Bright and white teeth play an important role in creating a likable appearance, just as the clean, white skin of the face has aesthetically positive psychological effects. According to a survey of men and women in their 20s and 30s conducted by healthcare research under the theme of "The effect of white teeth," 95% of respondents said that "clean teeth give trust" [1].

This is considered essential to a beautiful smile in the measure of success demanded by modern society, and interest in tooth whitening has recently been increasing in various age groups.

Generally, the discoloration of teeth is divided into extrinsic discoloration that is stained teeth on the surface of the teeth and intrinsic discoloration that the tooth themselves are discolored. The main reasons for the extrinsic discoloration are food and beverages with colors such as coffee and cola, etc., or tobacco, which are favorite products. And intrinsic discoloration is caused by aging, administration of antibiotics in the tetracycline system and systemic diseases, etc [2]. So depending on the cause of the discolored tooth, the method of improving the color tone of the tooth is being applied. Extrinsic discoloration can be removed by physical stimuli and chemical actions such as brushing and professional oral prophylaxis method, and intrinsic discoloration can be achieved by using chemicals such as peroxide to whitening teeth [3]. Peroxide is a representative substance of whitening teeth and is widely used as a principle to brighten the coloration of teeth by oxidizing the organic matter, which is the staining part of the hemorrhoids, using peroxide agents such as hydrogen peroxide (HP), and carbamide peroxide (CP)[4].

Tooth whitening is divided into in-office bleaching performed in dental clinics and at-home bleaching performed by patients themselves [5]. In-office bleaching can have the most definite whitening effect by applying heat or light to a high concentration of 35 to 50 percent HP, a strong oxidizer. But after whitening dentin hypersensitivity, reduction of surface microhardness of human enamel, gingiva swelling and pain are reported [6
-8], and the hassle of having to visit the dentist and the high cost of the procedure are weakness. So the procedure that came out to overcome these weaknesses is self-whitening. Self -whitening is divided into home-bleaching [9], which is performed by injecting gels containing 10% CP into a mouseguard and attaching them to teeth under the guidance of a dentist, and Over the counter (OTC) bleaching [10
-12] that is performed by individuals own self-implementation without the prescription of an expert. Recently, OTC whitening has been generalized [13]. This is because it is easy for anyone to purchase products online and offline and is cheaper than other whitening procedures. However, OTC whitening also uses a lower concentration of HP, so the risk of side effects is lower than that of professional whitening, but side effects such as temporary gingivitis or hypersensitivity are reported [14].

Meanwhile, the public’s desire for white teeth has influenced the development of whitening procedures as well as the development of various whitening agents.

Among them, a method of whitening teeth was developed by adding a separate drug to the toothpaste that is highly accessible to use. The whitening action of whitening toothpaste is done by various chemicals, which contain enzymes, detergents, and oxygen supplies [15]. Therefore, the role of whitening toothpaste is excellent in that it is possible only by the act of brushing teeth in everyday life with toothpaste containing special chemicals. Especially as the number of favorite foods such as coffee, tea, cola, and wine that cause extrinsic discoloration is increasing whitening toothpaste is recognized as a convenience and cost-effectiveness whitening method for consumers. However, most of the main ingredients of whitening toothpaste currently on the market are chemicals such as peroxide, which are still highly controversial about safety [16]. So research is being conducted on components that can replace peroxide. Recently, studies have been reported that have proven the whitening effect of teeth using natural materials such as fruits [11,17], and studies are also being conducted related to the color change in the color of teeth by special abrasive ingredients contained in whitening toothpaste [12,18,19], but are still lacking.

This study reports statistically significant results from in vitro experiments designed to check tooth whitening and the effect of prevention of tooth staining and eliminating of tooth staining by using artificial teeth colored with coffee, tea, and wine based on a whitening effect study [20] that has been demonstrated in vitro tests in hydrated silica and sodium hexametaphosphate (SHMP).

1. Research materials

The experimental toothpaste used in this study is as shown in Table 1. The abrasive ingredients commonly used in toothpaste were 14% of common silica for control groups and 13% of hydrated silica for experimental group 1 and groups 2. In addition, 7.5% of sodium metaphosphate was used in experimental group 2 only, and fluorine and tocopherol acetate components were used equally for all experimental and control group.

Table 1 . Composition of experimental toothpaste (unit: %)

Composition	Control group (A)	Test 1 (B)	Test 2 (C)
Sodium fluoride (KP) (1,000 ppm F)	0.22	0.22	0.22
Dental type silica (NF)	14.0
Hydrated silica (KQC)		13.0	13.0
Tocopherol acetate (KP)	0.05	0.05	0.05
Sodium hexametaphosphate (KQC)			7.5

KP: The Korean Pharmacopoeia, KQC: Korean Quasi-drugs Codex, NF: United States Pharmacopeia-National Formulary.

The HAP specimen in this study was colored by applying the Stookey’s method [22]. The detailed method is as follows.

(1) Specimen manufacture of HAP

① Make the dough with artificiality tooth powder (HAP powder, Bongmin chemical Co., Ltd., Seoul, Korea) and polyvinyl alcohol (Sigma, St. Louis, MO, USA) 3% solution and then dry it at 105℃ for 2 to 3 hours, then measure 0.30 g to 0.35 g of moderately ground powder.

② After applying a pressure of 4.2 tons for 1 minute with the pressure for Infrared spectroscopy, make a HAP specimen with a diameter of 12.5 mm and a thickness of 1.8 mm.

③ After sintering the produced HAP specimen at 1,000℃, make a square-shaped, phosphoric hydroxide specimen shape using a brass mould of 2×2 cm and epoxy resin to make the right size for the specimen staining device.

(2) Manufacture of staining solution: A specimen staining solution is added 7.4 g of mixed powder mixed with coffee, tea, and mucin powder at a ratio of 8:8:6 and 1.6 g of FeCl (1.6 g) to a sterilized 800 ml Trypticase Soy Broth (Becton, Sparks, MD, USA) solution and it is mixed with 30 ml of Sarcina lutea culture solution.

(3) Staining of artificiality tooth specimen

① The staining machine consisted of a fixture frame, an electric motor, a rotating plate and a staining fluid trough, and it was designed so that the specimen can be colored in the fluid trough and the drying process in the air, repeatedly.

② Specimen was sequentially etched for 60 seconds at 1% hydrochloric acid, 30 seconds in super-saturated sodium carbonate solution and 60 seconds at 1% phytic acid to facilitate staining before the start of the staining process.

③ Rinse the corroded specimen thoroughly with water and dry it. The manufactured staining solution was poured into the trough and the rotating plate with the specimen attached was fixed to the motor and colored for five days by the 37℃ culture medium and the staining solution was replaced every 12 hours.

④ Using the initial L value (lightness value) of the colored hydroxide specimen below 60, the L value was distributed so that there would be no statistically significant differences among all experimental groups.

2. Experimental method

The whitening effect evaluation is a method of in vitro evaluation [22] using artificial tooth, which is colored with coffee and tea, which are the component of the tooth staining, and compared the whitening effects of control toothpaste, experimental group 1 and experimental group 2.

(1) Measurement of the initial value of the staining specimen: The initial L, a, and b values of the colored HAP specimen were measured with the colorimeter (Minota CR-321, Minolta Camera Co., Osaka, Japan) and made it the experi-mental specimen

(2) Manufacture of experimental toothpaste: For the brushing experiment, the solutions of control, experimental group 1 and experimental group 2 were manufactured as shown in Table 2.

Table 2 . Composition of experimental solution (unit: g)

Composition	Control solution	Experimental group 1 solution	Experimental group 2 solution
Toothpaste	25	25	25
Purified water	40	40	40
Total	65	65	65

(3) Evaluation of whitening efficacy by brushing: The manufactured experimental solution was secured and levelled to the container of the brushing machine where the staining HAP specimen is fixed. After injecting the manufactured toothpaste slurry, a toothbrush capable of transferring 250 g load to the specimen was placed in the center of the specimen and brushing 5,400 times at 90 strokes per minute (equivalent to about 10 days brushing at 90 strokes per minute, 2 minutes per day, and 3 times per day) was carried out.

(4) Measurement of color change using a colorimeter: After naturally drying the finished brushing specimen at room temperature for 24 hours, the color change was measured with Minota CR-321 (Minolta Camera Co.), and the color change (DE) was analyzed by comparing it with the initial value.

The colorimetric effects of the staining ingredient of coffee, tea, and wine were also evaluated using artificiality tooth powder [23
-25]. After treating artificiality tooth powder (HAP powder; Bongmin chemical Co., Ltd.) with 25% toothpaste slurry, it was washed in water. In addition, the staining inhibiting ability was evaluated by treating them with coffee (Kroger) solution, tea (Lipton Breakfast) solution and wine (Gapsted 2017 Shiraz). The detailed method is as follows [21, 24].

(1) Control and experimental group toothpaste slurry treatment: Using 50 ml of a conical tube, add 0.2 g of HAP powder, 12.5 g of contrasting toothpaste and experimental toothpaste, respectively, and add purified water to make a 25% toothpaste slurry.

Then stir it strongly for 30 seconds with shaker and soak it well into the powder, and remove the upper solution through centrifugation for 4000 RPM for 15 minutes.

(2) Purified water washing: Remove the upper solution and add purified water to the HAP powder that was treated with contrasting and experimental toothpaste first to make 50 ml. Then, wash it with purified water twice in the same way as above and wash the remaining toothpaste without adsorbing the powder.

(3) Staining: Here, stir the staining solution of coffee, tea, and wine strongly in shaker for 30 seconds in the same way. Then, remove the supernatant with the same centrifugation and wash it with purified water twice.

(4) Measurement of color change using a colorimeter: Finally, dry the powder obtained after treatment in 70oC oven for 24 hours, and the change in L, a, and b was measured. Measure the values of L, a, and b of the HAP powder before staining and analyze the color change (DE) in which staining has been suppressed, and calculate the values of L, a, and b that have been colored only by water, not by control or experimental group toothpaste. In contrast to water, the relative ratio of color-protected by control, experimental group 1, experimental group 2 toothpaste is obtained.

3. Data analysis

The experimental results used IBM SPSS Statistics for Windows ver. 19.0 (IBM Co., Armonk, NY, USA) and all statistical significance levels were set at p=0.05. A one-way ANOVA test was performed in the in vitro study to determine the difference in the ability to remove the Stookey’s stain and inhibitory ability of the staining substance of coffee, tea, and wine between experimental group 1, experimental group 2, and control toothpaste. If the removal ability and inhibitory ability results from the used toothpaste were significant, the comparison was verified for the interaction verification and the student’s t-test was performed to verify the main effects of the toothpaste.

1. In vitro tooth whitening effect evaluation results

Figure 1 and Table 3 show the results of comparative evaluation of whitening effects (Stookey’s stain removal ability) using specimens of control toothpaste and experimental tooth-paste. The color change value (DE) was identified as 2.53±0.79, 9.91±3.13, 18.03±0.96, respectively, in the control group, the experimental group 1 and the experimental group 2. The whitening effect was about 3.9 times higher in the test group 1 toothpaste containing hydrated silica compared and about 7.1 times higher the experimental group 2 toothpaste containing SHMP to the control group (p<0.05).

Table 3 . Stookey’s stain in vitro tooth whitening effect evaluation results (n=3)

Group	DE	p-value
Control group	2.53±0.79a	<0.05
Experimental group 1	9.91±3.13b
Experimental group 2	18.03±0.96c

Values are presented as mean±standard deviation. By the independent one-way ANOVA test at α＝0.05. ^a,b,c: Same letters means there is no significant difference by student’s t (p<0.05).

Figure 1. Stookey’s stain in vitro tooth whitening effect evaluation results (n =3).

2. In vitro tooth staining inhibitory effect evaluation

Table 4 shows the results of a comparative evaluation of the staining substance adhesion inhibitory ability of coffee, tea and wine using artificiality tooth (HAP powder; Bongmin chemical Co., Ltd.) in control toothpaste and experimental toothpaste. Compared with the control group, experimental group 1 containing hydrated silica did not show much difference. However, in the second group of experiments containing hydrated silica and SHMP, coffee stain inhibitory ability (4.96±0.13) are about 5.5 times, tea stain inhibitory ability (2.56±0.03) are about 8.0 times, and wine stain inhibitory ability (1.76±0.09) are about 8.9 times higher (p<0.05).

Table 4 . Evaluation of staining substance inhibitory ability (n=3)

	Control group	Experimental group 1	Experimental group 1	p-value
Coffee DE	27.33±0.11a	27.81±0.36a	4.96±0.13b	<0.05
Tea DE	20.43±0.16a	20.77±0.21a	2.56±0.03b	<0.05
Wine DE	15.62±1.36a	15.95±0.21a	1.76±0.09b	<0.05

Values are presented as mean±standard deviation. By the independent one-way ANOVA test at α＝0.05. ^a,b: Same letters means there is no significant difference by student’s t (p<0.05).

Figure 2 and Table 5 show the results of converting the results of the staining of water pre-treated HAP powder to coffee, tea and wine into a denominator DE values of the HAP powder pre-treated and stained with control, experimental 1 and 2 group toothpaste into molecules. The experimental group 2 containing SHMP showed the staining substance adhesion inhibitory ability of 83.3%, 90.2%, and 89.0% respectively for staining substance of coffee, tea and wine compared to control (water) (p<0.05). However, no significant inhibits were observed in control and experimental group 1 that did not contain SHMP.

Table 5 . Against water, experimental toothpaste’ ability to inhibit staining substance adhesion to coffee, tea and wine (n=3) (unit: %)

	DE average % reduction vs. H2O
	Control group	Experimental group 1	Experimental group 2	p-value
Coffee	8.04	6.42	83.30	<0.05
Tea	21.80	20.47	90.19	<0.05
Wine	0.00	0.00	88.95	<0.05

By the independent one-way ANOVA test at α＝0.05. p<0.05.

Figure 2. Against water, experimental toothpaste’ ability to inhibit staining substance adhesion to coffee, tea and wine (unit: %).

Today, whitening toothpaste is chosen by the public as a convenience that can bring whitening effects along with daily tooth brushing without disturbing daily life. However, side effects such as tooth sensitivity symptoms and gingiva swelling and inflammation caused by peroxide, known as the main ingredient of whitening toothpaste currently on the market, are much debated today [6
-8]. Therefore, the need for safe ingredients to minimize side effects is emerging. This study was done with a well-known Stookey’s method in academia. The effect of staining removal ability was evaluated by using toothpaste mixed with hydrated silica and SHMP, which does not contain peroxide, using artificiality tooth colored with coffee, tea and wine. And staining inhibiting ability were observed using coffee, tea, and wine, known as extrinsic discoloration factors. This method was used by foreign multinationals companies such as Procter&Gamble and Glaxo-SmithKline [23,26,27] and it has the advantage of being able to quantitatively measure color changes by anti-staining effect that can be checked by the effect of the main ingredient, not by physical tooth brushing.

To evaluate the whitening effect of teeth according to the ability to remove the Stookey’s stain, the color change (DE) was measured before and after brushing with physical friction similar to tooth brushing 3 times a day, 2 minutes a day, during 10 days. This result is because the removal of the staining substance and the reattach of the staining substance were inhibited using hydrated silica and SHMP as a way to remove the stains attached to the teeth. Hydrated silica is manufactured through a sinking process similar to dental type silica. However, by changing the manufacturing conditions during the sinking process, the silica form was the same as the dental silica, but by increasing the aging time during the manufacturing process, the silica network strength could effectively remove the staining substance of the teeth by increasing the physical cleansing force when the same pressure was transmitted to the teeth. As a result, in-vitro experiments also, the experimental group 1 displayed statistically significant changes in the brightness of the specimen compared to the control group. And SHMP is a component that contains 10 to 12 pyrophosphates in repeating units, serving as a calcium phosphate surface active builder, a component of teeth, and characterized by strong binding affinity with minerals contained in tooth enamel or dentin. SHMP is known to play a role in detaching the pelicle protein that contains the tooth staining substance from the tooth surface while adsorbed inorganic matter on the tooth surface [28,29]. For this reason, the staining substance attached to the teeth is removed, resulting in the whitening effect of the teeth. In addition, SHMP is a substance that is characterized by adsorption of polyvalent ions and shows the effect of inhibiting the coloration of the staining substance by inertizing of chelating the polyvalent ions (Ca2+, Mg2+, Fe3+, etc.) that can make the color of the tooth staining substance more darker. Therefore, even if it contains equally high- cleaning the hydrated silica, in the results of whitening assessment of artificiality tooth specimen, experimental group 2 toothpaste containing 7.5% SHMP showed that the color change value was significantly higher than that of the group 1 of experiments. These results were consistent with the results of the in vivo study, which the authors preceded, showing the largest color difference in the test group containing hydrated silica and SHMP [20]. In addition, Ahn et al. [18] laboratory research also reported the best brightness change in the experimental group with SHMP added to 2.8% HP was shown.

Meanwhile, coffee, tea and wine, all foods that cause tooth stains, are foods that contain dark brown stains, combined with protein acquired pellicle, the pre-stage to the formation of the plaque on the surface of the teeth upon ingestion, causing discoloration of the teeth [25]. In addition, these staining substance have polyphenol components that contain abundant double bonds. These components are characterized by increasing the concentration of staining substance through coordination bond with metal ion, in the protein acquired pellicle stage, it can be removed by toothbrushing, but afterwards, when the protein acquired pellicle are solidified by the aggregation of bacteria and calcium and phosphorus of saliva in the mouth, they form a stain that cannot be removed with a simple toothbrush alone.

Compared to the control group in the assessment of staining inhibits ability, experimental group 2 containing SHMP showed high inhibition with coffee about 5.5 times, tea about 8.0 times, and wine about 8.9 times (p<0.05). And in the results of the assessment of the staining substance adhesion inhibitory ability, the experimental group 2 displayed 83.3%, 90.2%, and 89.0% stain substance adhesion inhibitory ability in coffee, tea and wine respectively (p<0.05). However, no significant inhibitory ability were observed in control and experimental group 1 that did not contain SHMP. This causes the SHMP component to have strong adhesion to the teeth because the molecular weight is higher than the calcium phosphate surface active builder, which has only one iteration unit, such as sodium pyrophosphate, potassium pyrophosphate, disodium dihydrogen pyrophosphate, etc. As a result, SHMP is present in calcium phosphate even if it washed with water during tooth brushing process, thus inhibiting tooth staining by fundamentally blocking access to external staining substance intended to be attached to the calcium ion. Therefore, experimental group 1, which consists mainly of physical cleaning power, confirmed that staining occurs similar to control toothpaste. But the experimental group 2, which contains SHMP, which can chemically inhibit stain, was found to have an excellent effect of inhibiting the attachment of the tooth staining substance compared to experimental group 1 or control.

Overall, experimental group 2 toothpaste containing hydrated silica and SHMP are whitening toothpaste with excellent stain removal ability, at the same time, for coffee, tea, and wine, which are the factors of stain in daily life, have been found to be particularly excellent. These results are thought to be expected to replace peroxide.

As a result of evaluating the tooth whitening effects of the Stookey’s stain removal ability on toothpaste mixed with the hydrated silica and SHMP, and comparing the stain inhibitory ability with the staining substance adhesion inhibitory ability of coffee, tea and wine known as extrinsic discoloration factors, the following conclusions were obtained.

1. As a result of evaluating tooth whitening effects with brushing machine using artificiality tooth specimen, about 3.9 times higher in the test group 1 (9.91±3.13) toothpaste containing hydrated silica compared and about 7.1 times higher in the test group 2 (18.03±0.96) toothpaste containing SHMP to the control group (2.53±0.79) (p<0.05).

2. As a result of evaluating the stain inhibitory ability using artificial tooth, which is colored with coffee, tea and wine, which are the staining component of the teeth. Compared to the control, experimental group 2 containing SHMP showed high staining inhibits ability of 5.5 times, 8.0 times and 8.9 times respectively for coffee, tea and wine (p<0.05).

3. As a result of evaluating the staining substance adhesion inhibitory ability, the experimental group 2 containing SHMP showed staining substance adhesion inhibitory ability of 83.3%, 90.2% and 89.0% of the staining substance of coffee, tea, and wine, respectively, compared to the control group (water) (p<0.05).

To sum up the above results, toothpaste containing hydrated silica and SHMP is whitening toothpaste with excellent stain removal ability and has been confirmed that the anti-stain ability is particularly good for coffee, tea, and wine, which are the factors of staining in daily life. These results showed the possibility of whitening toothpaste as a substitute for peroxide.

No potential conflict of interest relevant to this article was reported.