CN113456521B - Oral care composition containing peptide and preparation method thereof - Google Patents

Abstract

The invention discloses a peptide-containing oral care composition, which comprises antibacterial peptide and nano-hydroxyapatite; and further comprising a mono fatty acid polyethylene glycol sodium phosphate salt. In addition, the invention also discloses a corresponding preparation method, wherein the humectant and the ethanol are uniformly mixed; adding antibacterial peptide and mono fatty acid polyethylene glycol sodium phosphate; then adding nano hydroxyapatite. The composition has good effect in eliminating oral bacteria, and inhibiting enamel demineralization.

Description

Oral care composition containing peptide and preparation method thereof

Technical Field

The invention belongs to the technical field of oral care, and particularly relates to an oral care composition and a preparation method thereof. The oral care composition includes an antibacterial peptide and nano-hydroxyapatite.

Background

In oral diseases, caries is a chronic disease of bacterial infection that is susceptible to progressive destruction of the hard tissues of the tooth. The pathogenic mechanism of caries is: plaque microorganisms adhere to the tooth surface, breaking down the sugars to acids, causing organic breakdown and mineral demineralization. Streptococcus, Lactobacillus, and Actinomyces are the main pathogenic bacteria for dental caries. Among them, Streptococcus mutans is a well-known cariogenic bacterium, which not only ferments sugars to produce a large amount of acid, but also destroys enamel; the production of exopolysaccharides simultaneously promotes the formation and deposition of plaque biofilm. The bacterial plaque biomembrane is an animal factor for the formation of caries, and provides a natural and effective protective barrier for the survival and propagation of bacteria. Therefore, control of plaque biofilm is a focus and platform for anticaries research.

For caries, no perfect treatment means exists at present, and more attention is paid to prevention of early caries rather than invasive repair treatment. Prevention of caries using sodium fluoride, sodium hexafluorophosphate, etc. is the main prevention method. The above fluoride can react with calcium ions released from enamel into saliva to form calcium fluoride, deposit at enamel plaque interface, reduce enamel solubility, and promote remineralization of demineralized enamel, but it has a poor effect on progressive caries.

In recent years, related research reports of natural anti-caries medicaments are continuously increased, and the anti-caries efficacy of partial medicaments such as dark plum fruit, Chinese gall, honeysuckle flower, sarcandra glabra, propolis, angelica dahurica, liquorice, wild chrysanthemum, pseudo-ginseng and the like is mature, but the clinical application effect is not ideal, and particularly the antibacterial effect cannot be lasting. At present, no ideal anticariogenic medicament which has no toxic or side effect, is effective and does not influence the micro-ecological environment of the oral cavity is available. Thus, some researchers have focused on the transfer of antimicrobial peptides.

Antimicrobial peptides are a broad class of polypeptides found in organisms, and are usually produced by partially mutating cells in the body during resistance to invasion by foreign microorganisms. As the first line of defense of innate immunity, antimicrobial peptides have a broad spectrum of antimicrobial activity. These antimicrobial peptides have great potential in the development of antimicrobial therapeutics. The antibacterial peptide has small molecule, good water solubility and stability, and no antigenicity. The antibacterial peptide has antibacterial activity for inhibiting and killing microorganisms, and also has a certain inhibiting effect on bacterial plaque biomembranes.

Chinese patent application CN1771905A discloses a mouthwash consisting of honeysuckle, dandelion, zinc acetate, propolis, biological antibacterial peptide, menthol, essence and distilled water. The mouthwash has the functions of sterilization, decontamination, deodorization and the like for the oral cavity of a human, and has the characteristics of safety, no toxic or side effect and convenient use. However, this patent application does not describe the amino acid sequence of the biological antibacterial peptide.

Chinese patent application CN108704119A discloses an antibacterial peptide for killing oral cariogenic bacteria and inhibiting the formation of dental plaque and application thereof. The invention is based on the inability of conventional antimicrobial agents to sustain antimicrobial activity and lack of ability to bind to teeth. The antibacterial peptide is polymerized by an amino acid sequence shown as SEQ ID NO. 1 and a group with a function of combining with the surface of teeth, has the capacity of combining with the surface of teeth, and can be applied to the treatment and prevention of dental caries.

Further, Cao Ge et al synthesized the designed amino acid sequence of the antibacterial peptide by solid phase synthesis based on the aforementioned patent application, and studied the effect of hydroxyapatite-bound antibacterial peptide DPSAMP on the antibacterial properties of Streptococcus mutans and the oral cavity biofilm. The results show that the minimum inhibitory concentrations of the untreated antimicrobial peptides AMP and DPSAMP are 40 mug/mL and 80 mug/mL respectively; and after anaerobic culture of the DPSAMP-treated bovine enamel slabs with Streptococcus mutans, the Streptococcus mutans on the enamel surface was significantly less than that of AMP and the control group.

In conjunction with the foregoing studies, the inventors believe that when the antimicrobial peptide has a phosphate structure at the end, it has a strong binding effect with the enamel surface, thereby reducing the adsorption of bacteria to the enamel surface and inactivating the adsorbed pathogenic bacteria; hydroxyapatite conjugated antimicrobial peptides have a similar antimicrobial mechanism. Furthermore, hydroxyapatite, as it is known, has proven itself to have anticaries action, being more resistant to acid attack by maintaining calcium and phosphorus supersaturation on the enamel surface, remineralizing the enamel. However, the synthesis of the hydroxyapatite combined with the antibacterial peptide is complex, and the hydroxyapatite combined with the antibacterial peptide is inconvenient to use when the hydroxyapatite combined with the antibacterial peptide is applied to incisors in a coating mode.

Chinese patent application CN108704119A mentions that the novel antibacterial peptide can be applied in mouthwash and states that the concentration of the antibacterial peptide is 10-20 μ g/mL. But the cleaning effect on oral bacteria is not obvious when normal gargling is carried out under the concentration; and the inhibitory effect on enamel demineralization is not good.

Therefore, the inventor provides a peptide-containing oral care composition which has better oral bacteria removing effect and better enamel demineralization inhibiting effect on the basis of the prior art and a preparation method thereof.

Disclosure of Invention

In view of the above problems, the present invention aims to provide a peptide-containing oral care composition and a method for preparing the same.

In order to achieve the purpose, on one hand, the invention adopts the following technical scheme: a peptide-containing oral care composition, wherein the composition comprises an antibacterial peptide and nano-hydroxyapatite.

The composition according to the present invention further comprises a mono fatty acid polyethylene glycol sodium phosphate salt.

Preferably, the fatty acid is selected from the group consisting of C14-22 fatty acids; more preferably C16-20 fatty acids.

Preferably, the polyethylene glycol is selected from the group consisting of polyethylene glycol 100, polyethylene glycol 200, polyethylene glycol 400, polyethylene glycol 600, polyethylene glycol 800 and polyethylene glycol 1000.

In one embodiment, the mono fatty acid polyethylene glycol phosphate sodium salt is selected from polyethylene glycol 400 sodium mono-oleate phosphate.

The composition of the invention, wherein the antibacterial peptide is selected from H₄P₂O₇-Arg-Arg-Trp-Cys-Phe-Arg-Val-Cys-Tyr-Arg-Gly-Phe-Cys-Tyr-Arg-Lys-Cys-Arg。

The antibacterial peptide is synthesized according to the method of the embodiment of Chinese patent application CN 108704119A.

The composition of the invention, wherein the average particle size of the nano-hydroxyapatite is 10-30 nm; the specific surface area is 30-70m²/g。

Preferably, the average particle size of the nano hydroxyapatite is 15-25 nm; the specific surface area is 40-60m²/g。

In a specific embodiment, the nano-hydroxyapatite has an average particle size of 20 nm; the specific surface area is 50m²/g。

The composition of the invention, wherein the antibacterial peptide accounts for 0.1-0.5% by weight; and/or the nano hydroxyapatite accounts for 1.5 to 3.5 percent.

Preferably, the antibacterial peptide accounts for 0.2-0.4% by weight; and/or the nano hydroxyapatite accounts for 2-3 percent.

In one embodiment, the antimicrobial peptide is 0.3% by weight; and/or the nano hydroxyapatite is 2.5 percent.

The composition of the invention comprises 1.6-2.4 wt% of the sodium monofatty acid polyethylene glycol phosphate.

Preferably, the content of the mono fatty acid polyethylene glycol sodium phosphate is 1.8-2.2% by weight.

In one embodiment, the mono fatty acid polyethylene glycol sodium phosphate salt is 2% by weight.

The composition according to the present invention, wherein the composition further comprises a humectant.

Preferably, the humectant is selected from propylene glycol and/or glycerin.

The composition of the invention comprises 10-20 wt% of the humectant.

Preferably, the humectant is 12-18% by weight.

In one embodiment, the humectant is 15% by weight.

The composition according to the present invention, wherein the composition further comprises a surfactant.

Preferably, the surfactant is selected from polysorbate 80.

The composition provided by the invention is characterized in that the polysorbate 80 accounts for 2-6% in percentage by weight.

Preferably, the polysorbate 80 is 3-5% by weight.

In a specific embodiment, polysorbate 80 is 4% by weight.

The composition according to the present invention, wherein the composition further comprises ethanol.

The composition of the invention comprises 2-6% by weight of ethanol.

Preferably, the ethanol is 3-5% by weight.

In one embodiment, the ethanol is 4% by weight.

The composition according to the present invention, wherein the composition further comprises other adjuvants including, but not limited to, sweeteners, colorants, preservatives and flavors.

The amounts of these components are well known to those skilled in the art. For example, the sweetening agent, the coloring agent, the preservative and the essence are each used in an amount of 0 to 5wt% in terms of weight percentage.

Preferably, the sweetening agent, the coloring agent, the preservative and the essence are each used in an amount of 0 to 2wt% in terms of weight percentage.

The composition according to the present invention, wherein the composition is a mouthwash.

In a particular embodiment, the balance of the composition is water.

On the other hand, the invention also adopts the following technical scheme: a method of making a peptide-containing oral care composition comprising the steps of:

uniformly mixing the humectant and the ethanol; adding surfactant, other auxiliary agents and 10-20% of water; then adding the antibacterial peptide and the mono fatty acid polyethylene glycol sodium phosphate salt and uniformly mixing; then adding nano hydroxyapatite and 80-90% of water, mixing uniformly, and regulating pH value to 6-8.

Compared with the prior art, the oral care composition containing the peptide has better cleaning effect on oral bacteria and better inhibition effect on enamel demineralization.

Detailed Description

The following examples are merely illustrative of embodiments of the present invention and do not limit the scope of the invention.

Example 1

An oral care composition containing peptides comprises the following raw materials in percentage by weight:

15% of glycerol;

2% of polyethylene glycol monooleate 400 sodium phosphate;

nanometer hydroxyapatite (average particle diameter of 20 nm; specific surface area of 50 m)²/g） 2.5%；

H₄P₂O₇-Arg-Arg-Trp-Cys-Phe-Arg-Val-Cys-Tyr-Arg-Gly-Phe-Cys-Tyr-Arg-Lys-Cys-Arg；0.3%；

4% of ethanol;

polysorbate 804%;

1% of xylitol;

0.1 percent of sodium benzoate;

0.1% of essence;

the balance of deionized water.

The preparation method comprises the following steps:

mixing glycerol and ethanol uniformly; adding polysorbate 80, xylitol, sodium benzoate, essence and 10-20% deionized water (based on the total amount of deionized water); then adding the antibacterial peptide and the mono fatty acid polyethylene glycol sodium phosphate salt and uniformly mixing; nano-hydroxyapatite and 80-90% deionized water (based on the total amount of deionized water) were then added, mixed well, and pH was adjusted = 7.

Comparative example 1

An oral care composition containing peptides comprises the following raw materials in percentage by weight:

15% of glycerol;

nanometer hydroxyapatite (average particle diameter of 20 nm; specific surface area of 50 m)²/g） 2.5%；

H₄P₂O₇-Arg-Arg-Trp-Cys-Phe-Arg-Val-Cys-Tyr-Arg-Gly-Phe-Cys-Tyr-Arg-Lys-Cys-Arg；0.3%；

4% of ethanol;

806% of polysorbate;

1% of xylitol;

0.1 percent of sodium benzoate;

0.1% of essence;

the balance of deionized water.

The preparation method comprises the following steps:

mixing glycerol and ethanol uniformly; adding polysorbate 80, xylitol, sodium benzoate, essence and 10-20% deionized water (based on the total amount of deionized water); then adding the antibacterial peptide and mixing uniformly; nano-hydroxyapatite and 80-90% deionized water (based on the total amount of deionized water) were then added, mixed well, and pH was adjusted = 7.

Comparative example 2

An oral care composition containing peptides comprises the following raw materials in percentage by weight:

15% of glycerol;

2% of polyethylene glycol monooleate 400 sodium phosphate;

H₄P₂O₇-Arg-Arg-Trp-Cys-Phe-Arg-Val-Cys-Tyr-Arg-Gly-Phe-Cys-Tyr-Arg-Lys-Cys-Arg；0.3%；

4% of ethanol;

polysorbate 804%;

1% of xylitol;

0.1 percent of sodium benzoate;

0.1% of essence;

the balance of deionized water.

The preparation method comprises the following steps:

mixing glycerol and ethanol uniformly; adding polysorbate 80, xylitol, sodium benzoate, essence and 10-20% deionized water (based on the total amount of deionized water); then adding the antibacterial peptide and the mono fatty acid polyethylene glycol sodium phosphate salt and uniformly mixing; then 80-90% deionized water (based on total amount of deionized water) was added, mixed well, and pH was adjusted = 7.

Evaluation of oral bacteria removing Effect

(1) Test grouping

Group A: example 1 mouthwash is gargled for 1min, 10mL each time;

group B: comparative example 1 mouthwash is gargled for 1min, 10mL each time;

group C: comparative example 2 mouthwash is gargled for 1min, 10mL each time;

group D: rinsing with clear water for 1min, 10mL each time.

Each group had 5 males and 5 females with an average age of between 37 and 38 years.

(2) Fungus taking method

Before and after gargling, randomly weighing bacteria on the left and right sides of the horizontal mucosa of molar teeth of palate part with sterilized filter paper sheets, wherein the area of each bacteria is 1.0cm². The filter paper sheet is placed in a bacteria extraction area for 30s and taken out, and is placed in a 1mL PBS solution centrifuge tube.After fully mixing in a vortex mixer, 10 times of dilution, 10 μ L of the mixture was inoculated into an agar culture dish and cultured at 37 ℃ for 48 hours, and colony count (CFU/mL) was performed to calculate the total amount of oral bacteria.

The results are shown in Table 1.

TABLE 1

Evaluation of demineralization Effect of enamel

(1) Test grouping

Group A: example 1 mouthwash is gargled for 1min, 10mL each time; 1 time in the morning and at night;

group B: comparative example 1 mouthwash is gargled for 1min, 10mL each time; 1 time in the morning and at night;

group C: comparative example 2 mouthwash is gargled for 1min, 10mL each time; 1 time in the morning and at night;

group D: rinsing with clear water for 1min, 10mL each time; 1 time in the morning and at night.

(2) Test method

Each group is 5 male and 5 female, the average age is 32-33 years old, and the patients are orthodontic patients. The oral hygiene is maintained in the same way for four groups of other oral hygiene, i.e. brushing teeth 1 time respectively in the morning and evening, and using the same kind of toothpaste for 5min each time. Each group of experiments was continued for 3 months. Then, the degree of demineralization of enamel on the labial and buccal surfaces of all upper and lower incisors was observed, and the degree was classified into a grade 0 (smooth and transparent enamel surface, no demineralization), a grade 1 (light chalky spots appeared on the enamel surface, and the area was less than 50% of the area of the upper and the lower incisors of the lower teeth of the upper and the lower incisors of the lower teeth. The grades 1-3 are the manifestations of demineralized teeth. Demineralization rate = number of demineralised teeth/number of observed teeth.

See table 2 for results.

TABLE 2

It should be understood that the detailed description of the invention is merely illustrative of the spirit and principles of the invention and is not intended to limit the scope of the invention. Furthermore, it should be understood that various changes, substitutions, deletions, modifications or adjustments may be made by those skilled in the art after reading the disclosure of the present invention, and such equivalents are also within the scope of the invention as defined in the appended claims.

Claims (4)

1. A peptide-containing oral care composition, wherein the composition comprises an antibacterial peptide and nano-hydroxyapatite; and further comprising mono fatty acid polyethylene glycol sodium phosphate, a humectant, a surfactant and ethanol;

wherein the antibacterial peptide is selected from H₄P₂O₇-Arg-Arg-Trp-Cys-Phe-Arg-Val-Cys-Tyr-Arg-Gly-Phe-Cys-Tyr-Arg-Lys-Cys-Arg；

The average particle size of the nano hydroxyapatite is 10-30 nm; the specific surface area is 30-70m²/g；

The mono-fatty acid polyethylene glycol sodium phosphate is selected from polyethylene glycol 400 sodium monooleate phosphate; the surfactant is selected from polysorbate 80;

according to weight percentage, the antibacterial peptide is 0.1-0.5%; the nano hydroxyapatite accounts for 1.5 to 3.5 percent; the content of the mono-fatty acid polyethylene glycol sodium phosphate is 1.6-2.4%.

2. The composition of claim 1, wherein the composition further comprises sweeteners, colorants, preservatives and flavors.

3. The composition of claim 1, wherein the composition is a mouthwash.

4. A process for preparing a peptide-containing oral care composition according to any one of claims 1 to 3, comprising the steps of:

uniformly mixing the humectant and the ethanol; adding surfactant, other auxiliary agents and 10-20% of water; then adding the antibacterial peptide and the mono fatty acid polyethylene glycol sodium phosphate salt and uniformly mixing; then adding nano hydroxyapatite and 80-90% of water, mixing uniformly, and regulating pH value to 6-8.