CN113679632B

CN113679632B - Application of antibacterial peptide compound DM80Bu20 in preparation of antibacterial toothpaste

Info

Publication number: CN113679632B
Application number: CN202111022238.0A
Authority: CN
Inventors: 刘润辉; 褚夫江; 李毅苹; 耿帅峰
Original assignee: Haoyikang Biotechnology Guangzhou Co ltd
Current assignee: Haoyikang Biotechnology Guangzhou Co ltd
Priority date: 2021-09-01
Filing date: 2021-09-01
Publication date: 2022-09-27
Anticipated expiration: 2041-09-01
Also published as: WO2023029137A1; CN113679632A

Abstract

The invention provides application of an antibacterial peptide compound DM80Bu20 in preparation of antibacterial toothpaste and the antibacterial toothpaste. The research of the invention reveals that the antibacterial peptide compound DM80Bu20 can selectively inhibit the activity of most oral pathogenic bacteria, has no inhibition effect on oral probiotics, has low hemolytic activity and low cytotoxicity, high safety and good stability, and can be used for preparing oral care toothpaste products, further, the antibacterial toothpaste prepared by using the compound DM80Bu20 also has the effect of selectively inhibiting the oral pathogenic bacteria, in addition, the compound DM80Bu20 can realize the synergistic antibacterial effect with the antibacterial peptide compound DM80Bu20 by compounding specific functional components, the formation of dental calculus and dental plaque is reduced, the gingival bleeding is reduced, the dental caries and the oral ulcer are prevented, the halitosis is relieved, and the prepared toothpaste has good 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 particularly, relates to the application of an antibacterial peptide compound DM80Bu20 in preparing bacteriostatic toothpaste.

Background

Oral health is an important part of the whole body health, and oral diseases such as caries, periodontal disease and the like destroy tooth hard tissues and supporting tissues around the teeth, and besides affecting the functions of chewing, speaking, beauty and the like, social difficulties and psychological disorders are caused, and the main reason of the oral diseases is the formation of dental plaque. The effective solution to the formation of dental plaque is the first task in research and development of the functional oral care toothpaste product. With the concern of people on the health problem of teeth, nearly 50% of people think that the Chinese herbal toothpaste is safer than the traditional chemical toothpaste, so the Chinese herbal toothpaste is more and more popular, however, researchers have found that the antibacterial potential of the common Chinese herbal toothpaste in the market to the common oral bacteria is evaluated, the Chinese herbal components in the toothpaste have no selectivity on the inhibition and killing action of various oral bacteria, the strength of the bacterial action generally depends on the concentration of the medicine, in addition, the sensitivity of candida albicans to various Chinese herbal toothpastes is generally low, and the risk of candida albicans infection [ Chenhong hong, Dingxi, and Zhanghua ] is presumed to be caused by long-term use of the toothpastes, and the antibacterial potential of the five Chinese herbal toothpastes to the common oral bacteria is compared, Zhongguan microorganism , 2007.19 (4): p.351-353 ]. In addition, most of antibacterial active extracts contained in the existing Chinese herbal medicine toothpaste belong to unstable components in an acid toothpaste matrix, so that long-time stability and activity maintenance are difficult to guarantee; moreover, the existing antibacterial toothpaste has broad-spectrum antibacterial activity and no selective specificity to pathogenic bacteria and probiotics.

Antibacterial peptides (antibacterial peptides) are widely distributed in nature, are small molecular polypeptides which are produced by a host and resist pathogen infection, are also an important component in a biological natural immune system, and are used as novel antibacterial drug ingredients with development potential at the present stage, and a patent CN201910047525.3 discloses an oral composition containing probiotics and antibacterial peptides, which can improve the problem of oral inflammation, has the functions of resisting bacteria, diminishing inflammation, relieving pain and diminishing swelling, and has important application value in the aspect of treating oral diseases. Therefore, more novel antibacterial peptide active substances are needed to be found, the defect that the traditional Chinese herbal medicine toothpaste has no selective inhibition on various oral pathogenic bacteria and probiotics is overcome, and a material basis is provided for the development of oral antibacterial toothpaste products.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide the application of a novel antibacterial peptide compound DM80Bu20 in inhibiting oral harmful bacteria and the antibacterial toothpaste prepared by the antibacterial peptide compound DM80Bu20, the antibacterial peptide compound DM80Bu20 disclosed by the invention can selectively inhibit the activity of most oral pathogenic bacteria, has no inhibiting effect on oral probiotics, and has low hemolytic activity and low cytotoxicity, high safety and good stability; further, the compound DM80Bu20 is used for preparing compound toothpaste compounded with specific functional components, and the obtained antibacterial toothpaste product has good antibacterial stability.

The invention aims at providing the application of an antibacterial peptide compound DM80Bu20 in inhibiting oral harmful bacteria.

The invention also aims to provide application of the antibacterial peptide compound DM80Bu20 in preparing antibacterial toothpaste.

It is still another object of the present invention to provide a bacteriostatic toothpaste comprising the antibacterial peptide compound DM80Bu 20.

The above purpose of the invention is realized by the following technical scheme:

the invention provides application of an antibacterial peptide compound DM80Bu20 in inhibiting oral harmful bacteria.

The structural formula of the antibacterial peptide compound DM80Bu20 is as follows:

preferably, the oral cavity harmful bacteria comprise one or more of candida albicans, staphylococcus aureus, methicillin resistant staphylococcus aureus, streptococcus mutans, porphyromonas gingivalis.

Among them, candida albicans causes precancerous lesions or states such as candida stomatitis (chiorosis, antibiotic stomatitis, denture stomatitis, and candida leukoplakia), candida cheilitis, candida angular stomatitis, lozenge glossitis, leukoplakia, lichen planus, lupus erythematosus, etc., and since some helicobacter pylori is latent in candida albicans, active compounds effective against candida albicans are intensively developed, and the latent of helicobacter pylori in the oral microbial environment can be indirectly prevented or blocked. Staphylococcus aureus and MRSA are pathogenic bacteria of furuncle and carbuncle of maxillofacial region, loose connective tissue inflammation, oral cavity and maxillofacial wound infection, infantile angular cheilitis, periodontitis, and mixed infection of dental pulp root tip and periphery. Mutans streptococci are the main causative bacteria of caries and can cause secondary infections such as bacteremia, endocarditis, and the like. Porphyromonas gingivalis is a major pathogenic bacterium involved in periodontal disease. Streptococcus sanguis is one of bacteria which are planted in oral cavity earlier, resident bacteria in oral cavity, and is suspicious pathogenic bacteria for antagonizing caries and periodontal disease by producing hydrogen peroxide and haemagainin, and is a type of oral cavity beneficial bacteria.

The compound DM80Bu20 is an amino acid polymer simulating natural polypeptide, is a mimic of Host Defense Peptide (HDP) of a natural antibacterial agent, has an antibacterial mechanism similar to that of the HDP, has an action target of a bacterial cell membrane, and achieves the aim of sterilization by destroying the integrity of the cell membrane. According to the invention, the antibacterial activity experiment of the compound DM80Bu20 for oral bacteria is carried out, and the result shows that the antibacterial peptide compound DM80Bu20 has good antibacterial activity for Candida albicans, Staphylococcus aureus, MRSA, Streptococcus mutans and Porphyromonas gingivalis, and the MIC value is 12.5-25 g/mL; the probiotic streptococcus sanguis has no inhibitory activity under the test concentration, and the MIC value is more than 200 mug/mL; the compound is shown to be capable of selectively inhibiting most oral pathogenic bacteria, and has no inhibition effect on oral probiotics. In addition, a hemolytic experiment and a cytotoxicity experiment are carried out on the antibacterial peptide compound DM80Bu20, and the result shows that the compound DM80Bu20 has low hemolytic activity and low cytotoxicity and good safety.

In addition, the toothpaste developed by further adopting the compound DM80Bu20 has the bacteriostasis rate of 90-100% on Candida albicans, Staphylococcus aureus, MRSA (methicillin resistant Staphylococcus aureus), Streptococcus mutans, Porphyromonas gingivalis and Escherichia coli; the inhibition rate of the antibacterial toothpaste against streptococcus sanguis is lower than 10 percent, which shows that the antibacterial toothpaste can well inhibit harmful bacteria in the oral cavity without damaging beneficial bacteria and can effectively balance flora. In addition, after the antibacterial toothpaste disclosed by the invention is placed at 45 ℃ for 30 days and 90 days, the antibacterial effect is relatively reduced along with the prolonging of the storage period, but the whole antibacterial effect is still good, so that the antibacterial effect of the toothpaste disclosed by the invention is stable, and the toothpaste is beneficial to long-term storage. The antibacterial toothpaste provided by the invention can inhibit the generation of bacteria and the formation of dental calculus, reduce dental plaque, reduce gingival bleeding and inhibit bacteria for a long time by supplementing the defense components of harmful bacteria in the oral cavity through an external source.

Therefore, the application of the antibacterial peptide compound DM80Bu20 in preparing antibacterial toothpaste and the antibacterial toothpaste containing the antibacterial peptide compound DM80Bu20 are within the protection scope of the invention. The toothpaste is prepared by taking an antibacterial peptide compound DM80Bu20 as a main antibacterial active ingredient and assisting with other auxiliary materials.

As a preferred implementable method, the bacteriostatic toothpaste consists of the following components in parts by weight: 200.001-0.1 parts of antibacterial peptide compound DM80Bu200.001-45 parts of humectant, 20-25 parts of friction agent, 0.5-2 parts of adhesive, 0.5-3 parts of foaming agent, 0.5-1.5 parts of essence, 0.1-0.5 part of sweetening agent, 1-3 parts of chelating agent, 0.1-2 parts of preservative and 20-35 parts of deionized water.

Further, the toothpaste also comprises one or more of lysozyme, lactoferrin, probiotics, regenerated silicon and fructo-oligosaccharide. Wherein, the lysozyme inhibits pathogenic bacteria in the oral cavity and balances the flora in the oral cavity; lactoferrin can enhance mucosal immunity and reduce helicobacter pylori infection; the probiotics play a role in balancing oral flora and inhibiting pathogenic bacteria; the regenerated silicon can play a role in repairing caries, improving the hardness of teeth and protecting enamel; the fructo-oligosaccharide mainly promotes the proliferation of probiotics and balances the oral flora. The research of the invention shows that after the compound DM80Bu20 is compounded with lysozyme, lactoferrin, probiotics, regenerated silicon and fructo-oligosaccharide, the antibacterial effect on oral harmful bacteria can be synergistically enhanced, the generation of bacteria and the formation of dental calculus can be better inhibited, dental plaque is reduced, gingival bleeding is reduced, and the antibacterial effect is lasting.

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

Preferably, the humectant comprises one or more of sorbitol, propylene glycol, glycerol and polyethylene alcohol.

Preferably, the abrasive comprises one or more of silicon dioxide, hydrated silica, aluminium hydroxide, calcium bicarbonate, hydroxyapatite or calcium hydrogen phosphate.

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

Preferably, the foaming agent comprises one or more of sodium lauryl sulfate, sodium lauroyl sarcosinate, sodium lauroyl glutamate, fatty amidopropyl methyl betaine, cocamidopropyl methyl betaine, sodium cocoyl glutamate and polyoxyethylene hydrogenated castor oil 40.

Preferably, the flavour comprises mint flavour.

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

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

Preferably, the preservative comprises one or more of potassium sorbate, methyl paraben and propyl paraben.

Preferably, the probiotic comprises one or more of lactobacillus paracasei, lactobacillus rhamnosus, lactobacillus plantarum, lactobacillus salivarius.

Compared with the prior art, the invention has the beneficial effects that:

(1) the invention provides a new application of an antibacterial peptide compound DM80Bu20 in inhibiting oral harmful bacteria, wherein the antibacterial peptide compound DM80Bu20 has good antibacterial activity on Candida albicans, Staphylococcus aureus, MRSA, Streptococcus mutans and Porphyromonas gingivalis, the MIC value is 12.5-25 g/mL, the antibacterial peptide compound has no inhibitory activity on probiotic streptococcus sanguis under the test concentration, the MIC value is more than 200 mug/mL, the antibacterial peptide compound has a specific selective antibacterial effect, is safe and nontoxic, and can be used for preparing various oral care products.

(2) According to the invention, the antibacterial peptide compound DM80Bu20 and other functional components (lysozyme, lactoferrin, probiotics, regenerated silicon and fructo-oligosaccharide) are compounded to prepare the antibacterial toothpaste, the components synergistically inhibit harmful bacteria in the oral cavity, beneficial bacteria are protected, the balance of oral flora is effectively maintained, the formation of dental calculus and dental plaque is reduced, gingival bleeding is reduced, the prepared toothpaste has stable antibacterial effect, and long-term storage is facilitated.

Detailed Description

The present invention will be further described with reference to specific embodiments, but the present invention is not limited to the examples in any way. The starting reagents used in the examples of the present invention are those conventionally purchased, unless otherwise specified.

The compound DM80Bu20 is white powder, contains a structural skeleton similar to natural Host Defense Peptides (HDP), is different in that unnatural amino acids are introduced into the structure, so that the stability of the compound is improved, and the structural formula is shown as follows:

the test strains are:

candida albicans ATCC SC 5314;

staphylococcus aureus ATCC 6538;

methicillin-resistant Staphylococcus aureus (Methicillin-resistant Staphylococcus aureus 18908);

streptococcus mutans ATCC UA 159;

porphyromonas gingivalis ATCC 33277;

streptococcus sanguis ATCC 10556.

Example 1 DM80Bu20 in vitro bacteriostatic Activity test

1. Experimental method

(1) Culturing of bacterial strains

Candida albicans ATCC SC5314 was cultured in RPMI 1640 medium at 37 ℃ and 80% humidity, and 5% CO ₂ Incubating under the conditions of (a);

staphylococcus aureus ATCC 6538 in TSB medium at 37 deg.C and 5% CO ₂ Incubating under the conditions of (a);

methicillin-resistant Staphylococcus aureus (MRSA) Methicilin-resistant Staphylococcus aureus18908 in TSB medium at 37 deg.C and 5% CO ₂ Incubating under the conditions of (a);

porphyromonas gingivalis ATCC 33277, Hemeropherin (haemin, 5. mu.g/mL) and vitamin K (menadione, 1. mu.g/mL) are added into a BHI culture medium, and the mixture is cultured at 37 ℃ under strict anaerobic conditions;

streptococcus mutans Clarke Streptococcus mutans ATCC UA159 in culture medium Brain Heart Infusion Agar/Broth at 37 deg.C, 5% CO ₂ Culturing under the condition;

streptococcus sanguis ATCC 10556 in Brain Heart infusion agar/Broth medium at 37 deg.C and 5% CO ₂ Culturing under the condition.

(2) Preparation of Compounds

Compound DM80Bu20 powder was dissolved in DMSO to a concentration of 10mg/mL and stored in the refrigerator until use.

(3) Determination of Minimum Inhibitory Concentration (MIC)

The Minimal Inhibitory Concentrations (MIC) of compounds against different microorganisms were determined by the two-fold broth microdilution method, which is performed under the provisions of the Clinical and Laboratory Standards Institute (CLSI). The method is an effective method recognized by the Clinical and Laboratory Standardization Institute (CLSI), has wide and universal application range, can quickly realize detection of drug interaction effect in a short time, can obtain a reliable conclusion by further analyzing experimental data, and has stable result and high repeatability.

For Candida albicans ATCC SC5314, the test strain was adjusted to a concentration of 0.5 to 2.5X 10 using physiological saline ⁶ CFU mL ^-1 Thereafter, the bacterial suspension was diluted to 0.5-2.5X 10 in RPMI 1640 medium ⁴ CFU mL ^-1 Preparing the bacterial liquid to be detected. Adding 200 mu L of the bacterial liquid to be detected into the first hole of the flat-bottom 96-hole plate, and then adding 100 mu L of the bacterial liquid to be detected into each hole. Adding 2 mu L of stock solution (100 mu g/mL) of the compound to be detected into the first hole, uniformly blowing and beating the first hole, taking 100 mu L of uniform suspension, adding the second hole, uniformly blowing and beating the first hole, repeating the process until the last hole is reached, and discarding the redundant bacterial liquid. The 96-well plate was incubated at 37 ℃ for 24 hours, and the MIC was defined as the minimum drug concentration that inhibits bacterial growth observed visually. Three replicates of the experiment were performed and econazole was selected as a positive control.

For Staphylococcus aureus ATCC 6538 and methicillin-resistant Staphylococcus aureus18908, the test strains were adjusted to a concentration of 0.5-2.5X 10 using MH medium ⁵ And preparing CFU mL-1 into a bacterial liquid to be detected. Adding 200 mu L of the bacterial liquid to be detected into the first hole of the flat-bottom 96-well plate, and then adding 100 mu L of the bacterial liquid to be detected into each hole. Adding 2 mu L of stock solution (100 mu g/mL) of the compound to be detected into the first hole, uniformly blowing and beating the first hole, taking 100 mu L of uniform suspension, adding the second hole, uniformly blowing and beating the first hole, repeating the process until the last hole is reached, and discarding the redundant bacterial liquid. The 96-well plates were incubated at 37 ℃ and 5% CO2 for 24 hours, and the MIC was defined as the minimum drug concentration that inhibits bacterial growth observed visually. The experiment was performed in triplicate, andvancomycin was selected as a positive control.

For Streptococcus mutans ATCC UA159 and Streptococcus sanguis ATCC 10556, the test strains were adjusted to a concentration of 0.5 to 2.5X 10 using BHI medium ⁶ And preparing CFU mL-1 into a bacterial liquid to be detected. Adding 200 mu L of the bacterial liquid to be detected into the first hole of the flat-bottom 96-hole plate, and then adding 100 mu L of the bacterial liquid to be detected into each hole. Adding 2 mu L of stock solution (100 mu g/mL) of the compound to be detected into the first hole, uniformly blowing and beating the first hole, taking 100 mu L of uniform suspension, adding the second hole, uniformly blowing and beating the first hole, repeating the process until the last hole is reached, and discarding the redundant bacterial liquid. 96-well plates were incubated at 37 ℃ with 5% CO ₂ Incubated under conditions for 24 hours, and MIC was defined as the minimum drug concentration that inhibits bacterial growth observed by the naked eye. The experiment was repeated in three replicates and vancomycin was selected as the positive control.

For Porphyromonas gingivalis ATCC 33277, hemin (5. mu.g/mL) and vitamin K (1. mu.g/mL) were added to BHI medium to adjust the concentration of the test strain to 0.5-2.5X 10 ⁶ And preparing CFU mL-1 into a bacterial liquid to be detected. Adding 200 mu L of the bacterial liquid to be detected into the first hole of the flat-bottom 96-hole plate, and then adding 100 mu L of the bacterial liquid to be detected into each hole. Adding 2 mu L of stock solution (100 mu g/mL) of the compound to be detected into the first hole, uniformly blowing and beating the first hole, taking 100 mu L of uniform suspension, adding the second hole, uniformly blowing and beating the first hole, repeating the process until the last hole is reached, and discarding the redundant bacterial liquid. The 96-well plates were incubated at 37 ℃ for 24 hours under strictly anaerobic conditions, and the MIC was defined as the minimum concentration of drug that inhibits bacterial growth observed by the naked eye. The experiment was repeated in three replicates and chlorhexidine was selected as the positive control.

2. Results of bacteriostatic experiments

TABLE 1

The results in the table show that the compound DM80Bu20 has obvious bacteriostatic action on Candida albicans, Staphylococcus aureus, MRSA, Streptococcus mutans and Porphyromonas gingivalis, and the MIC value is 12.5-25 mug/mL; the probiotic streptococcus sanguis has no inhibitory activity under the test concentration, and the MIC value is more than 200 mug/mL; in addition, as part of helicobacter pylori is latent in candida albicans, active compounds effective against candida albicans are intensively developed, and the latent of helicobacter pylori in the oral microbial environment can be indirectly prevented and blocked.

EXAMPLE 2 Compound DM80Bu20 test for hemolytic Activity

1. Experimental methods

Fresh human blood was washed 3 times with Tris Buffer (TBS) and collected human red blood cells (hRBCs) were diluted to 5% (v/v) with TBS. Compound DM80Bu20 was diluted to 3.13-400. mu.g/mL on a 96-well plate using a two-fold gradient dilution method. Equal volumes of the hRBCs suspension and the compound DM80Bu20 solution were mixed and incubated at 37 ℃ for 1 hour. TBS was used as a blank control, and a mixed solution of Triton X-100(TBS) at 3.2. mu.g/ml and hRBCs at equal volumes was used as a positive control. After centrifugation, 80. mu.l of the supernatant from each well was transferred to another 96-well plate, and the OD at 405nm of the solution to be tested in the 96-well plate was read by a microplate reader, and the percentage of hemolysis was calculated by the following formula:

2. results of the experiment

Hemolytic Activity of DM80Bu20 (HC) ₅₀ ) 200. mu.g/mL, indicating that the compound DM80Bu20 has low hemolysis.

EXAMPLE 3 cytotoxicity Activity assay of Compound DM80Bu20

1. Experimental methods

Preparing single cell suspension with DMEM culture solution containing 10% fetal calf serum, and the cell density is 1 × 10 ⁶ CFU/mL, at 1X 10 per well ⁴ The individual cells were seeded in 96-well plates in a volume of 100. mu.L per well. The cells were cultured at 37 ℃ for 24 hours. After removal of the old medium, media containing different concentrations of compound DM80Bu20 were added, three duplicate wells being set for each concentration. After culturing the cells at 37 ℃ for 24 hours, 10. mu.L of MTT solution (5mg/mL in PBS) was added to each well, and incubation was continued for 4 hours to terminate the culture. Carefully suck off the culture in the holeThe supernatant was incubated, 150. mu.L of DMSO was added to each well, and the mixture was shaken on a shaker for 10 minutes to dissolve the crystals sufficiently. Cells treated without any compound DM80Bu20 were included as controls and a blank without seeding cells with DMSO alone in the same 96-well plate. Reading the OD value of the solution to be detected in the 96-well plate at 570nm by using a microplate reader, and calculating the cell survival rate of each well according to the following formula:

2. results of the experiment

As a result, the cytotoxicity (IC) of the compound DM80Bu20 was obtained ₅₀ ) It was 100. mu.g/mL.

EXAMPLE 4 acute eye irritation test

1. Materials and methods

1.1 test substance: sterile water is adopted to prepare 0.1 percent of compound DM80Bu20 aqueous solution which is colorless transparent liquid, and a tested prototype is taken for testing during the experiment.

1.2 Experimental animals and rearing environments

Experimental animals: 3 common-grade New Zealand white rabbits weigh 1.80-2.20kg, and are provided by a Dongxin Hua laboratory animal farm in the Huadu district of Guangzhou city, and the laboratory animals produce license numbers: SCXK (yue) 2019-: no.44007600007472, animal is quarantined for at least 3 days before experiment, and is ready for use after qualified.

A breeding environment: after the animals are purchased, the animals are raised in a single cage in an animal room in a common area of Guangzhou quality supervision and detection institute, and the temperature is as follows: 18-26 ℃, relative humidity: 40% -70%, the license number used by experimental animals: SYXK (Yue) 2018-.

Feed: provided by the Beijing department Australian cooperative feed Co., Ltd, the license number of feed production: SCXK (Jing) 2019-: 1112622000019812.

1.3 test methods

1.3.1 the test rabbits were examined for both eyes (including the use of sodium fluorescein) 24h before the start of the test and the eligible rabbits were examined for testing.

1.3.2 slightly pulling open the lower eyelid of one side of the rabbit, dripping 0.1ml of the test object into the conjunctival sac to make the upper and lower eyelids passively closed for 1s to prevent the loss of the picking house. The other eye was left untreated for self-control. Eyes are not washed within 24 hours after the test object is dripped.

1.3.3 the eyes of the animals were examined at 1h, 24h, 48h and 72h of instillation of the test substances. After 24h observation and recording, all animals' eyes were further examined with sodium fluorescein. The score for eye irritation response was recorded at each examination according to the criteria of acute eye irritation/corrosivity test-scoring of eye damage in cosmetic safety technology (2015 edition). The stimulation intensity of the test object to the eyes is judged according to the acute eye irritation/corrosivity test-eye irritation response grading of technical Specification for cosmetic safety (2015 edition) by evaluating the highest integral mean value and the recovery time of the stimulation response of the cornea, iris or conjunctiva of the animal at 24h, 48h or 72h observation time after the test object.

2. Results of the experiment

TABLE 3 results of acute eye irritation test of compound DM80Bu20 on rabbits

As can be seen from table 3, compound DM80Bu20 showed no irritation in the rabbit acute eye irritation test.

Example 5 acute skin irritation test

1. Materials and methods

1.1 test substance: a test sample was prepared as a colorless transparent liquid using sterile water to prepare a 0.1% aqueous solution of the compound DM80Bu 20.

1.2 Experimental animals and rearing environments

Experimental animals: 4 common-grade New Zealand white female rabbits with the weight of 1.80kg-2.20kg are provided by Dongxin Huadu district laboratory animal farms in Guangzhou city, and the laboratory animals produce license numbers: SCXK (yue) 2019-: no.44007600007434, animal was quarantined at least 3 days before testing and was ready for use after qualification.

A breeding environment: after the animals are purchased, the animals are raised in a single cage in an animal room in a common area of Guangzhou quality supervision and detection institute, and the temperature is as follows: 18 ℃ -26 ℃, relative humidity: 40% -70%, the license number used by experimental animals is as follows: SYXH (Guangdong) 2018-.

Feed: provided by the Kyowa cooperative fodder Co., Ltd of Beijing, Aurea, and the license number of the experimental animal fodder production: SCXK (Jing) 2019-: 1112622000019812.

1.3 test methods

1.3.1 both sides of the spinal column of the experimental animal were cut off 24h before the experiment, and the range of hair removal was 3cm × 3cm each.

1.3.2 apply 0.5ml of the test substance to one side of the skin, the area of the application is 2.5cm × 2.5cm, then cover with two layers of gauze (2.5cm × 2.5cm) and a layer of cellophane, and fix with non-irritant adhesive plaster and bandage. The other side of the skin was left untreated as a control. The application time is 4h by adopting a sealing test. After the test is finished, the residual test substance is removed by warm water.

1.3.3 observing the skin reaction of the applied part after removing the test objects for 1h, 24h, 48h and 72h, scoring the skin reaction according to the skin irritation/corrosivity test of technical Specification for cosmetic safety (2015 edition), comprehensively evaluating the average value of the animal integrals of the test objects, and judging the skin irritation intensity according to the highest integral average value of the observation time points of 24h, 48h and 72h and the skin irritation/corrosivity test of technical Specification for cosmetic safety (2015 edition). The test was observed for symptoms other than skin irritation.

2. Test results

TABLE 4 results of acute skin irritation test of rabbit with DM80Bu20

The results in table 4 show that the antimicrobial peptide compound DM80Bu20 is not irritating to rabbit skin.

EXAMPLE 6 Compound DM80Bu20 acute oral toxicity test

1. Materials and methods

1.1 test substances

Test groups: a test sample was prepared as a colorless transparent liquid using sterile water to prepare a 0.1% aqueous solution of the compound DM80Bu 20.

1.2 animal and feeding Environment

The preparation method comprises the following steps: 5.00g of the sample was weighed out and prepared to 10.0mL with pure water for use.

10 SPF-grade KM mice with the weight of 18-22 g are provided by Guangdong province medical experimental animal center, and the production license number of the experimental animals is as follows: SCXK (yue) 2018-: no. 44007200074743. The feed is provided by the medical experimental animal center of Guangdong province. After the animal is purchased, the animal is used after the quarantine of animal rooms of Guangzhou quality supervision and detection research institute is qualified, and the license number of the experimental animal is as follows: SYXK (yue) 2018-. Temperature: 20 ℃ to 26 ℃, relative humidity: 40 to 70 percent.

1.3 test methods

The test adopts a maximum test, only one dose group of 5000mg/kg body weight is set, namely 10 quarantine qualified mice are taken, and the number of the mice is half that of the mice. Animals were fasted overnight before the experiment without restriction of water. And (3) performing gavage on an empty stomach for 1 time, wherein the gavage volume is 10.0mL/kg of body weight, continuously fasting for 3-4 h after toxicant exposure, observing and recording the poisoning expression, death number and death time of the animals, wherein the observation period is 14d, and weighing the non-dead animals after the experiment is finished. Toxicity grading is judged according to the evaluation rule of an acute oral toxicity test.

2. Test results

After the infection, the toxic manifestation and death of the mice are not found in the observation period, and the surviving animals are dissected after the experiment is finished, so that no abnormality is seen with naked eyes. See table 5 for details.

TABLE 5 test results of acute oral toxicity of test substances to mice

The result shows that the compound DM80Bu20 has acute oral toxicity LD50 of more than 5000mg/kg body weight to female and male KM mice, and is of practical non-toxic grade.

EXAMPLE 7 preparation of antibacterial toothpaste

(1) This example provides a series of toothpastes containing compound DM80Bu20, the specific formulation of which is shown in table 6.

TABLE 6

Wherein the probiotics in toothpaste 4, 5, 7, and 8 are Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus acidophilus, Bacillus bifidus, and Lactobacillus salivarius, respectively.

(2) Preparation method of toothpaste

The current production process of a company adopts a one-step toothpaste making method to complete the preparation of toothpaste body:

s1, mixing the humectant, the foaming agent (liquid), the sweetening agent, the chelating agent and the preservative with water, and stirring for 5-15 minutes;

s2, mixing a friction agent, an adhesive and a foaming agent (solid) with the aqueous solution prepared in the S1, keeping the vacuum degree between-0.090 MPa and-0.096 MPa, and stirring for 20 to 50 minutes;

s3, adding essence and active ingredients, keeping the vacuum degree between-0.090 Mpa and-0.096 Mpa, and stirring for 10-50 minutes to obtain the toothpaste 1-9.

EXAMPLE 8 bacteriostatic Property test of toothpaste

1. Experimental methods

According to 7.3 in QB 2738-. The measurement results are shown in Table 7.

2. Results of the experiment

TABLE 7 antibacterial rate (%) of toothpaste after standing at room temperature for 24h to 1-9

As can be seen from table 7, the toothpaste containing the antibacterial peptide compound DM80Bu20 provided by the invention has the bacteriostatic rates of over 90% for candida albicans, staphylococcus aureus, MRSA (methicillin-resistant staphylococcus aureus), streptococcus mutans, porphyromonas gingivalis and escherichia coli, wherein the bacteriostatic rates of the toothpastes 2 and 3 for various oral harmful bacteria reach 99-100%; in addition to the antibacterial peptide compound DM80Bu20, other functional components (lysozyme, lactoferrin, probiotics, regenerated silicon and fructo-oligosaccharide) are added into the toothpaste 4-9 for compounding, so that the antibacterial effect of the toothpaste is improved, and the compounding of the lysozyme, the lactoferrin, the probiotics, the regenerated silicon and the fructo-oligosaccharide can effectively improve the antibacterial effect of the toothpaste, therefore, based on the expensive cost of the antibacterial peptide compound DM80Bu20, other compounded effective components can be adopted to improve the antibacterial effect of the toothpaste.

In addition, the bacteriostatic rate of the toothpaste with each formula to streptococcus sanguis is lower than 5 percent, which is mainly because other auxiliary materials in the toothpaste have certain bacteriostatic action. In general, the bacteriostatic toothpaste disclosed by the invention can well inhibit harmful bacteria in the oral cavity, has no harm to beneficial bacteria, and can effectively balance flora.

EXAMPLE 9 bacteriostatic stability test of toothpaste

1. Experimental methods

With reference to the evaluation method of the antibacterial and bacteriostatic effects of the QB 2738-. And (3) placing the toothpaste 1-9 at 45 ℃ for 30 days and 90 days, and then respectively carrying out a toothpaste antibacterial test. The measurement results are shown in Table 8.

2. Results of the experiment

TABLE 8 antibacterial ratio (%) after toothpaste 1-9 were left at 45 ℃ for 30 days and 90 days, respectively

As can be seen from the results in table 8, after the toothpaste formulations were left at 45 ℃ for 30 days and 90 days, the antibacterial effect was relatively reduced with the increase of the storage period, but the whole toothpaste still had a good antibacterial effect, indicating that the toothpaste of the present invention has a stable antibacterial effect and is beneficial to long-term storage.

EXAMPLE 10 toothpaste application Effect (mottle index measurement)

1. Test method

(1) Groups and subgroups of subjects

80 human subjects with all normal physical examination indexes; 12 natural anterior teeth are arranged in the mouth of a tested person, and no large-area filling or crown or other restorations exist; the teeth have extrinsic stains.

Randomly dividing the test group into a test group 1, a test group 2, a test group 3 and a test group 4, wherein each group comprises 20 people and halves of men and women; the sample used in the test group 1 is toothpaste 1, the sample used in the test group 2 is toothpaste 3, the sample used in the test group 3 is toothpaste 4, and the sample used in the test group 4 is toothpaste 6.

(2) Tooth brushing mode

The subjects dispensed toothpaste and toothbrush together, and brushed their teeth 1 time each day, 2 times each day for 1min, according to BASS brushing method.

(3) Experimental procedure

The test subjects carried out the detection of the color spot index before use (baseline), and after 3 months of continuous use, the detection of the color spot index was carried out, and the changes of the color spot index of the test subjects before and after the use of toothpaste 1, toothpaste 3, toothpaste 4 and toothpaste 6 were compared.

The color spot index scoring method has the following specific scoring rule:

score 0-no specks present;

1 point, color spots exist, the size of the color spots does not exceed 1/3 of the tooth surface, and the degree is light color spots (yellow or yellow brown);

2 points-stained, the size of the stain was greater than 1/3, not greater than 2/3, and moderate stain (medium brown);

score 3-there was a mottle, which was greater in size than 2/3 on the tooth surface, and the degree was severe (dark brown or black).

The tooth positions are checked to be the lip and the lingual surface of the mandibular anterior teeth and the labial surface of the maxillary anterior teeth. Each tooth stain index count is divided by the product of the stain area score and the stain level score for that tooth surface. The stain index score for each subject was the average of the individual tooth surface stain index scores, i.e.:

number of teeth checked by sum of color spot index ∑ (area score degree score)/total number of teeth checked

2. Test results

TABLE 9

Note: the reduction rate of stain index (average of stain index before use-average of stain index after use)/average of stain index before use × 100%.

As can be seen from the results in table 9: the toothpaste of the invention has the function of alleviating dental stains, and the effect of removing the dental stains is improved along with the increase of the content of DM80Bu 20; in addition, compared with toothpaste 1, toothpaste 4 and toothpaste 6 have higher reduction rate of stain index of a subject, which shows that after lysozyme, lactoferrin, probiotics, regenerated silicon and fructo-oligosaccharide are added into the toothpaste disclosed by the invention, the effect of removing exogenous stains can be effectively improved, so that the use cost of DM80Bu20 is reduced and a good effect of removing stains on tooth surfaces is realized by adding other active ingredients, wherein the reduction rate of the stain index of toothpaste 6 is the highest, which shows that the effect of adding lysozyme and DM80Bu20 after compounding is the best.

In addition, the subjects used the above toothpaste to effectively improve dental calculus, gingival bleeding, oral ulcer, halitosis, etc., wherein the improvement of toothpaste 4 and 6 was more significant than that of toothpaste 1.

It should be understood that the above-described embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. The application of the antibacterial peptide compound DM80Bu20 in preparing the antibacterial toothpaste is characterized in that the structural formula of the antibacterial peptide compound DM80Bu20 is as follows:

2. the use of claim 1, wherein the bacteriostatic action is the inhibition of oral harmful bacteria.

3. The use of claim 2, wherein the oral cavity deleterious bacteria comprise one or more of candida albicans, staphylococcus aureus, methicillin resistant staphylococcus aureus, streptococcus mutans, porphyromonas gingivalis.

4. A bacteriostatic toothpaste comprising the antibacterial peptide compound DM80Bu20 of claim 1.

5. The toothpaste according to claim 4, wherein the toothpaste comprises the following components in parts by weight: 200.001-0.1 parts of antibacterial peptide compound DM80Bu200.001-45 parts of humectant, 20-25 parts of friction agent, 0.5-2 parts of adhesive, 0.5-3 parts of foaming agent, 0.5-1.5 parts of essence, 0.1-0.5 part of sweetening agent, 1-3 parts of chelating agent, 0.1-2 parts of preservative and 20-35 parts of deionized water.

6. The toothpaste according to claim 5, wherein said toothpaste further comprises one or more ingredients selected from lysozyme, lactoferrin, probiotics, reconstituted silicon, fructo-oligosaccharides.

7. The toothpaste according to claim 6, wherein the total part of one or more of lysozyme, lactoferrin, probiotics, regenerated silicon and fructo-oligosaccharide in the toothpaste is 0.5-2 parts.

8. The toothpaste according to any one of claims 5 to 7, wherein the humectant comprises one or more of sorbitol, propylene glycol, glycerin and polyethylene alcohol; the friction agent comprises one or more of silicon dioxide, hydrated silica, aluminum hydroxide, calcium bicarbonate, hydroxyapatite or calcium hydrophosphate; the adhesive comprises one or more of sodium carboxymethylcellulose, hydroxyethyl cellulose, carrageenan, carbomer, polyvinylpyrrolidone or xanthan gum; the foaming agent comprises one or more of sodium lauryl sulfate, sodium lauroyl sarcosinate, sodium lauroyl glutamate, fatty amidopropyl methyl betaine, cocamidopropyl methyl betaine, sodium cocoyl glutamate and polyoxyethylene hydrogenated castor oil 40.

9. The toothpaste according to any one of claims 5 to 7, wherein the flavor comprises peppermint flavor; the sweetener comprises one or more of saccharin sodium, aspartame and stevioside; the chelating agent comprises one or more of pyrophosphate, phosphate and EDTA disodium; the preservative comprises one or more of potassium sorbate, methyl paraben and propyl paraben.

10. The toothpaste according to claim 6 or 7, wherein the probiotic bacteria comprise one or more of Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus salivarius.