WO2023029137A1

WO2023029137A1 - Application of antibacterial peptide compound dm80bu20 in preparation of antibacterial toothpaste

Info

Publication number: WO2023029137A1
Application number: PCT/CN2021/121158
Authority: WO
Inventors: 刘润辉; 褚夫江; 李毅苹; 耿帅峰
Original assignee: 好易康生物科技(广州)有限公司
Priority date: 2021-09-01
Filing date: 2021-09-28
Publication date: 2023-03-09
Also published as: CN113679632B; CN113679632A

Abstract

The present invention provides an application of an antibacterial peptide compound DM80Bu20 in preparation of an antibacterial toothpaste, and an antibacterial toothpaste thereof. According to the study of the present invention, the antibacterial peptide compound DM80Bu20 can selectively inhibit the activity of most of oral pathogenic bacteria, has no inhibitory effect on oral probiotics, has low hemolytic activity, low cytotoxicity, high safety, and good stability, and can be used for preparing an oral care toothpaste product; further, using the compound DM80Bu20 for preparing the antibacterial toothpaste also exhibits the effect of selectively inhibiting the oral pathogenic bacteria; in addition, by compounding specific functional components, the synergistic antibacterial effect with the antibacterial peptide compound DM80Bu20 can be achieved, the formation of dental calculuses and dental plaque is reduced, gum bleeding is reduced, dental caries and oral ulcer are prevented, halitosis and the like is reduced, and the prepared toothpaste is good in antibacterial stability.

Description

Application of Antibacterial Peptide Compound DM80Bu20 in Preparation of Antibacterial Toothpaste

technical field

The invention belongs to the technical field of oral care. More specifically, it relates to the application of antibacterial peptide compound DM80Bu20 in the preparation of antibacterial toothpaste.

Background technique

Oral health is an important part of overall health. Oral diseases such as caries and periodontal disease will destroy the hard tissues of the teeth and the supporting tissues around the teeth. In addition to affecting the functions of chewing, speech, and aesthetics, they will also cause social difficulties and psychological barriers , and the main cause of these oral diseases is the formation of dental plaque. Effectively solving the formation of dental plaque is the primary task of the research and development of functional oral care toothpaste products. As people pay more attention to dental health problems, nearly 50% of people think that Chinese herbal toothpaste is safer than traditional chemical toothpaste, so Chinese herbal toothpaste is becoming more and more popular. The evaluation of the antibacterial potential of common oral bacteria found that the Chinese herbal ingredients in toothpaste have no selective inhibitory and killing effects on various oral bacteria, and the strength of their effect on bacteria generally depends on the concentration of the drug. In addition, Candida albicans The sensitivity of various Chinese herbal toothpastes is generally low, and it is speculated that long-term use of these toothpastes may cause the risk of Candida albicans infection [Chen Hong, Ding Xi, and Zhang Xiuhua, Comparison of the antibacterial potential of five Chinese herbal toothpastes against common oral bacteria. Chinese Microecology Journal, 2007.19(4): p.351-353]. In addition, most of the antibacterial active extracts contained in the existing Chinese herbal toothpaste are unstable components in the acidic toothpaste matrix, and it is difficult to ensure long-term stability and activity; and probiotics without selection specificity.

Antibacterial peptides (antibacterial peptides) are widely distributed in nature. They are a kind of small molecular polypeptides produced by the host to resist pathogen infection, and are also an important part of the natural immune system of organisms. At this stage, they are also used as a new type of antibacterial peptide with development potential. Pharmaceutical ingredients, patent CN201910047525.3 discloses an oral composition containing probiotics and antimicrobial peptides, which can improve oral inflammation and have antibacterial, anti-inflammatory, pain-relieving and swelling effects. It can be seen that antimicrobial peptides have important applications in the treatment of oral diseases value. Therefore, there is an urgent need to find more new antibacterial peptide active substances, overcome the shortcomings of traditional Chinese herbal toothpaste for non-selective inhibition of various oral pathogens and probiotics, and provide a material basis for the development of oral antibacterial toothpaste products.

Contents of the invention

The present invention aims at the deficiencies of the prior art, and aims to provide a novel antimicrobial peptide compound DM80Bu20 in the application of inhibiting oral harmful bacteria and antibacterial toothpaste prepared therefrom. The present invention discloses that the antimicrobial peptide compound DM80Bu20 can selectively inhibit most Oral pathogenic bacteria are active, and have no inhibitory effect on oral probiotics, and have low hemolytic activity and low cytotoxicity, high safety and good stability; further, the compound DM80Bu20 is used to prepare a compound toothpaste compounded with specific functional components, and the obtained Antibacterial toothpaste products have good antibacterial stability.

The primary purpose of the present invention is to provide the application of the antimicrobial peptide compound DM80Bu20 in inhibiting oral harmful bacteria.

Another object of the present invention is to provide the application of antibacterial peptide compound DM80Bu20 in the preparation of antibacterial toothpaste.

Another object of the present invention is to provide a bacteriostatic toothpaste comprising the antimicrobial peptide compound DM80Bu20.

Above-mentioned purpose of the present invention is achieved through the following technical solutions:

The invention provides the application of the antimicrobial peptide compound DM80Bu20 in inhibiting oral harmful bacteria.

The structural formula of the antimicrobial peptide compound DM80Bu20 is:

Preferably, the oral harmful bacteria include one or more of Candida albicans, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Streptococcus mutans, and Porphyromonas gingivalis.

Among them, Candida albicans can cause candidal stomatitis (snow mouth disease, antibiotic stomatitis, denture stomatitis, candida leukoplakia), candida cheilitis, candida angular cheilitis, median rhomboid glossitis, leukoplakia, For precancerous lesions or conditions such as lichen planus and lupus erythematosus, since some Helicobacter pylori are latent in Candida albicans, the focus is on the development of active compounds that are effective against Candida albicans, which can also indirectly prevent and block the growth of Helicobacter pylori in oral microorganisms. Latent in the environment. Staphylococcus aureus and MRSA are the pathogenic bacteria of maxillofacial furunculosis, loose connective tissue inflammation, oral and maxillofacial wound infection, infantile angular cheilitis, periodontitis, and periapical mixed infection of dental pulp. Streptococcus mutans is the main pathogen of dental caries, and can cause secondary infections such as bacteremia and endocarditis. Porphyromonas gingivalis is the main pathogenic bacteria associated with periodontal disease. Streptococcus sanguinis is one of the bacteria that colonized the oral cavity earlier. The resident bacteria in the oral cavity antagonize the suspected pathogenic bacteria of dental caries and periodontal disease by producing hydrogen peroxide and sanitin. It is a kind of oral beneficial bacteria.

Compound DM80Bu20 is an amino acid polymer simulating natural peptides. It is a host defense peptide (HDP) mimic of natural antibacterial agents. Its antibacterial mechanism is similar to that of HDP. The target of action is the bacterial cell membrane. purpose of sterilization. The present invention carries out oral bacteria antibacterial activity experiment to compound DM80Bu20, and the result shows that this antimicrobial peptide compound DM80Bu20 has good antibacterial activity to Candida albicans, Staphylococcus aureus, MRSA, Streptococcus mutans, Porphyromonas gingivalis, The MIC value is 12.5-25g/mL; it has no inhibitory activity against the probiotic Streptococcus sanguis at the tested concentration, and the MIC value is greater than 200 μg/mL; it indicates that the compound can selectively inhibit most oral pathogens and has no inhibitory effect on oral probiotics effect. In addition, the antimicrobial peptide compound DM80Bu20 was tested for hemolysis and cytotoxicity, and the results showed that the compound DM80Bu20 had low hemolytic activity and low cytotoxicity, and was safe.

In addition, the toothpaste developed by further using the compound DM80Bu20 has a bacteriostatic rate of Candida albicans, Staphylococcus aureus, MRSA (methicillin-resistant Staphylococcus aureus), Streptococcus mutans, Porphyromonas gingivalis, and Escherichia coli. The bacteriostatic rate against Streptococcus sanguinis is lower than 10%, which shows that the bacteriostatic toothpaste of the present invention can well inhibit harmful bacteria in the oral cavity, has no harm to beneficial bacteria, and can effectively balance the flora. In addition, after placing the bacteriostatic toothpaste of the present invention at 45°C for 30 days and 90 days, with the prolongation of the storage period, the bacteriostatic effect is relatively reduced, but the overall still has a good bacteriostatic effect, indicating that the bacteriostatic toothpaste of the present invention The effect is stable, which is good for long-term storage. The bacteriostatic toothpaste of the present invention can inhibit the generation of bacteria and the formation of dental calculus by supplementing oral harmful bacteria defense components from external sources, reduce dental plaque, reduce gingival bleeding, and maintain bacteriostasis for a long time.

Therefore, the application of the above-mentioned antibacterial peptide compound DM80Bu20 in the preparation of antibacterial toothpaste, and a kind of antibacterial toothpaste containing the antibacterial peptide compound DM80Bu20 are all within the protection scope of the present invention. The toothpaste is prepared by taking the antibacterial peptide compound DM80Bu20 as the main antibacterial active ingredient and supplemented with other auxiliary materials.

As a preferably implementable method, the antibacterial toothpaste is composed of the following components by weight: 0.001-0.1 parts of antimicrobial peptide compound DM80Bu20, 30-45 parts of moisturizing agent, 20-25 parts of friction agent, 0.5 parts of adhesive ~2 parts, foaming agent 0.5~3 parts, essence 0.5~1.5 parts, sweetener 0.1~0.5 parts, chelating agent 1~3 parts, preservative 0.1~2 parts, deionized water 20~35 parts.

Further, the toothpaste also contains one or more components of lysozyme, lactoferrin, probiotics, regenerated silicon, and fructooligosaccharides. Among them, lysozyme inhibits oral pathogenic bacteria and balances oral flora; lactoferrin can enhance mucosal immunity and reduce Helicobacter pylori infection; probiotics can balance oral flora and inhibit pathogenic bacteria; It has the effect of repairing carious lesions, improving tooth hardness and protecting tooth enamel; fructooligosaccharides mainly promote the proliferation of probiotics and balance the oral flora. The research of the present invention shows that compounding lysozyme, lactoferrin, probiotics, regenerated silicon and fructooligosaccharides to the compound DM80Bu20 can synergistically enhance the antibacterial effect on harmful bacteria in the oral cavity, and can better inhibit the production of bacteria and dental calculus The formation of dental plaque, reduce gingival bleeding, long-lasting antibacterial.

Preferably, in the toothpaste, the total number of parts of one or more of lysozyme, lactoferrin, probiotics, regenerated silicon, and fructooligosaccharides is 0.5-2 parts.

Preferably, the humectant contains one or more of sorbitol, propylene glycol, glycerin and polyethylene glycol.

Preferably, the friction agent contains one or more of silicon dioxide, hydrated silica, aluminum hydroxide, calcium bicarbonate, hydroxyapatite or calcium hydrogen phosphate.

Preferably, the binder includes one or more of sodium carboxymethylcellulose, hydroxyethylcellulose, carrageenan, carbomer, polyvinylpyrrolidone or xanthan gum.

Preferably, the foaming agent comprises sodium lauryl sulfate, sodium lauroyl sarcosinate, sodium lauroyl glutamate, fatty amidopropyl methyl betaine, cocoamidopropyl methyl betaine, coconut oil One or more of sodium acyl glutamate, polyoxyethylene hydrogenated castor oil 40.

Preferably, the essence comprises peppermint essence.

Preferably, the sweetener contains one or more of sodium saccharin, aspartame and stevioside.

Preferably, the chelating agent contains one or more of pyrophosphate, phosphate, and disodium EDTA.

Preferably, the preservative contains one or more of potassium sorbate, methylparaben, and propylparaben.

Preferably, the probiotics comprise one or more of Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus plantarum and Lactobacillus salivarius.

Compared with prior art, the beneficial effect of the present invention is:

(1) The present invention provides antimicrobial peptide compound DM80Bu20 for the new application of suppressing oral harmful bacteria, this antimicrobial peptide compound DM80Bu20 has good effect on Candida albicans, Staphylococcus aureus, MRSA, Streptococcus mutans, Porphyromonas gingivalis Antibacterial activity, MIC value is 12.5 ~ 25g/mL, no inhibitory activity on the probiotic Streptococcus sanguis at the test concentration, MIC value is greater than 200μg/mL, it has specific selective antibacterial effect, and is safe and non-toxic, available In the preparation of various oral care products.

(2) In the present invention, the antibacterial peptide compound DM80Bu20 is compounded with other functional ingredients (lysozyme, lactoferrin, probiotics, regenerated silicon, fructooligosaccharides) to prepare antibacterial toothpaste, and each ingredient synergistically inhibits harmful bacteria in the oral cavity, protecting the beneficial bacteria, effectively maintain the balance of oral flora, reduce the formation of dental calculus and dental plaque, reduce gum bleeding, and the prepared toothpaste has a stable antibacterial effect, which is beneficial to long-term storage.

Detailed ways

The present invention will be further described below in combination with specific embodiments, but the examples do not limit the present invention in any form. Unless otherwise specified, the raw material reagents used in the examples of the present invention are conventionally purchased raw material reagents.

The compound DM80Bu20 of the present invention is a white powder, containing a structural skeleton similar to natural host defense peptides (Host defense peptides, HDP). The difference is that unnatural amino acids are introduced into the structure, thereby increasing the stability of the compound. Its structural formula is as follows Shown:

Tested strains:

Candida albicans ATCC SC5314;

Staphylococcus aureus ATCC 6538;

Methicillin-resistant Staphylococcus aureus18908;

Streptococcus mutans ATCC UA159;

Porphyromonas gingivalis ATCC 33277;

Streptococcus sanguis ATCC 10556.

Embodiment 1 DM80Bu20 in vitro antibacterial activity test

1. Experimental method

(1) Strain culture

Incubate Candida albicans ATCC SC5314 in RPMI 1640 medium at 37°C, 80% humidity, and 5% CO ₂ ;

Staphylococcus aureus ATCC 6538 was incubated in TSB medium at 37°C and 5% CO ₂ ;

Methicillin-resistant Staphylococcus aureus18908 was incubated in TSB medium at 37°C and 5% CO ₂ ;

Porphyromonas gingivalis ATCC 33277 was cultured in BHI medium with hemin (haemin, 5 μg/mL) and vitamin K (menadione, 1 μg/mL) at 37°C under strict anaerobic conditions;

Streptococcus mutans Clarke Streptococcus mutans Clarke Streptococcus mutans ATCC UA159 was cultured in Brain Heart Infusion Agar/Broth at 37°C and 5% CO ₂ ;

Streptococcus sanguis ATCC 10556 was cultured in medium Brain Heart Infusion Agar/Broth at 37°C and 5% CO ₂ .

(2) Compound preparation

The compound DM80Bu20 powder was dissolved in DMSO, adjusted to a concentration of 10 mg/mL, and stored in a refrigerator until use.

(3) Determination of minimum inhibitory concentration (MIC)

Using the Clinical and Laboratory Standards Institute (CLSI) regulations as a reference, the microbroth double dilution method was implemented to determine the minimum inhibitory concentration (MIC) of the compound against different microorganisms. This method is recognized as an effective method by the Clinical and Laboratory Standardization Institute (CLSI), widely used and widely used, and can quickly realize the detection of drug interaction effects in a short period of time, and can draw reliable conclusions through further analysis of experimental data. The results are stable and highly reproducible.

For Candida albicans ATCC SC5314, use physiological saline to adjust the concentration of the tested strain to 0.5-2.5×10 ⁶ CFU mL ^-1 , after that, dilute the bacterial suspension to 0.5-2.5×10 ⁴ CFU in RPMI 1640 medium mL ^-1 was prepared as the bacterial solution to be tested. Take 200 μL of the bacterial solution to be tested and add it to the first well of a flat-bottomed 96-well plate, and then add 100 μL of the bacterial solution to be tested to each well. Add 2 μL of the stock solution of the compound to be tested (100 μg/mL) to the first well, pipette and mix in the first well, take 100 μL of the homogeneous suspension, add it to the second well, pipette and mix, repeat to the last well, discard excess bacteria. The 96-well plate was incubated at 37°C for 24 hours, and MIC was defined as the minimum drug concentration that could inhibit bacterial growth by visual inspection. The experiment was repeated in three groups, and econazole was selected as a positive control.

For Staphylococcus aureus ATCC 6538 and methicillin-resistant Staphylococcus aureus 18908, use MH medium to adjust the tested strains to a concentration of 0.5-2.5×10 ⁵ CFU mL-1 to prepare the test bacterial solution. Take 200 μL of the bacterial solution to be tested and add it to the first well of a flat-bottomed 96-well plate, and then add 100 μL of the bacterial solution to be tested to each well. Add 2 μL of the stock solution of the compound to be tested (100 μg/mL) to the first well, pipette and mix in the first well, take 100 μL of the homogeneous suspension, add it to the second well, pipette and mix, repeat to the last well, discard excess bacteria. The 96-well plate was incubated at 37°C and 5% CO2 for 24 hours, and MIC was defined as the minimum drug concentration that could inhibit bacterial growth by visual inspection. The experiment was repeated three times, and vancomycin was selected as a positive control.

For Streptococcus mutans ATCC UA159 and Streptococcus sanguis ATCC 10556, use BHI medium to adjust the concentration of the tested strains to 0.5-2.5×10 ⁶ CFU mL-1, and prepare the bacterial solution to be tested. Take 200 μL of the bacterial solution to be tested and add it to the first well of a flat-bottomed 96-well plate, and then add 100 μL of the bacterial solution to be tested to each well. Add 2 μL of the stock solution of the compound to be tested (100 μg/mL) to the first well, pipette and mix in the first well, take 100 μL of the homogeneous suspension, add it to the second well, pipette and mix, repeat to the last well, discard excess bacteria. The 96-well plate was incubated at 37°C and 5% CO ₂ for 24 hours, and MIC was defined as the minimum drug concentration that could inhibit the growth of bacteria by visual inspection. The experiment was repeated three times, and vancomycin was selected as a positive control.

For Porphyromonas gingivalis ATCC 33277, use BHI medium to add hemin (5 μg/mL) and vitamin K (1 μg/mL) to adjust the concentration of the tested strain to 0.5-2.5×10 ⁶ CFU mL -1, prepared as the bacteria solution to be tested. Take 200 μL of the bacterial solution to be tested and add it to the first well of a flat-bottomed 96-well plate, and then add 100 μL of the bacterial solution to be tested to each well. Add 2 μL of the stock solution of the compound to be tested (100 μg/mL) to the first well, pipette and mix in the first well, take 100 μL of the homogeneous suspension, add it to the second well, pipette and mix, repeat to the last well, discard excess bacteria. The 96-well plate was incubated at 37°C for 24 hours under strict anaerobic conditions, and MIC was defined as the minimum drug concentration that could inhibit bacterial growth by visual inspection. The experiment was repeated three times, and chlorhexidine was selected as a positive control.

2. Antibacterial test results

Table 1

Table 1 is the antibacterial result of compound DM80Bu20 to oral bacteria, as can be seen from the results in the table, compound DM80Bu20 has significant inhibitory effect on Candida albicans, Staphylococcus aureus, MRSA, Streptococcus mutans, Porphyromonas gingivalis Bacteria effect, MIC value between 12.5-25μg/mL; no inhibitory activity on the probiotic Streptococcus sanguis at the test concentration, MIC value greater than 200μg/mL; in addition, because part of Helicobacter pylori is latent in Candida albicans, Therefore, it is important to develop active compounds that are effective against Candida albicans, which can also indirectly prevent and block the incubation of Helicobacter pylori in the oral microbial environment.

Embodiment 2 compound DM80Bu20 hemolytic activity test

1. Experimental method

Fresh human blood was washed three times with Tris buffer (TBS), and the collected human red blood cells (hRBCs) were diluted to 5% (v/v) with TBS. On a 96-well plate, compound DM80Bu20 was diluted to 3.13-400 μg/mL by two-fold serial dilution. After mixing equal volumes of hRBCs suspension and compound DM80Bu20 solution, they were incubated at 37°C for 1 hour. TBS was used as the blank control, and Triton X-100 (TBS) 3.2 μg/ml and hRBCs equal volume mixed solution were used as the positive control. After centrifugation, transfer 80 μl of the supernatant in each well to another 96-well plate, read the OD value of the solution to be tested in the 96-well plate at 405 nm with a microplate reader, and calculate the hemolysis percentage by the following formula:

2. Experimental results

The hemolytic activity (HC ₅₀ ) of DM80Bu20 was 200 μg/mL, indicating that the compound DM80Bu20 has low hemolytic activity.

Example 3 Cytotoxic activity test of compound DM80Bu20

1. Experimental method

Prepare a single cell suspension with DMEM medium containing 10% fetal bovine serum at a cell density of 1×10 ⁶ CFU/mL, and inoculate 1×10 ⁴ cells per well into a 96-well plate with a volume of 100 μL per well. Cells were incubated at 37°C for 24 hours. After the old medium was removed, medium containing different concentrations of compound DM80Bu20 was added, and three replicate wells were set for each concentration. After the cells were cultured at 37° C. for 24 hours, 10 μL of MTT solution (5 mg/mL, prepared in PBS) was added to each well, and the incubation was continued for 4 hours to terminate the culture. Carefully suck off the culture supernatant in the wells, add 150 μL DMSO to each well, and shake on the shaker for 10 minutes to fully dissolve the crystals. On the same 96-well plate, cells treated without any compound DM80Bu20 were included as a control group, and a blank group with no inoculation of cells but only DMSO was included. Read the OD value of the solution to be tested in the 96-well plate at 570nm with a microplate reader, and the cell viability of each well is calculated by the following formula:

2. Experimental results

As a result, the cytotoxicity (IC ₅₀ ) of the compound DM80Bu20 was 100 μg/mL.

Embodiment 4 acute eye irritation test

1. Materials and methods

1.1 Test object: 0.1% compound DM80Bu20 aqueous solution was prepared with sterile water, a colorless and transparent liquid, and the test prototype was used for testing during the experiment.

1.2 Experimental animals and feeding environment

Experimental animals: 3 ordinary New Zealand white rabbits, weighing 1.80-2.20kg, provided by Huadong Xinhua Experimental Animal Farm in Huadu District, Guangzhou City, experimental animal production license number: SCXK (Guangdong) 2019-0023, qualified quality Certificate No.: No.44007600007472, the animals should be quarantined for at least 3 days before the experiment, and they will be used after passing the test.

Breeding environment: After the animals are purchased, they are reared in single cages in the animal room of the Guangzhou Institute of Quality Supervision and Inspection, temperature: 18-26°C, relative humidity: 40%-70%, experimental animal use license number: SYXK (Guangdong) 2018 -0137.

Feed: Provided by Beijing Keao Xieli Feed Co., Ltd., feed production license number: SCXK (Beijing) 2019-0003, feed certificate: 1112622000019812.

1.3 Test method

1.3.1 Check the two eyes of the experimental rabbits (including the use of sodium fluorescein) 24 hours before the start of the test, and the qualified rabbits are used for the test.

1.3.2 Gently pull the lower eyelid of one eye of the rabbit, and drop 0.1ml of the test substance into the conjunctival sac, so that the upper and lower eyelids are passively closed for 1 second, so as to prevent the bag from being lost. The other eye was not treated as self-control. Do not rinse the eyes within 24 hours after instilling the test substance.

1.3.3 Check the eyes of the animals 1h, 24h, 48h and 72h after instilling the test substance. After 24 hours of observation and recording, the eyes of all animals were further examined with sodium fluorescein. In each inspection, the integral of eye irritation reaction is recorded according to the scoring standard of "Cosmetics Safety Technology" (2015 edition) acute eye irritation/corrosion test-eye damage. According to the "Safety and Technical Specifications for Cosmetics" (2015 Edition) acute eye irritation/ Corrosion Test - Eye Irritation Grading To determine the degree of eye irritation of the test substance.

2. Experimental results

table 3

Acute Eye Irritation Test Results of Compound DM80Bu20 on Rabbits

As can be seen from Table 3, the results of the acute eye irritation test of the compound DM80Bu20 on rabbits all showed no irritation.

Embodiment 5 acute skin irritation test

1. Materials and methods

1.1 Test substance: Prepare 0.1% compound DM80Bu20 aqueous solution with sterile water, colorless and transparent liquid, and use the prototype of the test substance for testing.

1.2 Experimental animals and feeding environment

Experimental animals: 4 ordinary New Zealand white female rabbits, weighing 1.80kg-2.20kg, provided by Dongxinhua Experimental Animal Farm in Huadu District, Guangzhou City, experimental animal production license number: SCXK (Guangdong) 2019-0023, quality Certificate of Conformity No.: No.44007600007434. Animals should be quarantined for at least 3 days before the test, and they will be used after passing the test.

Breeding environment: After the animals are purchased, they are reared in single cages in the animal room of the Guangzhou Institute of Quality Supervision and Inspection, temperature: 18°C-26°C, relative humidity: 40%-70%, experimental animal use license number: SYXH (Guangdong) 2018-0137.

Feed: Provided by Beijing Keao Xieli Feed Co., Ltd., experimental animal feed production license number: SCXK (Beijing) 2019-0003, quality certificate number: 1112622000019812.

1.3 Test method

1.3.1 Cut off the hair on both sides of the back and spine of the experimental animals 24 hours before the test, and the range of hair removal is 3cm×3cm.

1.3.2 Take 0.5ml of the test substance and apply it on one side of the skin, covering an area of 2.5cm×2.5cm, then cover it with two layers of gauze (2.5cm×2.5cm) and a layer of cellophane, and then use non-irritating adhesive tape and bandage be fixed. The skin on the other side was not treated as a control. The closed test was adopted, and the application time was 4 hours. After the test, remove the residual test substance with warm water.

1.3.3 After removing the test substance for 1h, 24h, 48h, and 72h, observe the skin reaction at the application site, and perform skin reaction scoring according to the "Safety and Technical Specifications for Cosmetics" (2015 Edition) skin irritation/corrosion test, and use the test substance The average value of the animal points is comprehensively evaluated, and the skin irritation intensity is determined according to the "Safety and Technical Specifications for Cosmetics" (2015 edition) skin irritation/corrosion test according to the highest point average value at each observation time point of 24h, 48h and 72h. During the test, observe whether there are other symptoms other than skin irritation.

2. Test results

Table 4

Acute Skin Irritation Test Results of Compound DM80Bu20 on Rabbits

The results in Table 4 show that the antimicrobial peptide compound DM80Bu20 has no irritation to rabbit skin.

Example 6 Compound DM80Bu20 Acute Oral Toxicity Test

1. Materials and methods

1.1 Test substance

Test group: a 0.1% aqueous solution of compound DM80Bu20 was prepared with sterile water, a colorless transparent liquid, and tested as the prototype of the test substance.

1.2 Animals and feeding environment

Preparation method: Weigh 5.00g of the sample, prepare it to 10.0mL with pure water, and set aside.

10 SPF grade KM mice, weighing 18g-22g, were provided by the Guangdong Provincial Medical Experimental Animal Center, experimental animal production license number: SCXK (Guangdong) 2018-0002, quality certificate number: No.44007200074743. The feed was provided by the Guangdong Medical Experimental Animal Center. After the animals are purchased, they will be used in the animal room of the Guangzhou Institute of Quality Supervision and Inspection after passing the quarantine inspection. The license number for the use of experimental animals: SYXK (Guangdong) 2018-0137, animal room. Temperature: 20℃～26℃, relative humidity: 40%～70%.

1.3 Test method

This test adopts a maximum test, and only one dose group of 5000mg/kg body weight is set up, that is, 10 mice that have passed the quarantine are taken, and the male and female are divided in half. Animals were fasted overnight without water restriction before the experiment. Gavage once on an empty stomach, with a volume of 10.0mL/kg body weight, continue to fast for 3h-4h after poisoning, observe and record the poisoning performance, death number and time of death of the animals, the observation period is 14 days, and no one died at the end of the test Animals are weighed. Judgment of toxicity grade according to the evaluation regulations of acute oral toxicity test.

2. Test results

After exposure, no poisoning or death was found in the mice during the observation period. After the experiment, the surviving animals were dissected, and no abnormalities were found with the naked eye. See Table 5 for details.

Table 5 The results of the acute oral toxicity test of the test substance on mice

The results showed that the acute oral toxicity of the compound DM80Bu20 to female and male KM mice was LD50>5000 mg/kg body weight, which was practically non-toxic.

The preparation of embodiment 7 antibacterial toothpaste

(1) This example provides a series of toothpastes containing the compound DM80Bu20, and the specific formulations are shown in Table 6.

Table 6

Among them, the probiotics in toothpaste 4, 5, 7, and 8 are Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus acidophilus, Bifidobacterium, and Lactobacillus salivarius, respectively.

(2) toothpaste preparation method

The company's current production process adopts a one-step paste method to complete the toothpaste paste preparation:

S1 Mix humectant, foaming agent (liquid), sweetener, chelating agent, preservative with water, and stir for 5-15 minutes;

S2 Mix the friction agent, adhesive, foaming agent (solid state) with the aqueous solution prepared in S1 above, keep the vacuum at -0.090Mpa～-0.096Mpa, and stir for 20-50 minutes;

Add essence and active ingredients in S3, keep the degree of vacuum at -0.090Mpa to -0.096Mpa, and stir for 10 to 50 minutes to obtain toothpaste 1 to 9.

The antibacterial performance test of embodiment 8 toothpaste

1. Experimental method

According to 7.3 of QB 2738-2012 "Evaluation Method for Antibacterial and Antibacterial Effects of Daily Chemical Products" as the detection basis, Candida albicans, Staphylococcus aureus, MRSA (methicillin-resistant Staphylococcus aureus), Streptococcus mutans, The inhibition rate of Porphyromonas gingivalis, Escherichia coli, Streptococcus sanguinis, etc. is an index for investigation. The toothpaste 1-9 prepared in Example 7 was placed at room temperature for 24 hours before the antibacterial test was performed. The measurement results are shown in Table 7.

2. Experimental results

Table 7 The bacteriostatic rate (%) of toothpaste 1-9 placed at room temperature for 24 hours

As can be seen from Table 7, the toothpaste comprising the antibacterial peptide compound DM80Bu20 prepared by the present invention is effective against Candida albicans, Staphylococcus aureus, MRSA (methicillin-resistant Staphylococcus aureus), Streptococcus mutans, Porphyromonas gingivalis bacteria and Escherichia coli have reached over 90%, and toothpaste 2 and 3 have a bacteriostatic rate of 99-100% against harmful bacteria in the oral cavity; The antibacterial effect of other functional ingredients (lysozyme, lactoferrin, probiotics, regenerated silicon, fructooligosaccharides) has been improved, indicating that lysozyme, lactoferrin, probiotics, regenerated silicon, fructooligosaccharides The compounding of DM80Bu20 can effectively improve its antibacterial effect. Therefore, based on the high cost of the antimicrobial peptide compound DM80Bu20, other active ingredients can be compounded to enhance its antibacterial effect.

In addition, the antibacterial rate of each formula toothpaste against Streptococcus sanguinis was lower than 5%, which was mainly due to the antibacterial effect of other auxiliary materials in toothpaste. Generally speaking, the antibacterial toothpaste of the present invention can well inhibit the harmful bacteria in the oral cavity without harming the beneficial bacteria, and can effectively balance the flora.

The antibacterial stability test of embodiment 9 toothpaste

1. Experimental method

With reference to the evaluation method of QB 2738-2012 antibacterial and antibacterial effects of daily chemical products, toothpaste 1-9 prepared in Example 7 was measured against Candida albicans, Staphylococcus aureus, MREA, Streptococcus mutans, Porphyromonas gingivalis, large coli Inhibition of Bacillus and Streptococcus sanguinis. After toothpastes 1-9 were placed at 45°C for 30 days and 90 days, toothpaste antibacterial tests were performed respectively. The measurement results are shown in Table 8.

2. Experimental results

Table 8 Antibacterial rate (%) after toothpaste 1-9 were placed at 45°C for 30 days and 90 days respectively

As can be seen from the results in Table 8, after the toothpastes of various formulas were placed at 45°C for 30 days and 90 days, with the prolongation of the storage period, the bacteriostatic effect was relatively reduced, but the overall still had a good bacteriostatic effect, indicating that the toothpaste of the present invention The antibacterial effect of toothpaste is stable, which is good for long-term storage.

Embodiment 10 toothpaste use effect (spot index measurement)

1. Test method

(1) Subject population and grouping

80 subjects with normal physical examination indicators; 12 natural anterior teeth in the subject population, and no large-area filling or crown restoration; teeth with exogenous stains.

It was randomly divided into test group 1, test group 2, test group 3 and test group 4, with 20 people in each group, half men and half women; wherein the sample used by test group 1 was toothpaste 1, and the sample used by test group 2 was toothpaste 3, The sample used in test group 3 was toothpaste 4, and the sample used in test group 4 was toothpaste 6.

(2) Brushing method

The subjects were uniformly distributed toothpaste and toothbrush, and brushed their teeth once a day in the morning and evening according to the BASS method, a total of 2 times, each time 1min.

(3) Experimental steps

The subjects were tested for stain index (baseline) before use, and then tested for stain index after 3 months of continuous use, and compared the changes in the stain index of the subjects before and after using toothpaste 1, toothpaste 3, toothpaste 4, and toothpaste 6.

Staining index scoring method, the specific scoring rules are as follows:

0 points - no stains exist;

1 point - there are stains, and the size of the stains does not exceed 1/3 of the tooth surface, and the degree is mild stains (yellow or yellowish brown);

2 points——There are stains, the stains exceed 1/3 of the tooth surface and do not exceed 2/3 of the tooth surface, and the degree of staining is moderate (medium brown);

3 points - There are stains, and the size of the stains exceeds 2/3 of the tooth surface, and the degree is severe stains (dark brown or black).

The teeth to be examined are the labial and lingual surfaces of the mandibular anterior teeth and the labial surface of the maxillary anterior teeth. Each tooth surface stain index count is divided into the product of the stain area score and the stain degree score for that tooth surface. The stain index score of each subject is the average of the stain index scores of each tooth surface, namely:

Stain index = ∑ (area scoring degree score) / total number of tooth surfaces checked

2. Test results

Table 9

Note: Reduction rate of stain index=(average value of stain index before use-average value of stain index after use)/average value of stain index before use×100%.

As can be seen from the results in Table 9: toothpaste of the present invention has a reducing effect on tooth stains, and as the content of DM80Bu20 increases, its effect of removing stains also improves; in addition, toothpaste 4 and 6 are compared with toothpaste 1 , which has a higher reduction rate of the tester's stain index, indicating that the addition of lysozyme, lactoferrin, probiotics, regenerated silicon, and fructooligosaccharides to the toothpaste of the present invention can effectively improve the effect of removing exogenous stains , so by adding other active ingredients, it is possible to reduce the use cost of DM80Bu20 and achieve a good effect of removing stains on the tooth surface. Among them, toothpaste 6 has the highest reduction rate of stain index, indicating the effect of adding lysozyme and DM80Bu20. most.

In addition, after the subjects used the above toothpaste, their dental calculus, bleeding gums, oral ulcers, bad breath, etc. were all effectively improved. Compared with the experimental group of toothpaste 1, toothpaste 4 and 6 had more obvious improvement.

Apparently, the above-mentioned embodiments of the present invention are only examples for clearly illustrating the present invention, rather than limiting the implementation of the present invention. For those of ordinary skill in the art, other changes or changes in different forms can be made on the basis of the above description. It is not necessary and impossible to exhaustively list all the implementation manners here. All modifications, equivalent replacements and improvements made within the spirit and principles of the present invention shall be included within the protection scope of the claims of the present invention.

Claims

Application of antibacterial peptide compound DM80Bu20 in the preparation of antibacterial toothpaste.
According to the described application of claim 1, it is characterized in that the bacteriostasis is to inhibit harmful bacteria in the oral cavity.
According to the described application of claim 2, it is characterized in that the harmful bacteria in the oral cavity comprise one of Candida albicans, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Streptococcus mutans, and Porphyromonas gingivalis. species or species of bacteria.
A kind of antibacterial toothpaste, it is characterized in that, described toothpaste comprises the antibacterial peptide compound DM80Bu20 described in claim 1.
The toothpaste according to claim 4, characterized in that the toothpaste is composed of the following components by weight: 0.001-0.1 part of antibacterial peptide compound DM80Bu20, 30-45 parts of moisturizing agent, 20-25 parts of friction agent, adhesive 0.5-2 parts, foaming agent 0.5-3 parts, essence 0.5-1.5 parts, sweetener 0.1-0.5 parts, chelating agent 1-3 parts, preservative 0.1-2 parts, deionized water 20-35 parts.
According to the described toothpaste of claim 5, it is characterized in that, described toothpaste also comprises one or more components in lysozyme, lactoferrin, probiotics, regenerative silicon, fructooligosaccharide.
The toothpaste according to claim 6, characterized in that, in the toothpaste, the total number of one or more components in lysozyme, lactoferrin, probiotics, regenerated silicon, and fructooligosaccharides is 0.5 to 2 parts .
According to the toothpaste according to claims 5-7, it is characterized in that, the humectant comprises one or more of sorbitol, propylene glycol, glycerin and polyethanol; the friction agent comprises silicon dioxide, hydrated silica, hydroxide One or more of aluminum, calcium bicarbonate, hydroxyapatite or calcium hydrogen phosphate; the binder includes sodium carboxymethylcellulose, hydroxyethyl cellulose, carrageenan, carbomer, polyethylene One or more of pyrrolidone or xanthan gum; the foaming agent includes sodium lauryl sulfate, sodium lauroyl sarcosinate, sodium lauroyl glutamate, fatty amidopropyl methyl betaine, coconut oil One or more of amidopropyl methyl betaine, sodium cocoyl glutamate, polyoxyethylene hydrogenated castor oil 40.
According to the toothpaste according to claims 5-7, it is characterized in that, the essence comprises peppermint essence; the sweetener comprises one or more of saccharin sodium, aspartame, and stevioside; the chelating agent comprises One or more of pyrophosphate, phosphate, and disodium EDTA; the preservative includes one or more of potassium sorbate, methylparaben, and propylparaben.
The toothpaste according to claim 6 or 7, wherein the probiotics comprise one or more bacteria of Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus plantarum and Lactobacillus salivarius.