Patent Description:
Dentin hyperesthesia, commonly referred to as 'sensitive dentin', is a common symptom experienced by <NUM>% to <NUM>% of the adult population. In particular, the case of periodontal disease, which is the most common disease in Korea, <NUM>% to <NUM>% of patients suffer from 'sensitive dentin' (Source: National Health Information Portal Medical Information;
http://health. kr/health/Main.

Dentin hyperesthesia can be defined as a pain caused by the exposure of dentinal tubules transmitting all external stimuli to the pulp nerve. This makes the pulp nerve to be more sensitive to the same stimulus. Although there are no nerves in the dentin itself, but we can perceive the changes because the cold temperature stimulus is transmitted through the dentinal tubules to the nerves inside the pulp.

In the dentin, which occupies the most part of the tooth, there are dentinal tubules that extend from the pulp to the enamel. These tubules are filled with liquid. The diameter increases toward the pulp and has a dense structure. Because of these distinct structures, external stimuli can be transmitted quickly to the pulp nerve. If the dentin surface is damaged and the number of exposed dentinal tubules increases, it may cause a more sensitive reaction to the same stimulus than usual.

Currently, there are two ways for dentin hyperesthesia, depending on the principle of action. One is to interfere with the signal transmission of the nerve that transmits pain, and the other is to block the exposed dentinal tubules to alleviate the symptoms.

Dipotassium phosphate (K<NUM>HPO<NUM>) has been widely used in a method for interfering with the signal transmission of nerves that transmit pain. However, dipotassium phosphate has a low pain-blocking effect and must be used repeatedly, and it is not an effective treatment method because it limits the sense of chewing.

Next, calcium hydrogen phosphate (CaHPO<NUM>), fluorine, oxalate, arginine (amino acid), and calcium carbonate (CaCO<NUM>) are used to block the dentinal tubules. In recent years, considering the inconvenience of receiving treatment at the dentist for dentin hypersensitivity, toothpastes for preventing or alleviating dentin sensitivity are on the market, including calcium phosphate, dental type silica, strontium chloride, calcium carbonate or tricalcium phosphate, as active ingredients. However, these are also foreign materials different from the original dentin, so there would be a gap created in the peripheral boundary area between dentin and the foreign materials. The nerve would be exposed again after the foreign material being separated from the sealing, and it would cause a recurrence of dentin sensitivity.

<CIT> relates to a novel peptide used to promote regeneration of dentin or dental pump and to treat dentine hyperesthesia. <CIT> relates to a tooth-cleaning composition having a high remineralizing effect on demineralized dental enamel. <CIT> relates to a liquid oral composition containing an emulsion and a method for improving the freeze-recoverability thereof.

Embodiments of the present inventive concepts may provide toothpaste composition for alleviating dentin hyperesthesia, comprising a peptide including an amino acid sequence of the following Formula <NUM>:.

K-Y-R1-R2-R3-R4-R5-R6-R7-R8     (Formula <NUM>).

Embodiments of the present inventive concepts may also provide a composition for oral care comprising a peptide including any one amino acid sequence of SEQ ID NOS: <NUM> to <NUM>.

Embodiments of the present inventive concepts, the toothpaste composition may include humectant <NUM>-<NUM> parts by weight, viscosity modifier <NUM>-<NUM> parts by weight, surfactant <NUM>-<NUM> parts by weight, flavoring agent <NUM>-<NUM> parts by weight, and diluting agent <NUM>-<NUM> parts by weight based on <NUM> parts by weight of the composition.

Embodiments of the present inventive concepts, the toothpaste composition may include <NUM>-<NUM> parts by weight of aminocaproic acid and <NUM>-<NUM> parts by weight of allantoin based on <NUM> parts by weight of the composition.

Embodiments of the present inventive concepts, the humectant may be D-sorbitol solution or sodium PCA solution, or concentrated glycerin, the viscosity modifier may be hydrous silicic acid, xanthan gum or CMC(Carboxymethyl Cellulose Sodium Salt), the surfactant may be sodium cocoylmethyltaurate, flavoring agent is green tea extract, chamomile extract, rosemary extract, myrrh tincture, rhatany tincture, chamomile tincture, mastic oil <NUM> HF-<NUM>, propolis extract, grapefruit seed extract, spearmint B71228 or peppermint oil <NUM>, and the diluting agent may be hydroxyapatite.

Embodiments of the present inventive concepts, the humectant may include <NUM>-<NUM>% by weight of the D-sorbitol solution, <NUM>-<NUM>% by weight of the Sodium PCA solution, and <NUM>-<NUM>% by weight of the concentrated glycerin.

Embodiments of the present inventive concepts, the viscosity modifier may include <NUM>-<NUM>% by weight of the hydrous silicic acid, <NUM>-<NUM>% by weight of xanthan gum, and <NUM>-<NUM>% by weight of the CMC.

The present inventive concepts may provide toothpaste composition that prevents or alleviates dentin hyperesthesia by sealing off exposed defects of the dentinal tubule through physiological remineralization of dentin.

Other aspects, advantages and salient features of the embodiments will become apparent to those skilled in the art from the following detailed description, which, taken in conjunction with the annexed drawings, discloses exemplary embodiments of the disclosure.

A-D shows the dentinal tubules treated only with purified water (comparative test example <NUM>-<NUM>), E-H is showing the dentinal tubules brushed using a toothpaste composition without the peptide comparing with the embodiment of the present invention (comparative test example <NUM>-<NUM>), and I-L shows the dentinal tubules brushed using a toothpaste composition for alleviating dentin hyperesthesia according to the embodiment of the present invention (scale bar: A, E, I, <NUM>; B, F, J, <NUM>; C, G, K, <NUM>; D, H, L, <NUM>). In each case, the dentin slices with exposed dentinal tubules were washed <NUM> times with distilled water after brushing once a day for <NUM> minute and then immersed in artificial saliva. After repeating this process for <NUM> weeks, the ability to seal off the dentin tubules was observed with a scanning electron microscope.

In this regard, the present embodiments may have different forms and should not be construed as limited to the descriptions set forth herein. Unless otherwise defined, all terms including technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skills in the art to which this disclosure belongs. In addition, terms to be described later are defined in consideration of contributions in the present disclosures, which may vary according to the intention of the user or practice.

The disclosure now will be described more fully hereinafter with reference to the accompanying drawings, in which illustrative embodiments of the disclosure are shown. This disclosure may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete and will fully convey the scope of the disclosure to those skilled in the art. However, as it is presented as an example, the present invention is not limited thereto and the present invention is defined only by the scope of the claims which will be described later.

It will be understood that the terms "comprises" and/or "comprising," when used in this specification, specify the presence of stated components, this means that it may contain more components, rather than exclude other components, unless there is a particularly contrary article.

Hereinafter, embodiments of the present invention are described in more detail.

An odontoblast may refer to a cell that synthesizes and secretes proteins and polysaccharides composing the matrix of the dentin. It is a columnar cell that is in contact with the predentin (uncalcified dentin) and forms a cell layer at the periphery of the dental pulp. It is a differentiated cell (becoming a cell derived from the mesenchymal ectoderm) involved in calcification of dentin. At the developmental stage, an odontoblast faces the enamel among the cells of the dental papilla, involved in calcification of dentin.

A peptide, included in the toothpaste composition for alleviating dentin hyperesthesia according to an embodiment of the present invention (hereinafter, 'odontoblast differentiation promoting peptide'), does not exhibit cytotoxicity, but it is possible to increase the expression level of the odontoblast differentiation marker genes DSPP, Dmp1 and Nestin. When transplanted in vivo with pulp tissue cells, the pulp tissue cells may form a dentin/dentin-like tissue.

Odontoblast differentiation promoting peptide includes mutant peptides having a sequence different from the amino acid sequence constituting the amino acid sequence and at least one amino acid residue, as long as it can promote dentin regeneration or treat dentin hyperesthesia.

Amino acid exchanges in proteins and polypeptides, which do not generally alter the molecular activity, are known in the art. The most commonly occurring exchanges are amino acid residues Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thy/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, Asp/Gly, in both directions. The peptide may include peptides that have improved structural stability against heat, pH, etc., or improved ability to promote regeneration of dentin or dental pulp due to alteration or modification of the amino acid sequence.

For example, although glutamine which is an acidic amino acid at position <NUM> of the peptide of SEQ ID NO: <NUM> of the present invention is substituted with a basic amino acid, lysine or arginine, the effects of the peptide of the present invention may be obtained as it is; although arginine which is a basic amino acid at position <NUM> or <NUM> of the peptide of SEQ ID NO: <NUM> is substituted with an acidic amino acid glutamine or a basic amino acid lysine, the effects of the peptide of the present invention may be obtained as it is; although lysine which is a basic amino acid at position <NUM>, <NUM>, or <NUM> of the peptide of SEQ ID NO: <NUM> is substituted with a basic amino acid arginine or an aromatic amino acid tyrosine, the effects of the peptide of the present invention may be obtained as it is; although asparagine which is an acidic amino acid at position <NUM> of the peptide of SEQ ID NO: <NUM> is substituted with a neutral amino acid serine, the effects of the peptide of the present invention may be obtained as it is; and although tyrosine which is an aromatic amino acid at position <NUM> of the peptide of SEQ ID NO: <NUM> is substituted with a basic amino acid lysine, the effects of the peptide of the present invention may be obtained as it is.

As such, although the acidic amino acids, basic amino acids, or aromatic amino acids constituting the peptide of the present invention are substituted with amino acids having the same properties, or substituted with different acidic amino acids, basic amino acids, neutral amino acids, or aromatic amino acids, respectively, the effects of the peptide of the present invention may be obtained as it is. Therefore, it is apparent that a peptide variant having a sequence including one or more amino acid residues different from those of the amino acid sequence constituting the peptide of the present invention is also included in the scope of the peptide of the present invention.

Further, although arbitrary amino acids are added at the N-terminus or C-terminus of the peptide of the prevention, the effects of the peptide of the present invention may be obtained as it is. Therefore, a peptide prepared by adding arbitrary amino acids at the N-terminus or C-terminus of the peptide of the present invention is also included in the scope of the peptide of the present invention. For example, a peptide prepared by adding <NUM> to <NUM> amino acids at the N-terminus or C-terminus of the peptide of the present invention may be exemplified, for another example, a peptide prepared by adding <NUM> to <NUM> amino acids at the N-terminus or C-terminus of the peptide of the present invention may be exemplified, and for still another example, a peptide prepared by adding <NUM> to <NUM> amino acids at the N-terminus or C-terminus of the peptide of the present invention may be exemplified.

The mRNA of the DSPP gene in MDPC-<NUM> cells treated with the odontoblast differentiation promoting peptide, compared to the mRNA level of the DSPP gene in MDPC-<NUM> cells (control) not treated with the odontoblast differentiation promoting peptide, was all <NUM> times or more (Tables <NUM> to <NUM>).

As reported up to now, it is known that as the mRNA level of DSPP is increased, odontoblast differentiation and dentin regeneration are promoted, and therefore, it can be seen that <NUM> kinds of peptides increases the mRNA level of Dspp gene, which in turn may exhibit the effect of promoting odontoblast differentiation and dentin regeneration (<NPL>; <NPL>).

The peptide included in the toothpaste composition for alleviating dentin hyperesthesia may be used in a single form of the peptide or a polypeptide form of <NUM> or more repeats of the peptide, and the peptide may also be used in a complex form of a drug having a therapeutic effect on dentin or dental pulp diseases linked at the N-terminus or C-terminus of the peptide.

The present inventors synthesized a peptide (SEQ ID NO: <NUM>) showing the effect of promoting regeneration of dentin or dental pulp tissues by a <NUM>-fluorenylmethyloxycarbonyl (Fmoc) method, and they synthesized peptides of respective groups (Tables <NUM> to <NUM>) by substituting the amino acids of the synthesized peptide.

First, peptides of Group <NUM> were synthesized by using the peptide of SEQ ID NO: <NUM> or by substituting any amino acid at positions <NUM> to <NUM> of the peptide of SEQ ID NO: <NUM> with lysine or arginine (Table <NUM>).

Next, peptides of Group <NUM> were synthesized by substituting any amino acid at positions <NUM> to <NUM> of the peptide of SEQ ID NO: <NUM> with lysine or arginine or by substituting an amino acid at position <NUM> of the peptide of SEQ ID NO: <NUM> with serine (Table <NUM>).

Next, peptides of Group <NUM> were synthesized by substituting any amino acid at positions <NUM> to <NUM> of the peptide of SEQ ID NO: <NUM> with lysine or arginine or by substituting an amino acid at position <NUM> of the peptide of SEQ ID NO: <NUM> with tyrosine (Table <NUM>).

Next, peptides of Group <NUM> were synthesized by substituting any amino acid at positions <NUM> to <NUM> of the peptide of SEQ ID NO: <NUM> with lysine or arginine, by substituting an amino acid at position <NUM> of the peptide of SEQ ID NO: <NUM> with serine, by substituting an amino acid at position <NUM> of the peptide of SEQ ID NO: <NUM> with tyrosine, or by substituting an amino acid at position <NUM> of the peptide of SEQ ID NO: <NUM> with lysine (Table <NUM>).

Next, peptides of Group <NUM> were synthesized by substituting an amino acid at position <NUM> of the peptide of SEQ ID NO: <NUM> with arginine, by substituting an amino acid at position <NUM> of the peptide of SEQ ID NO: <NUM> with glutamine, or by substituting any amino acid at positions <NUM> to <NUM> of the peptide of SEQ ID NO: <NUM> with lysine or arginine (Table <NUM>).

Next, peptides of Group <NUM> were synthesized by substituting an amino acid at position <NUM> of the peptide of SEQ ID NO: <NUM> with arginine, by substituting an amino acid at position <NUM> of the peptide of SEQ ID NO: <NUM> with glutamine, by substituting any amino acid at positions <NUM> to <NUM> of the peptide of SEQ ID NO: <NUM> with lysine or arginine, or by substituting an amino acid at position <NUM> of the peptide of SEQ ID NO: <NUM> with serine (Table <NUM>).

Next, peptides of Group <NUM> were synthesized by substituting an amino acid at position <NUM> of the peptide of SEQ ID NO: <NUM> with arginine, by substituting an amino acid at position <NUM> of the peptide of SEQ ID NO: <NUM> with glutamine, by substituting any amino acid at positions <NUM> to <NUM> of the peptide of SEQ ID NO: <NUM> with lysine or arginine, by substituting an amino acid at position <NUM> of the peptide of SEQ ID NO: <NUM> with tyrosine, or by substituting an amino acid at position <NUM> of the peptide of SEQ ID NO: <NUM> with lysine (Table <NUM>).

Next, peptides of Group <NUM> were synthesized by substituting an amino acid at position <NUM> of the peptide of SEQ ID NO: <NUM> with arginine, by substituting an amino acid at position <NUM> of the peptide of SEQ ID NO: <NUM> with glutamine, by substituting any amino acid at positions <NUM> to <NUM> of the peptide of SEQ ID NO: <NUM> with lysine or arginine, by substituting an amino acid at position <NUM> of the peptide of SEQ ID NO: <NUM> with serine, by substituting an amino acid at position <NUM> of the peptide of SEQ ID NO: <NUM> with tyrosine, or by substituting an amino acid at position <NUM> of the peptide of SEQ ID NO: <NUM> with lysine (Table <NUM>).

Next, peptides of Group <NUM> were synthesized by substituting an amino acid at position <NUM> of the peptide of SEQ ID NO: <NUM> with lysine, by substituting an amino acid at position <NUM> of the peptide of SEQ ID NO: <NUM> with glutamine, or by substituting any amino acid at positions <NUM> to <NUM> of the peptide of SEQ ID NO: <NUM> with lysine or arginine (Table <NUM>).

Next, peptides of Group <NUM> were synthesized by substituting an amino acid at position <NUM> of the peptide of SEQ ID NO: <NUM> with lysine, by substituting an amino acid at position <NUM> of the peptide of SEQ ID NO: <NUM> with glutamine, by substituting any amino acid at positions <NUM> to <NUM> of the peptide of SEQ ID NO: <NUM> with lysine or arginine, or by substituting an amino acid at position <NUM> of the peptide of SEQ ID NO: <NUM> with serine (Table <NUM>).

Next, peptides of Group <NUM> were synthesized by substituting an amino acid at position <NUM> of the peptide of SEQ ID NO: <NUM> with lysine, by substituting an amino acid at position <NUM> of the peptide of SEQ ID NO: <NUM> with glutamine, by substituting any amino acid at positions <NUM> to <NUM> of the peptide of SEQ ID NO: <NUM> with lysine or arginine, by substituting an amino acid at position <NUM> of the peptide of SEQ ID NO: <NUM> with tyrosine, or by substituting an amino acid at position <NUM> of the peptide of SEQ ID NO: <NUM> with lysine (Table <NUM>).

Lastly, peptides of Group <NUM> were synthesized by substituting an amino acid at position <NUM> of the peptide of SEQ ID NO: <NUM> with lysine, by substituting an amino acid at position <NUM> of the peptide of SEQ ID NO: <NUM> with glutamine, by substituting any amino acid at positions <NUM> to <NUM> of the peptide of SEQ ID NO: <NUM> with lysine or arginine, by substituting an amino acid at position <NUM> of the peptide of SEQ ID NO: <NUM> with serine, by substituting an amino acid at position <NUM> of the peptide of SEQ ID NO: <NUM> with tyrosine, or by substituting an amino acid at position <NUM> of the peptide of SEQ ID NO: <NUM> with lysine (Table <NUM>).

First, MDPC-<NUM> cells, which are mouse-derived odontoblasts, were cultured in DMEM medium containing <NUM>% FBS, <NUM>% CO2 and <NUM>.

Next, the cultured MDPC-<NUM> cells were dispensed into a <NUM>-well plate at <NUM>×<NUM> <NUM> cells per well, incubated for <NUM> hours. And then using Lipofectamine Plus™ reagent, the cultured cells were transformed by introducing a recombinant (pGL3 vector) the DSPP promoter and luciferase gene were introduced. The transformed MDPC-<NUM> cells were treated with the peptides of groups <NUM> to <NUM> synthesized in Example <NUM>, respectively, and cultured for <NUM> hours. Then luciferase activity was measured, and the average level was compared (<FIG>). As a control, transformed MDPC-<NUM> cells without an odontoblast differentiation promoting peptide were used.

<FIG> is a graph showing the effect of each peptide provided in the present invention on the expression of DSPP, an odontoblast differentiation marker gene, for each group. As shown in <FIG>, each peptide provided in the present invention showed a value of about <NUM> times or more of the luciferase activity level measured in the control group as a whole, but the difference was shown for each group, and the peptide of group <NUM> was the highest. And the next highest level of luciferase activity was from group <NUM> peptide.

Therefore, it was verified that the peptides provided by the present invention exhibit an effect of activating the DSPP promoter.

The MDPC-<NUM> cells cultured in Example <NUM>-<NUM> were treated with the peptides of each group synthesized in Example <NUM>, then cultured for <NUM> hours. The mRNA level of the DSPP, an odontoblast differentiation marker gene, expressed in the MDPC-<NUM> cells were measured, and the measured mRNA level of each DSPP gene was converted into a relative ratio to the mRNA level of the DSPP gene measured in control (Tables <NUM> to <NUM>). In addition, the average value of the mRNA level of the DSPP gene measured according to the peptides of each group was compared (<FIG>). At this time, as a control, MDPC-<NUM> cells that were not treated with the peptide promoting differentiation of odontoblast were used.

The expression level of the DSPP gene was measured through RT-PCR and real-time PCR analysis: Specifically, total RNA was extracted from the MDPC-<NUM> cells using TRIzol reagent. <NUM>µg of the total RNA, <NUM>µl of reverse transcriptase, and <NUM>µg of oligo (oligo; dT) were used to synthesize cDNA. The synthesized cDNA was used in a real-time polymerase chain reaction. The real-time polymerase chain reaction was performed on an ABI PRISM <NUM> sequence detection system (Applied Biosystems) and an SYBR GREEN PCR Master Mix (Takara, Japan). The real-time polymerase chain reaction was performed under conditions of <NUM>, <NUM>; <NUM>, <NUM> sec; <NUM>, <NUM> sec for <NUM> cycles. Results were analyzed by a comparative cycle threshold (CT) method. At this time, the Gapdh gene was used as the internal control group, and the measured value was repeated three times. The mean value and standard deviation value thereof were used.

As shown in Tables <NUM> to <NUM>, compared to the mRNA level of the DSPP gene measured in the control group, it was confirmed that the mRNA levels of the DSPP gene of the experimental group treated with the peptide were all <NUM> times or more. In particular, it was confirmed that all the peptides of group <NUM> showed a value of <NUM> times or more in the mRNA level of the DSPP gene, and all peptides of group <NUM> showed a value of <NUM> times or more in the mRNA level of the DSPP gene.

In addition, <FIG> is a graph comparing the expression level of the DSPP gene, an odontoblast differentiation marker, in MDPC-<NUM> cells treated with an odontoblast differentiation promoting peptide. As shown in <FIG>, when the peptide for promoting differentiation of odontoblast was treated, the mRNA level of the DSPP gene, which is a marker for odontoblast differentiation, is increased. Similar to that of <FIG>, it was confirmed that a value of about <NUM> times or more compared to the level of DSPP gene mRNA was measured in the control group.

From the results of Example <NUM>-<NUM>, it was confirmed that the odontoblast differentiation promoting peptide could increase the mRNA level of the DSPP gene, and in particular, the peptides of groups <NUM> and <NUM> can increase the mRNA level of the DSPP gene by at least <NUM> times or more.

Accordingly, it was confirmed whether the peptides of groups <NUM> and <NUM> can also increase the mRNA levels of the Dmp1 and Nestin genes, which are other odontoblast differentiation marker genes.

The following primers were used with the method from Example <NUM>-<NUM>. The peptides of groups <NUM> and <NUM> were used, thereby affecting the expression levels of Dmp1 and Nestin genes. The effect of the differentiation promoting peptide was measured, and the average level was compared (<FIG>). At this time, as a control group, MDPC-<NUM> cells without a peptide promoting differentiation of odontoblast were used.

<FIG> is a graph showing the results of comparing the expression levels of the odontoblast differentiation marker DSPP, Dmp1, and Nestin genes in MDPC-<NUM> cells treated with the peptides of groups <NUM> and <NUM>. As shown in <FIG>, when the odontoblast differentiation promoting peptide was treated, the expression levels of the odontoblast differentiation marker DSPP, Dmp1, and Nestin genes were all increased, but the level of increase for each gene was different. It was confirmed that the peptide of group <NUM> was more effective.

Since each differentiation marker gene is known to be involved in the differentiation of odontoblasts and dentin calcification, the peptides provided in the present invention were analyzed whether they promote the dentin regeneration.

Human dental pulp cells were separated from wisdom teeth of <NUM> adults (aged <NUM>-<NUM>) at the School of Dentistry, Seoul National University. In detail, all experiments were performed after the approval of the Institutional Review Board and obtaining the informed consent from patients. Wisdom teeth were fractured according to a method of <NPL>)) to expose the dental pulps, and dental pulp tissues were separated with forceps. Each of the separated dental pulp tissues was chopped into small pieces with a razor blade, put in a <NUM>-mm dish, covered with a coverslip, and then cultured in a Dulbecco's modified Eagle's medium.

Next, the obtained dental pulp tissue cells were dispensed into a <NUM>-well plate so the number of cells per well was to be about <NUM>×<NUM><NUM>, cultured for <NUM> hours. Then the peptides of groups <NUM> or <NUM> were treated at a concentration of <NUM> or <NUM> ug/ml. And it was incubated again for <NUM>, <NUM> or <NUM> days. The cultured cells were washed with PBS, <NUM>µl of MTT solution was added, and then reacted at about <NUM> for <NUM> hours. After the reaction was completed, the MTT solution was removed, <NUM>µl of DMSO was added, and absorbance was measured at a wavelength of <NUM> (<FIG>). At this time, as a control, pulp tissue cells cultured without the peptide were used.

<FIG> is a graph showing the cytotoxicity of a peptide that promotes the differentiation of oblasts to dental pulp tissue cells. As shown in <FIG>, it was confirmed that the survival rate of pulp tissue cells was at the same level as that of the control group even when the odontoblast differentiation promoting peptide was added.

Mixing purified water and D-sorbitol solution.

Tricalcium phosphate, aminocaproic acid, allantoin, hydrous silicic acid, sodium PCA solution, hydroxyapatite, peptide (SEQ ID NO: <NUM>), enzyme-treated stevia, and xylitol were added and stirred in a stirrer for about <NUM> minutes (stirring conditions: PADDLE <NUM>~ <NUM> rpm, DISPERSE <NUM>~<NUM> rpm, HOMO <NUM>~<NUM> rpm).

Add (concentrated) glycerin and xanthan gum and stir for about <NUM> minutes (Stirring condition: PADDLE <NUM>~<NUM> rpm, DISPERSE <NUM>~<NUM> rpm, HOMO <NUM>~<NUM> rpm).

(concentrated) Glycerin, Carboxymethyl Cellulose Sodium Salt (CMC) added and stirred for about <NUM> minutes (Stirring condition: PADDLE <NUM> ~<NUM> rpm, DISPERSE <NUM>~<NUM> rpm, HOMO <NUM>~<NUM> rpm).

Add sodium cocoylmethyltaurate and stirring for about <NUM> minutes (Stirring conditions: PADDLE <NUM>~<NUM> rpm, DISPERSE <NUM>~<NUM> rpm),.

Prepared purified water of the same volume as in Example <NUM>.

Among the ingredients of Example <NUM>, all ingredients other than those that did not contain an odontoblast differentiation promoting peptide (SEQ ID NO: <NUM>) were prepared to be contained the same.

MALDI-TOF analysis of toothpaste composition for alleviating dentin hyperesthesia according to Example <NUM>.

Observation of the dentinal tubule permeability of the toothpaste composition for alleviating dentin hyperesthesia according to Example <NUM>.

Cut the crown of the extracted person's tooth horizontally with a diamond saw to expose the dentinal tubules, and then wash twice for about <NUM> minutes with a phosphate buffer solution.

The previously cut tooth was reacted with <NUM> ethylenediaminetetraacetic acid (EDTA, pH <NUM>) solution for about <NUM> minutes and then washed twice for about <NUM> minutes with a phosphate buffer solution.

Added <NUM>% of the fluorescent dyeing reagent to the toothpaste composition for alleviating dentin hyperesthesia containing the odontoblast differentiation promoting peptide (SEQ ID NO: <NUM>), mixed well, and then brushed the cut tooth exposed to the dentinal tubules for about <NUM> minute.

The brushed cut tooth was washed <NUM> times with distilled water and then cut lengthwise to a thickness of about <NUM> so that the dentinal tubules of the cut tooth looked long using a diamond saw, and the degree of penetration was observed with a fluorescence microscope (<FIG>).

Observation of the sealing ability of the dentinal tubules of the toothpaste composition for alleviating dentin hyperesthesia according to Example <NUM>.

The composition of artificial saliva is shown in Table <NUM> below.

<IMG> The purified water was added to the final concentration of each component in Table <NUM> and mixed, and potassium phosphate (K2HPO4) was added last.

<IMG> The pH of artificial saliva is measured near <NUM>, similar to human saliva.

The extracted human tooth was cut horizontally using a diamond saw to make a <NUM> thick, dentin specimen with exposed dentinal tubules.

<IMG> The dentin specimen was reacted for about <NUM> minutes in a <NUM>% phosphoric acid solution to expose the dentinal tubules completely, and then washed three times with purified water for about <NUM> minutes. Then, the dentin specimen was washed <NUM> times in an ultrasonic cleaner for about <NUM> minutes to expose the dentinal tubules completely.

Thereafter, washed three times with a phosphate buffer solution and stored.

Using the toothpaste composition for alleviating dentin hyperesthesia according to Example <NUM>, the specimen was brushed for about <NUM> minute to the dentinal tubule specimen, washed <NUM> times with distilled water, and then reacted to the artificial saliva for about <NUM> hours.

After repeating this process for <NUM> weeks, washed three times with distilled water, dried, and observed the degree of dentinal tubule blockade with a scanning electron microscope (S-<NUM>, HITACHI, Tokyo, Japan) (<FIG>, I-L).

Using the purified water prepared in Comparative Example <NUM>-<NUM>, brushed the dentinal tubule specimen for about <NUM> minute, washed <NUM> times with distilled water, and then reacted for about <NUM> hours in artificial saliva.

After repeating this process for <NUM> weeks, the specimens were washed with distilled water <NUM> times, dried, and observed the degree of dentinal tubule blockade with a scanning electron microscope (<FIG>, A-D).

Using the toothpaste prepared in Comparative Example <NUM>-<NUM>, brushed the dentinal tubule specimen for about <NUM> minute, washed <NUM> times with distilled water, and then reacted for about <NUM> hours in artificial saliva.

After repeating this process for <NUM> weeks, the specimens were washed with distilled water <NUM> times, dried, and observed the degree of dentinal tubule blockade with a scanning electron microscope (<FIG>, E-H).

Through Test Example <NUM>, the stability of the peptide contained in the toothpaste composition for alleviating dentin hyperesthesia according to the embodiment of the present invention can be confirmed. <FIG> is a result of measuring the molecular weight through MALDI-TOF analysis to confirm the stability of the peptide contained in the toothpaste composition for alleviating dentin hyperesthesia according to an embodiment of the present invention. In <FIG> shows the molecular weight of the peptide itself contained in the toothpaste composition for alleviating dentin hyperesthesia according to an embodiment of the present invention. In <FIG> shows the molecular weight of the peptide contained in the state of the toothpaste composition for alleviating dentin hyperesthesia according to an embodiment of the present invention.

Referring to <FIG>, it was observed that the peptide contained in the toothpaste composition according to Example <NUM> had the same peak (molecular weight of about <NUM> Da) as the measured value of the molecular weight, and through this, the stability of mixed dentin differentiation promoting peptide in the toothpaste composition was confirmed (see <FIG>).

According to Test Example <NUM>, as a result of observing the dentinal tubule permeability of the toothpaste composition for alleviating dentin hyperesthesia according to Example <NUM> with a fluorescence microscope, as shown in <FIG>, in the case of the tooth treated with the composition, fluorescence was strongly observed on the dentin surface. In addition, penetration of the fluorescent staining reagent was observed along the lower side of the exposed dentinal tubules.

Next, the results of comparing Test Example <NUM> and Comparative Test Examples <NUM>-<NUM> and <NUM>-<NUM> are as shown in <FIG> is a set of images comparing the sealing ability of the dentinal tubules of toothpaste composition for alleviating dentin hyperesthesia according to Example <NUM>, Comparative Example <NUM>-<NUM>, and Comparative Example <NUM>-<NUM>. And in more detail, A-D shows the dentinal tubules of the dentin treated only with purified water (Comparative Example <NUM>-<NUM>), and E-H shows the toothpaste composition without odontoblast differentiation promoting peptide (Comparative Example <NUM>-<NUM>), and I-L shows the dentinal tubules reacted with the composition, including a peptide for oral care that prevents or alleviates dentin hyperesthesia according to an embodiment of the present invention. One (size bar: A, E, I, <NUM>; B, F, J, <NUM>; C, G, K, <NUM> ; D, H, L, <NUM>).

As can be seen from <FIG>, it could be observed that the dentinal tubules were blocked by remineralization in the dentinal tubules by reacting with the toothpaste composition for alleviating dentin hyperesthesia according to an embodiment of the present invention.

<FIG> is an enlarged image of the dentinal tubules blocked by the composition including a peptide for oral care that prevents or alleviates dentin hyperesthesia according to an embodiment of the present invention and shows the results of remineralization in the blocked dentinal tubules and dentin surfaces.

As above, the toothpaste composition for alleviating dentin hyperesthesia according to an embodiment of the present invention forms a thin film on the dentin and at the same time strongly binds to the phosphate-calcium ions released from tricalcium phosphate, and remineralizes the exposed dentinal tubules and dentin surfaces. In other words, the toothpaste composition for alleviating dentin hyperesthesia according to an embodiment of the present invention induces remineralization not only on the surface of the exposed dentinal tubules but also inside the dentinal tubule, thereby exhibiting the effect of alleviating and/or preventing dentin hyperesthesia.

Claim 1:
Toothpaste composition for alleviating dentin hyperesthesia, comprising a peptide consisting of an amino acid sequence of the following Formula <NUM>:

        K-Y-R1-R2-R3-R4-R5-R6-R7-R8     (Formula <NUM>)

wherein R1 is arginine(R), lysine(K) or glutamine(Q);
R2 is arginine(R) or glutamine(Q);
R3, R4, and R5 are arginine(R) or lysine(K), respectively;
R6 is asparagine(N) or serine(S); and
R7 and R8 are lysine(K) or tyrosine(Y), respectively,
wherein said toothpaste composition includes <NUM>-<NUM> parts by weight of the peptide, <NUM>-<NUM> parts by weight of tricalcium phosphate, <NUM>-<NUM> parts by weight of hydroxyapatite, and <NUM>-<NUM> parts by weight of purified water,
wherein said toothpaste composition forms a thin film on the surface of said dentin and induces remineralization on said surface of said dentin and dentinal tubule by binding with the phosphate-calcium ions released from said tricalcium phosphate.