordinate and growth rate as a vertical coordinate.

Growth rate calculation formula:

the formula for calculating the bacteriostasis rate is as follows:

2.2 study of action mechanism of conditioning liquid and commercial mouthwash of the invention on oral flora

2.2.1 Effect of Conditioning solutions of the invention and commercial mouthwashes on bacterial cell wall integrity

And (3) taking 30mL of bacterial suspension which is activated and cultured to logarithmic phase, placing the bacterial suspension in a sterile centrifuge tube, centrifuging for 10min at 4 ℃ under the condition of 4000r/min, and collecting thalli precipitates. Washing the collected thallus precipitate for 3 times by using a sterilized PBS buffer solution, re-suspending in 5mL of PBS buffer solution, and respectively adding 5mL of blank auxiliary materials into the thallus suspension to serve as negative control, wherein the conditioning solution is an experimental group; taking a bacterial suspension added with 5mL of cetylpyridinium chloride gargle and Kolojie gargle as a positive control; taking PBS bacterial suspension added with 5mL of sterile water as a blank control; and simultaneously placing the bacterial suspension in a constant temperature incubator, sampling at regular time, sampling at intervals of 1 hour until 8 hours, centrifuging for 10 minutes at 4 ℃ under the condition of 4000r/min, taking the supernatant, measuring the OD value of the supernatant at 520nm by using an enzyme labeling instrument according to the operation method of the alkaline phosphatase kit, calculating the content of extracellular alkaline phosphatase, and carrying out experiment parallel determination for 3 times.

2.2.2 Effect of the Conditioning liquid of the invention and commercial mouth washes on the integrity of the bacterial cell membranes (content of small molecular substance potassium ion in the extracellular supernatant of the bacteria)

And (3) taking 30mL of bacterial suspension which is activated and cultured to logarithmic phase, placing the bacterial suspension in a sterile centrifuge tube, centrifuging for 10min at 4 ℃ under the condition of 4000r/min, and collecting thalli precipitates. Washing the collected thallus precipitate for 3 times by using a sterilized PBS buffer solution, re-suspending in 5mL of the PBS buffer solution, and respectively adding 5mL of blank auxiliary material gargle serving as a negative control into the thallus suspension, wherein the conditioning solution is an experimental group; taking a bacterial suspension added with 5mL of cetylpyridinium chloride gargle and Kolojie gargle as a positive control; taking PBS bacterial suspension added with 5mL of sterile water as a blank control; and (3) simultaneously placing the bacterial suspensions in a constant-temperature incubator, sampling at regular time, sampling at intervals of 1 hour until 8 hours, centrifuging for 10 minutes at 4 ℃ under the condition of 4000r/min, taking supernate, detecting the content of potassium ions in the bacterial suspensions by using an enzyme labeling instrument according to the operation method of a potassium ion kit, and performing parallel determination for 3 times.

2.2.3 Effect of Conditioning solutions of the invention and commercial mouthwashes on the content of bacterial cells (protein content in extracellular supernatants of bacteria)

And (3) taking 30mL of bacterial suspension which is activated and cultured to logarithmic phase, placing the bacterial suspension in a sterile centrifuge tube, centrifuging for 10min at 4 ℃ under the condition of 4000r/min, and collecting thalli precipitates. Washing the collected thallus precipitate for 3 times by using a sterilized PBS buffer solution, re-suspending in 5mL of PBS buffer solution, and respectively adding 5mL of blank auxiliary materials into the thallus suspension to serve as negative control, wherein the conditioning solution is an experimental group; taking a bacterial suspension added with 5mL of cetylpyridinium chloride gargle and Kolojie gargle as a positive control; taking PBS bacterial suspension added with 5mL of sterile water as a blank control; and simultaneously placing the bacterial suspension in a constant-temperature incubator, sampling at regular time, sampling at intervals of 1 hour until 8 hours, centrifuging for 10 minutes at 4 ℃ under the condition of 4000r/min, and determining the protein content of the supernatant, namely the extracellular soluble protein content. The assay was performed with reference to the total protein quantification kit instructions. The experiment was repeated 3 times and the average was calculated.

2.3 in vivo Effect of Conditioning liquid of the invention on oral flora

2.3.1 materials and methods

2.3.1.1 Material

Traditional Chinese medicine oral conditioning liquid: prepared by the method of example 21

2.3.1.2 study object

19 healthy adults aged 20-60 years had good health condition of the subjects, clear consciousness, normal handling behavior ability, no serious systemic diseases such as autoimmune diseases and the like, no oral treatment and antibiotic use in 3 months, and non-pregnant or lactating women. The study was approved by the ethical committee of Tianjin Chinese medicine university and subjects signed an informed consent.

2.3.2 methods of investigation

2.3.2.1 sample Collection

The subject collected 30mL of saline gargle in 50mL of sterile tube, frozen at-20 ℃ and stored in-80 ℃ refrigerator under protection of ice bag (transfer time is not more than 4 hours) on the first day and before tooth brushing and mouth rinsing after the eighth morning after applying the test sample (the conditioning solution, the conditioning solution blank adjuvant solution and the non-test sample in the invention) for one week. The samples were sent to Beijing Nuo Yoghuan bioinformatics technology, Inc. for high-throughput sequencing of the oral microorganisms 16SrDNA and ITS (transport time was no longer than 4 hours). The samples to be tested are used as the conditioning solution and the blank auxiliary material solution of the conditioning solution prepared by the invention, and the samples which do not use any samples to be tested are respectively marked as a conditioning solution group, a blank auxiliary material group and an unabated group of the conditioning solution prepared by the invention, wherein volunteers of the blank auxiliary material group and the unabated group are volunteers without oral problems.

2.3.2.2 sequencing data processing

Splitting each sample data from off-line data according to the Barcode sequence and the PCR amplification primer sequence, cutting off the Barcode and the primer sequence, splicing reads of each sample by using FLASH (V1.2.7), and obtaining a spliced sequence which is original Tags data (Raw Tags); the Raw Tags are filtered to obtain high-quality tag data (Clean Tags). Referring to the Tags quality control flow of QIIME (V1.9.1), the following operations were performed: a) and (5) intercepting Tags: truncating Raw Tags from the first low-quality base site of consecutive low-quality values (default quality threshold of 19) base numbers to a set length (default length value of 3); b) tags length filtration: and further filtering the Tags after the Tags are intercepted, wherein the length of continuous high-quality bases is less than 75% of the length of the Tags. The Tags obtained through the above processing needs to be processed to remove a chimera sequence, the Tags sequence is compared with a database through a UCHIME algorithm to detect the chimera sequence, and the chimera sequence is finally removed to obtain final Effective data (Effective Tags).

2.3.2.3 bioinformatics analysis

According to the characteristics of the amplified 16S and ITS regions, the small fragment library is constructed for double-End sequencing by using a double-End sequencing (Paired-End) method based on an Illumina NovaSeq sequencing platform. Performing reading splicing filtration, OTUs (operational Taxonomic units) clustering, performing species annotation and abundance analysis, and revealing the species composition of the sample; further Alpha Diversity analysis (Alpha Diversity) can mine the differences between samples.

(1) OTU clustering and species annotation

All Effective Tags of all samples were clustered by Upearse software (Upearse v7.0.1001), the sequences were clustered into OTUs with 97% Identity as default, and the sequences with the highest frequency of occurrence among the OTUs were selected as representative sequences of the OTUs. Species annotation analysis (with a threshold of 0.8-1) was performed on OTUs representative sequences by using a Mothur method and a SSUrRNA database of SILVA (http:// www.arb-SILVA. de /) to obtain the colony composition of each sample in kingdom, phylum, class, order, family, genus, and species at each classification level. The phylogenetic relationship of OTUs representative sequences was obtained by alignment using MUSCLE (Version3.8.31) software. And finally, carrying out homogenization treatment on the data of each sample by taking the data with the minimum data amount in the sample as a standard, wherein the subsequent Alpha diversity analysis is based on the data after the homogenization treatment.

(2) Sample complexity analysis (Alpha Diversity)

Alpha Diversity is used to analyze microbial community Diversity within a sample. The complexity of the sample species Diversity was first analyzed by taking statistics of the Alpha Diversity analysis index (shannon) of different samples at the 97% consistency threshold.

(3) Clustering analysis of species abundance

According to the species annotation result, the different species of each sample or group among groups under various classification levels (phylum, class, order, family, genus and species) are selected, and the result of the species level is displayed by default.

2.3.2.4 statistical analysis of data

The experimental result is expressed by x +/-s, the test is carried out by SPSS 25.0 software among groups, the abundance data of bacteria before and after the conditioning solution is used are compared and analyzed by adopting a paired t test, the difference significance between the groups is judged by adopting variance analysis, the comparison between the groups adopts LSD test, the difference P is less than 0.05, and the difference P is less than 0.01, so that the statistical significance is achieved.

3. Results of the experiment

3.1 in vitro Effect of the Conditioning liquid of the invention on oral flora

The conditioning liquid, the blank auxiliary materials and the commercial mouthwash of the invention have the influence on pathogenic bacteria C.albicans and F.nuclear and probiotics W.cibaria and S.salivarius

The effect of the conditioning solution of the present invention, the blank excipients and the commercial mouth rinse on oral flora was examined by the broth dilution method, and the time-growth rate curves thereof are shown in fig. 13, fig. 14, fig. 15 and fig. 16.

The result shows that compared with a control group, the conditioning liquid has obvious inhibiting effect on oral cavity pathogenic bacteria, namely candida albicans and fusobacterium nucleatum, and basically has no influence on the growth of oral cavity probiotic bacteria, namely weissella antrorum and streptococcus salivarius, so that after the conditioning liquid is used, the proportion of the oral cavity probiotic bacteria can be increased, the effect of adjusting the balance of oral flora is achieved, the blank auxiliary material has weaker inhibiting effect on the oral cavity pathogenic bacteria and stronger inhibiting effect on the oral cavity probiotic bacteria, and the blank auxiliary material shows the tendency that the blank auxiliary material cannot adjust the balance of the oral flora; the rest of mouthwash has obvious inhibition effect on pathogenic bacteria and probiotics in the oral cavity, and after the mouthwash is applied to the oral cavity, the number of the pathogenic bacteria is reduced, the probiotics is reduced, and the effect of regulating the oral flora cannot be achieved.

3.2 research on action mechanism of conditioning liquid and commercial mouthwash on oral flora

3.2.1 Effect of Conditioning liquid of the invention and commercial mouth washes on the integrity of bacterial cell walls (AKP enzyme Activity)

The determination results of the activity change of alkaline phosphatase in the extracellular supernatant of the bacterium suspension after each mouthwash acts on pathogenic bacteria candida albicans, fusobacterium nucleatum and porphyromonas gingivalis are shown in the following figure 17, figure 18 and figure 19.

As can be seen from the figure, the activities of the AKP enzyme in the extracellular supernatant of Candida albicans, Fusobacterium nucleatum and Porphyromonas gingivalis under the action of the mouthwash are all increased to different degrees; and the AKP enzyme activity in the supernatant of the thalli of the blank control group is basically unchanged, namely the cell wall of the blank control group is complete. The traditional Chinese medicine conditioning liquid acts on various time points of candida albicans, fusobacterium nucleatum and porphyromonas gingivalis, the AKP enzyme activity in the supernatant is obviously improved (P is less than 0.01) compared with that of a negative control group AKP enzyme activity, and the effect that the main effect natural active substance composition of the huangdu in the conditioning liquid destroys the integrity of the cell walls of the pathogenic bacteria candida albicans, fusobacterium nucleatum and porphyromonas gingivalis is stronger; compared with a blank control group, the AKP enzyme activity at each time point after the conditioning liquid and the high-dew cleaning mouthwash act has very significant difference (P is less than 0.01), and after 5 hours of action of the blank auxiliary material mouthwash and 4 hours of action of the cetylpyridinium chloride mouthwash, the AKP enzyme activity is significantly higher than that of the blank control group (P is less than 0.01); after each group of mouth wash acts on candida albicans, fusobacterium nucleatum and porphyromonas gingivalis thalli, the most obvious increase of AKP enzyme activity in the supernatant is the conditioning liquid, then the mouth wash is the high-dew cleaning mouth wash, the least obvious mouth wash is the cetylpyridinium chloride mouth wash, and the strong and weak sequence of the way of increasing the activity of the AKP enzyme in the extracellular direction and playing the bacteriostatic action by destroying the cell wall integrality of the candida albicans, the fusobacterium nucleatum and the porphyromonas gingivalis is the traditional Chinese medicine conditioning liquid > high-dew cleaning mouth wash > cetylpyridinium chloride mouth wash in sequence.

3.2.2 Effect of the Conditioning liquid of the invention and commercial mouth washes on the integrity of bacterial cell membranes (content of small molecular substance potassium ion in extracellular supernatant of bacteria)

The content change of potassium ion in the extracellular supernatant of the bacteria suspension after each collutory acts on pathogenic bacteria such as Candida albicans, Fusobacterium nucleatum and Porphyromonas gingivalis is shown in figure 20, figure 21 and figure 22.

As can be seen from the figure, under the action of the mouthwash, the potassium ion content of candida albicans, fusobacterium nucleatum and porphyromonas gingivalis thalli in the extracellular supernatant is increased to different degrees; in a certain time range, the longer the action time of the mouthwash is, the more obvious the potassium ion leakage phenomenon is; the potassium ion content in the extracellular supernatant of the thalli of the blank control group is basically unchanged, namely the cell membrane of the blank control group is complete. The conditioning liquid acts on various time points of candida albicans, fusobacterium nucleatum and porphyromonas gingivalis, the content of potassium ions in extracellular supernatant is obviously increased (P is less than 0.01) compared with the content of potassium ions in a negative control mouth wash group, and the condition shows that the natural active substance composition with the main effects of the xanthate in the conditioning liquid has stronger effect of destroying the integrality of cell membranes of the candida albicans, the fusobacterium nucleatum and the porphyromonas gingivalis; compared with a blank control group, the potassium ion content of each time point after the conditioning solution, the cetylpyridinium chloride mouthwash and the high-dew cleaning mouthwash act has very significant difference (P is less than 0.01), but under the action of the conditioning solution, the initial potassium ion content in the supernatant is the highest, and the change of the potassium ion content in the supernatant is the most significant, namely the conditioning solution plays the most significant role in bacteriostasis by destroying the cell membrane integrality of candida albicans, fusobacterium nucleatum and porphyromonas gingivalis.

3.2.3 Effect of Conditioning solutions of the invention and commercial mouthwashes on the content of bacterial cells (protein content in extracellular supernatants of bacteria)

The results of measuring the protein content change in the extracellular supernatant of the bacterial suspension after each mouthwash acts on pathogenic bacteria candida albicans, fusobacterium nucleatum and porphyromonas gingivalis are shown in fig. 23, fig. 24 and fig. 25.

As can be seen from the figure, the protein content of Candida albicans, Fusobacterium nucleatum and Porphyromonas gingivalis thalli in the extracellular supernatant liquid is increased to different degrees under the action of the mouthwash; the protein content in the supernatant of the blank control group thalli is basically unchanged. Along with the prolonging of the action time of the conditioning liquid, the protein content in the extracellular supernatant of candida albicans, fusobacterium nucleatum and porphyromonas gingivalis is increased most obviously, and the protein content in the extracellular supernatant is obviously increased (P is less than 0.05) compared with the protein content of a mouthwash blank auxiliary material when the conditioning liquid acts on each time point of the candida albicans, the fusobacterium nucleatum and the porphyromonas gingivalis, so that the main efficacy natural active substance composition in the conditioning liquid has stronger effect of inhibiting the synthesis of the proteins of pathogenic bacteria candida albicans, fusobacterium nucleatum and porphyromonas gingivalis; compared with a blank control group, the protein content of each time point after the action of the traditional Chinese medicine conditioning solution has very significant difference (P <0.01), and the protein content of each time point after the action of the cetylpyridinium chloride mouthwash and the high-dew cleaning mouthwash group has significant difference (P < 0.05); under the action of the conditioning liquid, the initial protein content in the supernatant is the highest, namely the conditioning liquid has stronger bacteriostatic action by inhibiting the synthesis way of the proteins of candida albicans, fusobacterium nucleatum and porphyromonas gingivalis. 3.3 in vivo Effect of Conditioning liquid of the invention on oral flora

3.3.1 Flora diversity analysis- -alpha diversity analysis

Alpha diversity analysis refers to the diversity within a particular area or ecosystem, and measures the diversity of species within a community. The Shannon index is used to estimate microbial diversity, with a greater Shannon index value indicating greater species diversity and a Shannon index difference (. DELTA.) reflecting the variation in diversity. The results are shown in table 12, and the Shannon index difference (delta) of the conditioning solution has no significant difference (P >0.05) with the non-dried preparation group before and after the conditioning solution is applied, and the variation of the Shannon index difference of the oral flora of each volunteer is within the fluctuation range of the non-dried preparation group; before and after the blank auxiliary material is applied for one week, the Shannon index is obviously reduced (delta is negative), the Shannon index difference is obviously lower than that of an unaged group (P is less than 0.05), the diversity of oral flora is reduced by the auxiliary material solution, and the auxiliary material components are common components of oral cavity mouth wash products. The experimental result shows that the conditioning liquid has mild effect, wherein the composition of the extracts of the Chinese goldthread and the ledum bugle plays a role in maintaining the stability of the microecological diversity of the oral cavity, can maintain the diversity of oral flora in a normal range, and plays a potential role in protecting the normal physiological function of the oral cavity.

TABLE 12 alpha diversity study of oral microbial flora before and after use of the conditioner of the invention

Note: Δ ═ shannon index value (after use) -shannon index value (before use).

3.3.2 structural analysis of the flora

3.3.3.1 based on species-level species relative abundance analysis

Studies have shown that the most prominent pathogenic bacteria causing oral malodor are moraxella (Solobacterium moorei), and suspected pathogenic bacteria are porphyromonas gingivalis, Prevotella intermedia (Prevotella intermedia), Fusobacterium nucleatum (Fusobacterium nuclear), etc.; the main pathogenic bacteria inducing periodontitis (gingivitis) are Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum, Prevotella melanogenes (Prevotella melaninogenica), Fossistana sp. Candida albicans is a conditionally pathogenic fungus present in the oral cavity that induces oral ulcers. Researches find that oral probiotics such as streptococcus salivarius and Lactobacillus salivarius have an inhibiting effect on pathogenic bacteria related to oral odor, and the quantity of oral probiotics such as Lactobacillus salivarius, Lactobacillus reuteri and Lactobacillus fermentum is increased to have a treatment effect on periodontitis (gingivitis). Researches show that the Weissella cibaria and the streptococcus salivarius have bacteriostatic activity on Candida albicans.

The statistical results are shown in fig. 26 and fig. 27, after the traditional Chinese medicine conditioning liquid is applied, the relative abundance values of pathogenic bacteria related to periodontitis (gingivitis), namely, porphyromonas gingivalis, fusobacterium nucleatum, prevotella intermedia, fosfomentin and prevotella melanogenes are reduced to different degrees; the relative abundance value of the most main pathogenic bacterium, namely the Moraxella, related to the oral odor is reduced; the relative abundance value of the conditional pathogenic fungus candida albicans related to the oral ulcer is obviously reduced; for the oral cavity probiotics, the relative abundance values of the other oral cavity probiotics are increased except that the relative abundance value of the Weissella cibaria is slightly reduced. The traditional Chinese medicine conditioning liquid has an inhibiting effect on main pathogenic bacteria of periodontitis (gingivitis), oral odor and oral ulcer, and can also up-regulate the proportion of probiotics related to the two oral diseases, so that the traditional Chinese medicine conditioning liquid has a certain prevention and treatment effect on the periodontitis (gingivitis), the oral odor and the oral ulcer by regulating the balance of oral flora.

3.3.3.2 analysis of relative abundance of Candida albicans in oral cavity of volunteer before and after use of the conditioning liquid

As shown in Table 13, the changes in relative abundance of Candida albicans, a pathogenic bacterium for oral ulcer, before and after use of the conditioning solution of the present invention were compared. Wherein 9, 10 and 12 volunteers are oral health volunteers, the initial relative abundance value of the candida albicans is low, and the relative abundance of the candida albicans has no obvious change after the conditioning liquid is used; 8. 11, the initial relative abundance value of the candida albicans is obviously higher when the oral ulcer is suffered, and the relative abundance value of the candida albicans is obviously reduced after the conditioning liquid is used. The result shows that the conditioning liquid has small influence on normal oral bacteria, but can obviously reduce the relative abundance level of the abnormally high conditioned pathogens, and is beneficial to the balance of the oral bacteria and the recovery of the abnormal state.

TABLE 13 comparison of the relative abundance of Candida albicans before and after use of the conditioning fluid of the present invention

Note: q: before using the conditioning solution, H: after the conditioning liquid is used

Example 30 evaluation of product Effect

1. Experimental methods

(1) A preparation period: volunteer screening work was performed 14d prior to the trial. Excluding pregnant women, lactating women and patients with mental diseases, and hormone therapy and antibiotic therapy are being performed. Wherein, the male is 7 cases, and the female is 12 cases; the age is 18-80 years old, wherein 5 persons have bad breath (halitosis), 4 persons have canker sore, 2 persons have periodontal and gingival swelling and pain, 1 person has pharyngitis discomfort, and 7 persons have other daily cleanings.

(2) The gargle comprises: in the liquid mouth wash prepared in example 21, the mass ratio of the scutellaria baicalensis extract to the eucommia ulmoides leaf extract in terms of crude drug was 1:1, and the sum of the two extracts in terms of crude drug mass accounted for 1.6% (W/V) of the oral flora polymorphism regulator as a whole.

(3) The gargling method comprises the following steps: gargling 3 times a day, gargling 1min each time after eating in the morning, noon and evening, 15mL each time, and no food can be eaten within 1h after gargling. During the test period, the consumption of sticky or high-sugar food was avoided, and chewing gum, drinking alcohol and drinking beverage were not possible.

(4) And (3) test period: in 19 cases, the treatment results were observed by using 7 days continuously according to the above-mentioned method.

2. Results of the experiment

Oral malodor (halitosis): all 5 people were eliminated;

oral ulcer: when the oral cavity is kept clean, burning pain at the ulcer part after gargling is relieved, the pain is obviously relieved after 3-4 times of use, the burning pain basically disappears after 2 days of use, the color of the ulcer part becomes light, the red swelling disappears, 4 persons of oral cavity ulcer are basically healed, and normal tooth brushing can be resumed after 3 days without generating touch pain;

periodontal, gingival swelling and pain: the swelling and pain can be obviously relieved after the medicine is used, and meanwhile, the halitosis caused by inflammation is obviously relieved;

pharyngitis: although the problem of oral cleaning can be solved by tooth brushing, sore throat and sticky and greasy tongue coating still exist, the sore feeling is reduced after the mouthwash is used, and the sticky and greasy tongue coating is obviously improved.

And others: daily cleaning is used for cleaning oral cavities after meals, the mouth smell is clear, the mouth feel is fresh, sweet and comfortable, and the oral cavity cleaning agent is a convenient choice for oral cavity nursing such as tooth brushing and the like.