CN108210385B - A jelly-like mouthwash with anti-caries, antibacterial and strong root tooth-fixing effects - Google Patents

Abstract

The invention discloses a jelly-shaped mouthwash with the functions of preventing caries, antibacterial and strong root and tooth fixation. The mouthwash comprises moisturizing ingredients 3%-5%, solvent 91%-95% and acid-base buffer 0.25%-0.75% , anti-caries ingredient 0.2%-0.6%, strong root ingredient 0.05%-0.15%, flavor ingredient 0.2%-0.6%, antiseptic ingredient 0.1%-0.2%, fragrance 0.25%-0.35%, tooth-fixing ingredient 0.01%-0.03 %, the gel component 0.4%-0.8%, and the anti-caries component is selected from one of compounds LX-S-06, LX-S-18, XQH-2-92, XQH-3-6, XQH-3-7 Or more, the strong root ingredient is quercetin. The present invention integrates the functions of preventing caries, antibacterial, and strong root and tooth-fixing, and is comfortable to use, without any toxic and side effects, and is suitable for long-term use.

Description

A jelly-like mouthwash with anti-caries, antibacterial and strong root tooth-fixing effects

technical field

The invention relates to the technical field of oral health care, in particular to a mouthwash, in particular to a jelly-like mouthwash with anti-caries and antibacterial properties, strong roots and teeth, and a preparation method thereof.

Background technique

Caries, commonly known as tooth decay, is a bacterial infection. The etiology of caries can be explained by the "quadruple factor theory", that is, the four interrelated factors of bacteria, oral microenvironment, host and time. Caries is mainly caused by cariogenic microorganisms, which form a microenvironment that destroys enamel by forming dental plaque biofilms in difficult-to-clean parts such as tooth crowns. The prevalent parts of caries are very hidden, mainly including pit and fissures, adjacent surfaces and neck, and these parts are also difficult to clean by traditional brushing. Oral health can be protected with oral care cleansers while also eliminating bad breath, and with anti-caries ingredients, it inhibits the growth of cariogenic bacteria and strengthens teeth.

Caries is a disease that progressively destroys the hard tissue of the tooth. Once the caries occurs, it cannot be stopped, but the development of the disease can only be delayed. However, caries is a disease that prevention is greater than cure. Through standardized daily brushing habits and regular oral cleaning, the effect of preventing caries can be achieved. According to the latest statistics from the Fourth National Oral Health Epidemiological Survey, the rates of 5-year-old and 12-year-old children brushing their teeth twice a day were 24.1% and 31.9%, respectively, and the usage rates of fluoride toothpaste were 42.1% and 55%, respectively. The rate of brushing twice a day was 36.1%, the usage rate of fluoride toothpaste was 61.0%, and the caries rates of 5-year-old and 12-year-old children in my country were 70.9% and 34.5%, respectively. This shows that the habit of brushing teeth of the population in my country is generally not good, and the incidence of caries in children is relatively high.

At present, oral care products include toothpaste, dental floss, tooth paste, mouthwash, etc., among which mouthwash plays an increasingly important role in daily oral care. Commonly used mouthwashes contain bactericidal ingredients such as triclosan, metronidazole, tinidazole, hydrogen peroxide, etc., but they can only non-specifically kill oral cariogenic microorganisms, and cause great damage to the root of the teeth, making them less effective as daily oral care; Essential oil mouthwashes are not effective in preventing caries and are mostly used for fresh breath. The development of mouthwash that is convenient to use, has a pleasant taste, can effectively prevent caries, and can be used for a long time is of great significance to the oral care of modern people.

SUMMARY OF THE INVENTION

In view of the problems existing in the above-mentioned prior art, especially the traditional mouthwash can only freshen the breath or non-specifically kill the oral cariogenic microorganisms, but usually does not have the problems of preventing dental caries and strengthening the teeth and other effects, the present invention provides a The mouthwash has the functions of anti-caries, antibacterial, strong root and tooth fixation, and has low toxicity and good user experience. It is expected to replace the existing mouthwashes on the market and protect people's teeth.

Specifically, the present invention relates to the following technical solutions:

First of all, the present invention discloses a jelly-like mouthwash with anti-caries, antibacterial and strong root tooth-fixing effects. The mouthwash includes moisturizing ingredients 3%-5%, solvent 91%-95%, acid-base buffer 0.25%- 0.75%, anti-caries ingredient 0.2%-0.6%, strong root ingredient 0.05%-0.15%, flavor ingredient 0.2%-0.6%, antiseptic ingredient 0.1%-0.2%, fragrance 0.25%-0.35%, tooth-fixing ingredient 0.01% -0.03%, gel component 0.4%-0.8%, the anti-caries component is selected from compounds LX-S-06, LX-S-18, XQH-2-92, XQH-3-6, XQH-3-7 One or more of the strong root ingredients are quercetin.

In a specific embodiment, the mouthwash contains 3.8% moisturizing ingredient, 93.7% solvent, 0.5% acid-base buffer, 0.4% anti-caries ingredient, 0.1% strong root ingredient, 0.4% flavoring ingredient, 0.15% antiseptic ingredient, and fragrance. 0.33%, solid tooth component 0.02%, gel component 0.6%.

Preferably, the moisturizing ingredients in the mouthwash are glycerol and propylene glycol, the solvent is purified water, the acid-base buffer is sodium citrate and sodium malate, and the anti-caries ingredients are selected from compounds LX-S-06, LX-S-18, One or more of XQH-2-92, XQH-3-6, XQH-3-7, strong root ingredient is quercetin, flavor ingredient is xylitol and trehalose, preservative ingredient is sodium benzoate, flavoring agent It is the essence and honeysuckle extract, the tooth-fixing ingredient is sodium fluoride, and the gel ingredient is carrageenan and konjac gum.

In a more preferred embodiment, the mouthwash contains 3% glycerin, 0.8% propylene glycol, 93.7% purified water, 0.25% sodium citrate, 0.4% anti-caries ingredient, 0.1% quercetin, 0.3% xylitol, and trehalose. 0.4%, sodium benzoate 0.15%, essence 0.15%, honeysuckle extract 0.18, sodium fluoride 0.02, carrageenan 0.3%, konjac gum 0.3%.

In a preferred embodiment of the present invention, the anti-caries component is selected from compound LX-S-06 or LX-S-18, and its configuration in jelly-like mouthwash is more effective than other anti-caries components; preferably, the anti-caries component is selected from compound Two or more of LX-S-06, LX-S-18, XQH-2-92, XQH-3-6, and XQH-3-7. The combination of LX-S-06 or LX-S-18 with other anti-caries ingredients is more effective. For example, in a preferred embodiment, the anti-caries ingredients are LX-S-06 and LX-S-18, and one or more of XQH-2-92, XQH-3-6, and XQH-3-7; another preferred In the embodiment of , the anti-caries ingredients are LX-S-06, LX-S-18, XQH-2-92, XQH-3-6, XQH-3-7.

The anti-caries components in the mouthwash of the present invention are LX-S-06, LX-S-18, XQH-2-92, XQH-3-6, XQH-3-7 and quercetin, whose structural formulas are shown in Table 1.

Table 1. Structural formulas of LX-S-06, LX-S-18, XQH-2-92, XQH-3-6, XQH-3-7, quercetin

Secondly, the present invention discloses the preparation method of the above-mentioned jelly-like mouthwash, comprising:

Step 1: Mix the weighed anti-caries ingredients, flavoring ingredients, antiseptic ingredients, flavoring agents and tooth-fixing ingredients and grind them thoroughly;

Step 2: Add a standard amount of moisturizing ingredients to the specified purified water, and add the gel ingredients to the solution;

Step 3: add the mixture obtained in step 1 to the solution obtained in step 2; add a standard amount of acid-base buffer to the mixed solution, and adjust the pH to 6.6-7.1;

Step 4: The obtained gel is fully sterilized at high temperature and then packaged.

The invention reasonably groups the used components, effectively cooperates, and has a simple and convenient preparation process, and is suitable for the processing and preparation of the jelly-like mouthwash.

In addition, the present invention also discloses two novel compounds with resistance to Streptococcus mutans, namely (S)-2-(1,3-dimethyl-2-oxo-1,2-dihydroquinoxaline-6 -sulfonamido)-3-(methylthio)-N-(5-nitrothiazol-2-yl)propanamide (ie compound LX-S-06) and (R)-methyl(2-(1 -Methyl-3-(((5-nitrothiazole-2-)amino)methyl)-2-oxo-1,2-dihydroquinoxaline-6-sulfonamido)-3-phenylpropane acyl)carbamate (ie compound LX-S-18).

The structural formula of compound LX-S-06 is:

The structural formula of compound LX-S-18 is:

Further, the application of compounds LX-S-06 and LX-S-18 is also the protection scope of the present invention, and the application includes compounds LX-S-06 and LX-S-18 in the preparation of oral Streptococcus mutans biofilm inhibitors In the preparation of caries prevention and treatment drugs, and in the preparation of functional toothpaste, mouthwash, oral ulcer film, denture cleaner, chewing gum or disinfectant.

The present invention has achieved the following beneficial effects:

(1) Aiming at the disadvantages of traditional mouthwashes, the present invention includes an anti-caries and antibacterial component and a strong root tooth-fixing component. The anti-caries component can specifically fight against bacteria that cause caries, and the strong root component can stabilize the root, strengthen the cementum, and increase the number of teeth. The bone volume of the groove bone integrates the functions of anti-caries and antibacterial and strong root and tooth fixation.

(2) The present invention not only fully exerts the efficacy of each component and effectively avoids toxic and side effects through the rational combination of various components, but also enhances the anti-caries effect of the mouthwash through reasonable compatibility, effectively improving the bactericidal and bacteriostatic properties of the mouthwash and Anti-inflammatory analgesic effect. In addition to the anti-caries component and the strong root component, the tooth-fixing component of the present invention can reduce the solubility of enamel hydroxyapatite, enhance the ability of the tooth to resist caries, and can also increase the integrity and hardness of the enamel, thereby achieving the effect of preventing caries; In the formula of the present invention, under the premise of ensuring good anti-caries, antibacterial, strong root and tooth-fixing effects, the color, fragrance and taste of the present invention are improved, and then a mouthwash with good use experience is obtained. The present invention uses glycerin and propylene glycol. As moisturizing ingredients, enhance the experience of mouthwash, use sodium citrate and sodium malate as acid-base buffers to adjust pH buffer system, reduce irritation and adjust oral senses, use xylitol and trehalose as flavoring ingredients, and use essence and honeysuckle as flavoring ingredients. The extract is a fragrance component, which improves the taste and refreshes the breath; in addition to the above components, the present invention also includes a tooth-fixing component sodium fluoride and an antiseptic component sodium benzoate, as well as gel components carrageenan and konjac gum, which are combined to form a jelly-like mouthwash , the jelly-like form is not only hygienic and convenient to use, but also can effectively form a long-term stable product. The mouthwash of the invention can effectively remove the odor in the oral cavity, keep the breath fresh for a long time, and has a good bactericidal and antibacterial effect; it can also quickly improve oral problems, maintain the balance of oral bacteria, and effectively prevent and improve gingival swelling and pain caused by bacteria. , bleeding gums, bad breath and other diseases, and at the same time can achieve strong teeth, avoid tooth loosening and reduce root damage. The invention has the advantages of convenient use, comfortable taste, remarkable anti-caries effect, no toxic and side effects, and can be used for a long time.

(3) The present invention also provides a preparation method of an anti-caries efficacy mouthwash, the preparation method is simple, low in cost, and convenient for industrialization.

(4) The present invention also designs and synthesizes two new compounds LX-S-06 and LX-S-18 with resistance to Streptococcus mutans, which not only have good anti-Streptococcus mutans effect, but also have less toxicity and are comparable to other anti- Streptococcus mutans compounds XQH-2-92, XQH-3-6, XQH-3-7 are used together, and the effect is better.

Description of drawings

Figure 1. ALP color development kit was used to observe the results with a high-power microscope (10×) after dyeing. After dyeing, the color of the experimental group was darker than that of the control group, indicating that the strong tooth component contained in the present invention has a high-efficiency osteopromoting effect;

Figure 2. The results of the experimental group and the control group using the anti-caries special effect mouthwash of the present invention, after using the special stain for dental plaque, the experimental group can see that there is slight plaque attachment on the tooth surface; and the control group shows that the plaque is widely attached on the tooth surface and in the gingival sulcus.

Detailed ways

It should be noted that the following detailed description is exemplary and intended to provide further explanation of the invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It should be noted that the terminology used herein is for the purpose of describing specific embodiments only, and is not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly dictates otherwise, the singular is intended to include the plural as well, furthermore, it is to be understood that when the terms "comprising" and/or "including" are used in this specification, it indicates that There are features, steps, operations, and/or combinations thereof.

It should be noted that the compounds with anti-Streptococcus mutans activity used in the present invention are LX-S-06 and LX-S-18. In addition, the compound with anti-Streptococcus mutans activity used in the present invention is also selected from XQH-2-92, XQH-3-6 and XQH-3-7. Among them, compound XQH-3-6 and its preparation and application are disclosed in invention patent CN106588911A; compound XQH-3-7 and its preparation and application are disclosed in invention patent CN106699751A; compound XQH-2-92 and its preparation and application are disclosed in Patent CN106588813A has been published; the contents of the three patents are incorporated into this application. The present invention will detail the preparation method and application of LX-S-06 and LX-S-18.

Example 1: (S)-2-(1,3-dimethyl-2-oxo-1,2-dihydroquinoxaline-6-sulfonamido)-3-(methylthio)-N- Preparation of (5-nitrothiazol-2-yl)propionamide (compound LX-S-06)

Preparation of Intermediate 3-Methylquinoxalin-2(1H)-one (1)

Weigh 10.81 g (0.10 mol) of o-phenylenediamine into a 500 mL round-bottomed flask, add 250 mL of absolute ethanol, and stir until all dissolved to obtain a pale yellow solution. 12.77 g (0.11 mol) of ethyl pyruvate was added dropwise to the solution under stirring at room temperature. After about 10 min, a large amount of solid was instantly generated, and the reaction was continued for 5 h, and the reaction ended. After filtration, the solid was collected and recrystallized with ethanol. After vacuum drying, a light white cotton floc-like product was obtained. The yield was 90.4%, and m.p.=241.0～242.7°C.

Preparation of intermediate 1,3-dimethylquinoxalin-2(1H)-one (2)

Weigh 8.01g (0.05mol) of compound 1, 13.80g (0.10mol) of anhydrous potassium carbonate and 0.4g of n-tetrabutylammonium bromide (TBAB) and place them in a 500ml round-bottomed flask, add 200ml of acetone to obtain a suspension liquid. 9.48ml (0.10mol) of dimethyl sulfate was added dropwise to the suspension under stirring at room temperature. After the dropwise addition, the mixture was transferred to an oil bath at 65°C for heating and refluxing for 6h, and the reaction was completed by TLC detection. The solvent was evaporated under reduced pressure, 50 ml of water and 100 ml of ethyl acetate were added for repeated extraction and washing 3 times, the ethyl acetate layers were combined and concentrated, 80-100 mesh silica gel was added to mix the samples, and the samples were subjected to flash column chromatography (eluent: petroleum ether). /ethyl acetate=6:1-3:1) separation and purification to obtain white flashing crystals, the product has a pungent odor, the yield is 75%, and m.p.=76.0～78.0°C.

Preparation of intermediate 1,3-dimethyl-2-oxo-1,2-dihydroquinoxaline-6-sulfonyl chloride (3)

Weigh 8.70g (0.05mol) of compound (2) into a 250ml round-bottomed flask, add 20ml of anhydrous dichloromethane (DCM) to dissolve it completely, slowly add 10ml of chlorosulfonic acid dropwise under stirring in an ice bath, and add dropwise After completion, stirring was continued for 5 min, and then transferred to a 100° C. oil bath to continue the reaction for 12 h, and the reaction was completed by TLC detection. The reaction solution was cooled to room temperature, diluted with 100 ml of DCM, and 50 ml of cooled distilled water was slowly added under stirring in an ice bath to remove unreacted chlorosulfonic acid. , further purified by acetone-ether system and directly put into the next reaction, the yield is 55%.

Intermediate (R)-Methyl 2-(1,3-dimethyl-2-oxo-1,2-dihydroquinoxaline-6-sulfonamido)-4-(methylthio)butyric acid methyl Preparation of ester (4)

0.012mol of methionine methyl ester hydrochloride was weighed and suspended in 50ml of anhydrous DCM, 4.18ml (0.03mol) of anhydrous TEA and 0.12g of DMAP were added successively, and the DCM solution of compound 2 was slowly added dropwise after stirring at room temperature for 10min ( 2.72g of compound 3 was dissolved in 10ml of DCM for use). After the dropwise addition was completed, the reaction was continued for 5 h, and the TLC detection reaction was completed. DCM was removed under reduced pressure, the solid residue was dissolved in 50 ml of ethyl acetate, washed with 1 mol/L citric acid solution (2 × 20 ml), saturated NaHCO ₃ solution (2 × 20 ml) and saturated NaCl solution (2 × 20 ml) successively, and the organic The layer was dried over anhydrous magnesium sulfate, mixed with samples, and separated and purified by flash column chromatography (eluent: petroleum ether/ethyl acetate=3:1-2:1) to obtain a pale yellow solid with a yield of 67%. Mp=149.5～150.5℃, ¹ H-NMR (DMSO-d ₆ , 400MHz, ppm): δ1.720-1.880 (m, 2H, CH ₂ ), 1.924 (s, 1H, CH ₃ ), 2.285-2.370 ( m, 1H, SCH ₂ ), 2.380-2.440 (m, 1H, SCH ₂ ), 3.470 (s, 3H, N=CCH ₃ ), 3.390 (s, 3H, OCH ₃ ), 3.642 (s, 3H, NCH ₃ ), 4.011(m, 1H, CH), 7.719(d, J=9.0Hz, 1H, ArH), 7.779(dd, J=1.8, 9.0Hz, 1H, ArH), 8.022(d, J=1.8Hz, 1H, ArH), 8.501(d, J=7.2Hz, 1H, NH). ESI-MS: 400.5[M+H] ⁺ .

Intermediate (R)-Methyl 2-(1,3-dimethyl-2-oxo-1,2-dihydroquinoxaline-6-sulfonamido)-4-(methylthio)butyric acid methyl Preparation of Ester (R)-2-(1,3-dimethyl-2-oxo-1,2-dihydroquinoxaline-6-sulfonamido)-4-(methylthio)butanoic acid (5)

Weigh 1.12g (0.02mol) of solid potassium hydroxide, add 25ml of distilled water to dissolve it for later use. Weigh 3.25 g (0.01 mol) of compound (4), add 50 ml of anhydrous ethanol to dissolve, stir at room temperature for 5 min, add pre-prepared potassium hydroxide solution, continue to stir for 4 h, and TLC detects the end of the reaction. The solvent was evaporated under reduced pressure, 25 ml of distilled water was added, and the pH was adjusted to about 2 with 4N hydrochloric acid, a large amount of solid was precipitated, filtered, and the filter cake was washed with water and recrystallized with tetrahydrofuran to obtain an off-white product with a yield of 71%. Mp=120.0～121.0℃, ¹ H-NMR (DMSO-d ₆ , 400MHz, ppm): δ 1.700-1.880 (m, 2H, CH ₂ ), 1.926 (s, 1H, CH ₃ ), 2.300-2.370 ( m, 2H, SCH ₂ ), 2.380-2.440 (m, 2H, CH ₂ ), 3.470 (s, 3H, N=CCH ₃ ), 3.638 (s, 3H, NCH ₃ ), 3.895 (m, 1H, CH) ,7.709(d,J＝9.0Hz,1H,ArH),7.904(dd,J＝1.8,8.4Hz,1H,ArH),8.047(d,J＝2.4Hz,1H,ArH),8.308(d,J =7.8Hz,1H,NH),12.68(s,1H,COOH).ESI-MS:386.6[M+H] ⁺ .

Target (R)-2-(1,3-dimethyl-2-oxo-1,2-dihydroquinoxaline-6-sulfonamido)-4-(methylthio)-N-(5 - Preparation of nitrothiazol-2-yl)butanamide (LX-S-06)

In an ice-salt bath, the intermediate 5 (0.37g, 1mmol) obtained in the previous step was dissolved in 15ml of anhydrous tetrahydrofuran, and 2-amino-5-nitrothiazole (0.17g, 1.2mmol), 2-amino-5-nitrothiazole (0.17g, 1.2mmol), 2-amino-5-nitrothiazole (0.17g, 1.2mmol), HOBt (0.23 g, 1.7 mmol) and EDCI (0.33 g, 1.7 mmol), the mixed solution was stirred under an ice bath for 15 minutes, N-methylmorpholine (0.2 ml, 1.7 mmol) was added, and the ice bath was removed, The reaction was continued to be stirred at room temperature for 6 hours, until the completion of the reaction was monitored by TLC, and the complete reaction of the intermediate 5 was completed. The reaction solution was washed with 5% KHSO ₄ (3×10ml), saturated NaHCO ₃ solution (3×10ml) and saturated brine (3×10ml) respectively, the aqueous phase was extracted twice with dichloromethane, and the organic phase was washed with After drying with magnesium sulfate, the crude product was obtained after evaporation and concentration. The crude product was purified and separated by silica gel column (eluent: petroleum ether: ethyl acetate=4:1) to obtain pure product with a yield of 66.5%. ¹ H-NMR (400MHz, DMSO-d ₆ , ppm) δ: 2.03 (s, 3H, N=C-CH ₃ ), 2.22 (s, 3H, S-CH ₃ ), 2.32-2.38 (m, 2H, SCH ₂ ), 2.39-2.43 (m, 2H, CH ₂ CHC=O), 3.33 (s, 3H, N-CH ₃ ), 3.82-3.84 (m, 1H, CHC=O), 7.73 (s, 1H , ArH), 7.88-7.89 (d, J=8.4Hz, 1H, ArH), 8.07-8.10 (d, J=9.0Hz, 1H, ArH), 8.33 (d, J=7.8Hz, 1H, NHSO ₂ ) , 8.86-8.87 (s, 1H, CH-N=C), 9.61 (s, 1H, NHC=O); ESI-MS: 535.5[M+Na] ⁺ .

Embodiment 2: (R)-methyl (2-(1-methyl-3-(((5-nitrothiazole-2-) amino) methyl)-2-oxo-1,2-dihydroquinoline Preparation of oxaline-6-sulfonamido)-3-phenylpropionyl)carbamate (compound LX-S-18)

Intermediate (R)-methyl 2-(1,3-dimethyl-2-oxo-1,2-dihydroquinoxaline-6-sulfonamido)-3-phenylpropionic acid methyl ester (6 ) preparation

The preparation method of the intermediate (6) is the same as that of the intermediate (4) in Example 1, and the yield is 69%. Mp=151.0～153.0℃, ¹ H-NMR (DMSO-d ₆ , 400MHz, ppm): δ 2.473 (s, 3H, N=CCH ₃ ), 2.751 (dd, J=9.0, 13.8 Hz, 1H, CH ₂ ), 2.93 (dd, J=6.0, 13.8 Hz, 1H, CH ₂ ), 3.34 (s, 3H, OCH ₃ C=O), 3.63 (s, 3H, NCH ₃ ), 4.08 (d, J=7.8 Hz,1H,CH),7.05-7.14(m,5H,5ArH),7.57(d,J=9.0Hz,1H,ArH),7.69(dd,J=2.4,9.0Hz,1H,ArH),7.85( d, J=2.4Hz, 1H, ArH), 8.63 (d, J=9.0Hz, 1H, NH). ESI-MS: 416.7[M+H] ⁺ .

Intermediate (R)-Methyl 2-(3-bromomethyl)-1-methyl-2-oxo-1,2-dihydroquinoxaline-6-sulfonamido)-3-phenylpropionic acid Preparation of methyl ester (7)

Under the ice bath, the intermediate 6 (0.42 g, 1 mmol) obtained in the previous step was placed in a reaction flask, and 5 ml of glacial acetic acid solution in which anhydrous sodium acetate (0.1 g, 1.2 mmol) was dissolved was added dropwise to the flask. After that, the reaction was carried out for 5 minutes, and then 3 ml of glacial acetic acid solution in which bromine (0.05 ml, 1 mmol) was dissolved was added. The mixed solution was reacted for 3 hours at room temperature under the protection of N ₂ gas, 10 ml of water and 10 ml of dichloromethane were added to terminate the reaction, 20 ml of dichloromethane was added to extract the above mixed solution, and finally dried with anhydrous MgSO ₄ , and ethyl acetate was used. The ester: petroleum ether (1:1-2:1) was separated by column chromatography to obtain a brown-red solid product with a yield of 55%. Mp=133.0～134.0℃, ¹ H-NMR (DMSO-d ₆ , 400MHz, ppm): δ 2.473 (s, 3H, N=CCH ₃ ), 2.751 (dd, J=9.0, 13.8 Hz, 1H, CH ), 2.914 (dd, J=6.0, 13.8 Hz, 2H, CH ₂ ), 3.34 (s, 3H, CH ₃ O), 3.66 (s, 3H, NCH ₃ ), 4.012 (td, J=6.6, 9.0 Hz , 1H, O=C-CH), 4.72 (2H, s, CH ₂ Br), 7.05-7.15 (m, 5H, ArH), 7.66 (d, J=9.0Hz, 1H, ArH), 7.69 (dd, J=2.4, 9.0Hz, 1H, ArH), 7.87 (d, J=2.4Hz, 1H, ArH), 8.63 (d, J=9.0Hz, 1H, NHSO ₂ ). ESI-MS: 494.8 [M+H ] ⁺ .

Target (R)-methyl 2-(1-methyl-3(((5-nitrothiazol-2-yl)amino)methyl)-2-oxo-1,2-dihydroquinoxaline- Preparation of 6-sulfonamido)-3-phenylpropionic acid methyl ester (LX-S-18)

Under the ice bath, the intermediate 7 (0.5g, 1mmol) obtained in the previous step was dissolved in 10ml of acetone, and the ground anhydrous potassium carbonate powder (0.28g, 2mmol) and 2-amino-5-nitrothiazole were added successively. (0.17 g, 1.2 mmol), the mixture was placed under nitrogen protection at room temperature and reacted for 6 hours until the reaction was completed as monitored by TLC. After the solvent was evaporated, 20ml of dichloromethane was added for extraction, washed with saturated ammonium chloride (3×10ml) and saturated brine (3×10ml) respectively, dried and concentrated, then washed with ethyl acetate:petroleum ether (2:1-3 : 1) column chromatography to obtain a light gray solid product with a yield of 48%. Mp=177.0～178.0℃, ¹ H-NMR (DMSO-d ₆ , 400MHz, ppm): δ2.88-2.90 (dd, J=9.0, 13.8 Hz, 2H , PhCH ₂ CH), 3.22 (dd, J =6.0, 13.8Hz, 1H , O=CCH-NH), 3.54(s, _3H , CH3O), 3.64(s, 3H, _NCH3 ), 4.55(td, J=6.6, 9.0Hz, 2H, N=CC H ₂ NH), 5.02 (1H, dd, J ₁ =1.8 Hz, J ₁ =3.0 Hz, NH-C=N), 7.27-7.38 (m, 5H, 5ArH), 7.79 (S, 1H, ArH), 7.80(d, J=9.0Hz, 1H, ArH), 7.89(d, J=2.4Hz, 1H, ArH), 8.56(s, 1H, C=NCH). ESI-MS: 559.6[M+ H] ⁺ .

Example 3: Antibacterial activity test of compound LX-S-06

Preparation of Streptococcus mutans

(1) The medium for culturing Streptococcus mutans is Brain heart infusion medium (brand OXOID, item number CM1135), and the main components of the medium are Brain infusion solids 12.5g/L, Beef heartinfusion solids 5.0g/L , Proteose peptone 10.0g/L, Glucose 2.0g/L, Sodiumchloride 5.0g/L, Di-sodium phosphate 2.5g/L, pH 7.4±0.2. For solid medium, add 1.5% agar powder. Sterilize with damp heat at 115°C for 30 minutes, and then use after cooling.

(2) The culture medium of Streptococcus mutans biofilm is brain heart infusion-sucrose medium. The preparation method is as follows: sucrose is made into a 20% stock solution and sterilized by filtration with a 0.22 μm sterile filter, and sucrose with a final concentration of 1% is added to the brain heart infusion medium.

(3) Bacterial liquids of Streptococcus mutans type strain UA159 and clinical strain UA246 were inoculated on the brain heart infusion solid medium and streaked, and cultured in anaerobic inversion at 37°C for 24 hours, and an obvious single colony appeared.

(4) Pick a single colony of Streptococcus mutans UA159 and UA246 strains with a sterile inoculating loop, inoculate it into a test tube containing the brain heart infusion liquid medium, and anaerobic static culture at 37 ° C, to the test tube The inner liquid is cloudy.

(5) The absorption value (OD600) of Streptococcus mutans UA159 and UA246 at the wavelength of 600nm was measured by UV-Vis spectrophotometer.

(6) Compound LX-S-06 was accurately weighed with an analytical balance, dissolved in dimethyl sulfoxide, prepared into a stock solution with a concentration of 1024 mg/L, and sterilized by filtration using a sterile filter with a specification of 0.22 μm. Store at -20°C until use.

Activity test results of compound LX-S-06 on planktonic cells of Streptococcus mutans

(1) According to the method described above, prepare Streptococcus mutans bacteria liquid and compound LX-S-06, and use brain heart for Streptococcus mutans UA159 and UA246 bacteria liquids that have been cultured to logarithmic phase (OD ₆₀₀ = 0.8-1.0). The infusion liquid medium was diluted to a final concentration of 5×10 ⁵ cfu/ml for use.

(2) The minimum inhibitory concentration of compound LX-S-06 on planktonic cells of Streptococcus mutans UA159 and UA246 was detected by the micro-broth dilution method. The final concentration of Streptococcus mutans in each well of a sterile 96-well plate is 5×10 ⁵ cfu/ml, and the stock solution of compound LX-S-06 prepared according to the above method is added to the first well and adjusted to the final concentration 256 μg/mL, mix well, then pipette 150 μL to the 2nd well, mix and then pipette 150 μL to the 3rd well, so serially dilute to the 11th well, and pipette 150 μL from the 11th well and discard. So far, the 1st to 11th wells are the experimental group with the drug solution added, and the 12th well is the growth control group without drug addition. The drug concentrations from the 1st well to the 12th well were 256, 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, and 0 μg/mL, respectively. The minimum inhibitory concentration (MIC) was defined as the lowest concentration that completely inhibited bacterial growth in the small wells.

(3) After the bacterial solution in the well was evenly spread on the brain heart infusion agar solid medium, anaerobic inversion was cultured at 37°C for 24 hours, and the lowest concentration without bacterial growth was set as the minimum bactericidal concentration (MBC).

(4) Use a microplate reader to detect the absorbance value of each well of the 96-well plate at a wavelength of 600 nm, and calculate the inhibition rate of cells at each drug concentration. The calculation formula was inhibition rate=(1-experimental group/growth control group)×100%, and the obtained data were calculated using SPSS statistical software to calculate the half-maximum inhibitory concentration (IC ₅₀ ).

(5) The experimental procedure of the antibacterial activity of nitazoxanide (NTZ) in the control group is the same as that of the compound LX-S-06, and the experimental results are shown in Table 2.

Table 2. Activity detection results of LX-S-06 and NTZ against Streptococcus mutans UA159 and UA246

MIC: minimum inhibitory concentration; MBC: minimum bactericidal concentration; IC ₅₀ : half inhibitory concentration

It can be seen from Table 2 that the compound LX-S-06 has better bacteriostatic and bactericidal activities against Streptococcus mutans UA159 and UA246 than the positive control nitazoxanide.

Example 4: Antibacterial activity test of compound LX-S-18

The steps are the same as in Example 3, and the results are shown in Table 3.

Table 3. Activity detection results of LX-S-18 and NTZ against Streptococcus mutans UA159 and UA246

MIC: minimum inhibitory concentration; MBC: minimum bactericidal concentration; IC ₅₀ : half inhibitory concentration

From Table 3, we can see that the compound LX-S-18 has better bacteriostatic and bactericidal activities against Streptococcus mutans UA159 and UA246 than the positive control drug nitazoxanide.

Embodiment 5: Preparation method one of jelly-like mouthwash with anti-caries and antibacterial, strong root and tooth-fixing effects

The specific components of mouthwash and their mass fractions are shown in Table 4. The specific implementation steps are as follows:

Step 1: Mix the weighed anti-caries ingredients, flavoring ingredients, antiseptic ingredients, flavoring agents and tooth-fixing ingredients and grind them thoroughly. The specific formula is LX-S-06, LX-S-18, XQH-2-92, XQH-3-6 or XQH-3-70.4% as an anti-caries ingredient, 0.1% quercetin as a strong root ingredient, and a flavor ingredient. Xylitol and trehalose are 0.3% and 0.1% respectively, the preservative ingredient ammonium benzoate is 0.15%, the flavoring agent essence 0.15% and the honeysuckle extract 0.18%, and the tooth-fixing ingredient sodium fluoride is 0.2%.

Step 2: take a standard amount of moisturizing ingredients and add them to the specified purified water, the mass percentages of the moisturizing ingredients glycerin and propylene glycol are 3% and 0.8% respectively, and the mass percentage of the solvent is 93.7%; the gel ingredients carrageenan and konjac are added. Gum was added to the solution, and its proportions were 0.3% and 0.3%, respectively.

Step 3: add the mixture obtained in step 1 to the solution obtained in step 2; add a standard amount of acid-base buffer to the mixed solution, and adjust the pH to 6.6-7.1.

Step 4: The obtained gel is fully sterilized at high temperature and then packaged.

Table 4. Composition and content of the anti-caries efficacy mouthwash obtained by preparation method 1

Embodiment 6: the preparation method two of the jelly-like mouthwash with anti-caries and antibacterial, strong root and tooth-fixing effects

In order to obtain the mouthwash with the best efficacy, the components can be formulated according to the ratio shown in Table 5 to obtain the target product:

Table 5. Composition and content of the anti-caries efficacy mouthwash obtained by preparation method 2

Embodiment 7: Preparation method three of jelly-like mouthwash with anti-caries and antibacterial, strong root and tooth-fixing effects

In order to obtain the mouthwash with the best efficacy, the components can be formulated according to the ratio shown in Table 6 to obtain the target product:

Table 6. Composition and content of the anti-caries efficacy mouthwash obtained by preparation method three

Embodiment 8: the preparation method 4 of the jelly-like mouthwash with anti-caries and antibacterial, strong root and tooth-fixing effects

In order to obtain the mouthwash with the best efficacy, the components can be formulated according to the ratio shown in Table 7 to obtain the target product:

Table 7. Composition and content of the anti-caries efficacy mouthwash obtained by preparation method 4

Example 9: Safety Test of Anti-caries Mouthwash—Cytotoxicity Test

A supplementary safety test was designed for this invention to further test the safety of the anti-caries ingredients. Therefore, the present invention uses normal cells (mouse macrophage RAW246.7) as the detection object, and nitazoxanide (NTZ) as the positive control. If the IC ₅₀ value is greater than the positive control, the cytotoxicity of the compound is very low.

Experimental materials: mouse normal cells (macrophage RAW246.7), 96-well plate, CCK-8 reagent, nitazoxanide (NTZ), test compound.

Experimental method: The mouse macrophage RAW246.7 cells were divided into 3 groups, cultured in a 96-well plate with 5×10 ³ cells per well, and cultured at 37°C and 5% CO ₂ to 70%~ After 80% confluence, the drugs nitazoxanide, LX-S-06, LX-S-18, XQH-2-92, XQH-3-6 and XQH-3-7 were added respectively. Cell proliferation was detected by CCK-8 method.

Experimental results and analysis: It can be seen from the results shown in Table 8 that several antibacterial components XQH-2-92, XQH-3-6, XQH-3-7, LX-S-06, LX-S provided by the present invention -18 was less toxic to mouse macrophage RAW246.7 than NTZ. Since the antibacterial active ingredient in the present invention does not enter the body as an additive of toothpaste, and because the cytotoxicity of the active ingredient used in the present invention is smaller than that of the clinically used oral drug nitazoxanide, the present invention It has higher safety performance.

Table 8. Cytotoxicity test results of anti-caries ingredients and nitazoxanide on mouse macrophage RAW246.7

Example 10: Detection of osteopromoting effect of anti-caries mouthwash

Experimental materials: bacterial culture dish, isolated teeth, ophthalmic scissors, centrifuge tube, 6-well plate, 4% paraformaldehyde, ALP color development kit.

Experimental protocol: In the Department of Oral and Maxillofacial Surgery of Shandong University Stomatological Hospital, healthy third molar teeth of patients aged 12-18 years were selected to be collected, and the teeth were sent to the laboratory within 4 hours. Rinse the isolated teeth 3-4 times with 10 times PBS in a sterile petri dish on an ultra-clean test bench. Using a sterile scalpel, carefully scrape 1/3 of the periodontal ligament in the root, rinse with 10 times PBS while scraping, and use ophthalmic scissors to cut the scraped tissue to the smallest size. The scraped periodontal ligament tissue was transferred to a centrifuge tube, the rotational speed was set to 1000 r/min, and the centrifugation time was 2 min. The supernatant was discarded, and type I collagenase (1 mL) and Dispase II enzyme (1 mL) were added in the dark. Digest in a constant temperature water bath at 37°C for 40-50 minutes, gently shake the centrifuge tube once every 5 minutes until the tissue is digested into cotton wool. The rotation speed was set to 1000 r/min, the centrifugation time was 5 min, the supernatant was discarded, 2 mL of complete culture medium was added, and the cells were resuspended. Inoculated in a 6-well plate, added 2 mL of complete culture medium to each well, and cultured in a 37°C, 5% CO ₂ incubator. After 5 days, the medium was changed for the first time. The medium was changed every 3 days thereafter. Cells were passaged when the cells were 80% confluent in a 6-well plate. Human periodontal ligament stem cells hPDLSCs were inoculated in 6-well plates, and after 70% to 80% confluence, an experimental group and a blank control group were set up. The experimental group received 5 μM quercetin, and the culture medium was discarded after culturing for 14 days, and 4% paraformaldehyde was used. Fixed at room temperature for 15 min, rinsed twice with PBS (pH=4.2), stained with ALP chromogenic kit, stained in a 37°C incubator for 15 min, rinsed 3 times with PBS, scanned and observed under a microscope.

Experimental results and analysis: After 14 days of osteogenic induction, the results under the microscope after staining with the ALP chromogenic kit are shown in Figure 1. It can be seen in Fig. 1 that the color of the experimental group is darker than that of the control group after dyeing, indicating that the strong tooth component quercetin contained in the present invention has a high-efficiency osteopromoting effect.

Embodiment 11: Anti-caries Mouthwash Anti-caries Effectiveness Test—Dental Plaque Staining Experiment

Under normal conditions, dental plaque is a biofilm formed by food and bacteria and attached to the surface of the teeth, which cannot be recognized by the naked eye. Plaque is visualized and clearly visible using a plaque indicator.

Experimental materials: the jelly-like anti-caries mouthwash of the present invention, a special stain for dental plaque (Plaque Indicator, CHROM-O-RED), and a medical cotton ball.

The experimental scheme is as follows:

1. Select 4 subjects with similar oral health status, divide the subjects into experimental group and control group, and ensure that the number of men and women in each group is equal. The experimental group used the jelly-shaped anti-caries mouthwash of Example 5 (LX-S-06) of the present invention, and the control group used the jelly-shaped anti-caries mouthwash (complete with distilled purified water) that did not contain an anti-caries and tooth-fixing ingredient;

2. Each group used mouthwash once a day in the morning, noon and evening, with a dosage of 20 ml each time, and the duration of use was 1 minute, and the dental plaque was detected after two weeks.

3. Detection steps of dental plaque staining method:

Ⅰ. Apply a straw or a small cotton ball dipped in plaque stain evenly on the tooth surface and leave it for one minute;

Ⅱ. After the dyeing agent is firmly combined in the mouth, rinse off the residual dyeing agent with water;

Ⅲ. Check the staining of teeth;

Ⅳ. Staining distribution location and chromaticity are the expression of plaque distribution location and quantity.

Experimental results and analysis: The results are shown in Figure 2. It can be seen from Figure 2 that in the experimental group, there is only a small amount of plaque attached to the adjacent teeth (the area that is difficult to clean by brushing), most of the tooth surfaces are white and rich in color, and the tissues around the teeth are healthy and full; Red plaque, gingival sulcus and tooth neck show a large amount of unremoved calculus and soft scale. By comparing the typical samples of the experimental group and the control group, it is proved that the present invention has obvious anti-caries effect. Example five (LX-S-18 or XQH-2-92 or XQH-3-6 or XQH-3-7) has an anti-caries effect similar to Example five (LX-S-06), with LX-S-06 And LX-S-18 is optimal.

Example 12: Taste test of anti-caries mouthwash

Experimental scheme: 16 subjects were selected and divided into juvenile group, youth group, middle-aged group, and elderly group, and the number of men and women in each group was equal. The five (LX-S-06), six and seven anti-caries mouthwashes of the present invention were used as the experimental group, which were followed by the experimental group one, the experimental group two, and the experimental group three. The saliva was the control group, and the taste evaluation was recorded separately.

Experimental results: The results are shown in Table 9.

Table 9. Results of the testers' evaluation of the effects of various mouthwashes

Example 13: Effect detection of long-term use of anti-caries mouthwash

Experimental scheme: 16 patients with oral diseases such as caries, more plaque biofilm or more calculus, swollen gums, oral ulcers and bad breath were selected, and the number of men and women was equal, and the average age was 31 years old. The subjects were randomly divided into 4 groups, with 4 people in each group. The experimental group used the fifth embodiment of the present invention (LX-S-06),

Example 6, Example 7 Anti-caries efficacy mouthwash, the control group used conventional market-sold mouthwash. The method of use is the gargle method, and the effect is calculated after 3 weeks of use.

Evaluation criteria: see Table 10.

Table 10. Evaluation criteria for testers' oral problems after using the anti-caries mouthwash of the present invention

Experimental results and analysis: The results are shown in Table 11.

Table 11. Performance test results after long-term use of the anti-caries mouthwash provided by the present invention

The above enumerated embodiments are only to illustrate the basic principles and specific effects of the present invention, and do not limit the present invention. For those of ordinary skill in the art, in principle, the content claimed in the present invention can be adjusted and adjusted appropriately. Improved for better results.

Claims (6)

1.A jelly-like mouthwash with the efficacies of preventing caries and resisting bacteria and strengthening root and teeth is characterized by comprising the following components: 3 to 5 percent of moisture retention component, 91 to 95 percent of solvent, 0.25 to 0.75 percent of acid-base buffer agent, 0.2 to 0.6 percent of caries prevention component, 0.05 to 0.15 percent of root strengthening component, 0.2 to 0.6 percent of seasoning component, 0.1 to 0.2 percent of antiseptic component, 0.25 to 0.35 percent of flavoring agent, 0.01 to 0.03 percent of tooth fixing component and 0.4 to 0.8 percent of gel component, wherein the caries prevention component is more than two of compounds LX-S-06, LX-S-18, XQH-2-92, XQH-3-6 and XQH-3-7 and at least comprises any one of LX-S-06 and LX-S-18, and the root strengthening component is quercetin;

the structural formula of the LX-S-06 is shown as

The structural formula of the LX-S-18 is shown as

The structural formula of the XQH-2-92 is shown in the specification

The structural formula of the XQH-3-6 is shown in the specification

The structural formula of the XQH-3-7 is shown in the specification

The jelly-like mouth wash comprises the moisturizing components of glycerin and propylene glycol, the solvent is purified water, the acid-base buffer agent is sodium citrate and sodium malate, the seasoning components are xylitol and trehalose, the preservative component is sodium benzoate, the flavoring agent is essence and honeysuckle extract, the tooth fixing component is sodium fluoride, and the gel component is carrageenan and konjac glucomannan.

2. The jelly-like mouthwash having caries preventing, antibacterial and teeth strengthening effects according to claim 1, wherein the composition of the jelly-like mouthwash is: 3.8% of moisture-keeping component, 93.7% of solvent, 0.5% of acid-base buffer agent, 0.4% of anticaries component, 0.1% of root-strengthening component, 0.4% of seasoning component, 0.15% of preservative component, 0.33% of flavoring agent, 0.02% of tooth-fixing component and 0.6% of gel component.

3. The compound with the anti-streptococcus mutans effect is characterized in that the compound is LX-S-06 and LX-S-18 respectively, and the structural formulas of the compounds are respectively

4. Use of the compound having anti-streptococcus mutans efficacy of claim 3 in the preparation of an oral streptococcus mutans biofilm inhibitor.

5. Use of the compound having an anti-streptococcus mutans effect according to claim 3 for the manufacture of a medicament for preventing and treating dental caries.

6. Use of the compound having an anti-streptococcus mutans effect according to claim 3 for preparing toothpaste, mouthwash, denture cleanser, dental ulcer patch, or disinfectant.

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