Search

CN-122016682-A - Test method for in-vitro evaluation of acid-resistant calcium locking effect of oral care product

CN122016682ACN 122016682 ACN122016682 ACN 122016682ACN-122016682-A

Abstract

The invention belongs to the technical field of efficacy evaluation of oral care products, and discloses a test method for in-vitro evaluation of acid-resistant calcium locking effect of an oral care product. The method takes isolated bovine enamel as a model, firstly uses a sample for pretreatment, then uses a pH 4.5 phytic acid solution for ore removal for 1h at a time, determines the concentration of calcium ions in ore removal liquid, calculates the calcium locking multiple/proportion, and can determine whether the toothpaste has the acid-resistant calcium locking effect through statistical test, the experimental period is greatly shortened, the time from sampling to quantitative result is only about 4h, compared with the traditional pH circulation model, the method has the advantages of low cost, no need of expensive animal experiments or complex buffer systems, good reproducibility, adoption of one-time phytic acid ore removal, fixed condition, high stability and batch difference reduction.

Inventors

  • SONG TAO
  • ZHANG DAWEI
  • SU FEI
  • XIA QIANG

Assignees

  • 挪亚检验认证集团有限公司

Dates

Publication Date
20260512
Application Date
20260304

Claims (9)

  1. 1.A test method for in vitro evaluation of acid and calcium locking effects of oral care products is characterized by comprising the following specific steps: step one, preparing an acidic solution Weighing a proper amount of phytic acid solution, adding the phytic acid solution into deionized water, adjusting the pH to 4.5, fixing the volume to 1000mL, and shaking uniformly for later use; Step two, sample configuration (pretreatment) The toothpaste is prepared into uniform slurry according to the mass ratio of 1:3 (toothpaste: deionized water), and the non-toothpaste is directly used according to the original or specified times of the instruction; Blank control, deionized water; Step three, grouping Randomly dividing the purchased 5mm multiplied by 5mm intact insert module teeth into 2 groups for standby, wherein at least 3 tooth samples in each group are parallel to each other; step four, sample processing tooth specimen Immersing the isolated bovine tooth specimen in the solutions of the sample group and the blank control group respectively, soaking for 1 hour at the constant temperature of 37 ℃, and then washing with deionized water and storing; step five, acid etching treatment Placing the treated isolated bovine tooth specimen into the phytic acid solution in the first step, and carrying out primary ore removal for 1 hour; Step six, measuring the content of the calcium element After the acid etching is finished, measuring the calcium content in the phytic acid solution of the sample group and the blank group by adopting a flame atomic absorption spectrometry; seventh, data processing and statistical analysis Data processing, namely, calcium locking multiple of a sample group, namely, a blank control group/a sample group, and calcium locking proportion of the sample and the control group, namely, the blank control group, the sample group/the sample group 100%; Statistical analysis, namely adopting a data statistical method t-test to judge whether the two groups of data have significance difference, wherein the significance level alpha=0.05; step eight, judging the data result When the calcium content of the sample group is smaller than that of the blank control group, and meanwhile, the t-test comparison is carried out on the parallel data of the sample group and 3 groups of the blank control group, and the P is smaller than 0.05, the statistical difference exists, and the product can be judged to have the effect of resisting acid and locking calcium.
  2. 2. The method according to claim 1, wherein when the sample is prepared (pretreated) in the second step, and the sample is compared with the control, a negative control is prepared, namely a slurry which is the same as the sample base but does not contain fluorine active substances.
  3. 3. The method for in vitro evaluation of calcium locking effect of oral care products according to claim 1, wherein the step four is characterized in that the step four of washing with deionized water and storing is carried out by taking out the immersed isolated bovine tooth specimen, washing with deionized water for 3 times and 1 minute each time, and then placing the washed isolated bovine tooth specimen in sterile water for temporary storage.
  4. 4. The method for in vitro evaluation of calcium locking resistance of oral care products according to claim 1, wherein the one-time demineralization in the fifth step is carried out for 1 hour by soaking at a constant temperature of 37 ℃ for 1 hour.
  5. 5. The method for in vitro evaluation of calcium locking resistance of oral care products according to claim 1, wherein the flame atomic absorption spectrometry in the sixth step comprises the steps of adding lanthanum solution as a releasing agent after digestion of a sample, and measuring absorbance at 422.7 nm, wherein the absorbance and calcium concentration are in a linear relationship within a standard curve range, and the calcium content in the sample can be quantitatively calculated through a calcium carbonate standard curve.
  6. 6. The method according to claim 1, wherein the t-test in step seven and in step eight is a statistical method for comparing whether there is a significant difference in the average of two samples.
  7. 7. The method according to claim 1, wherein the quality control is further performed during the in vitro evaluation of the anti-acid and anti-calcium-locking effects of the oral care product: The test of each batch is inserted with positive control of sodium fluoride (0.05% NaF), the calcium locking proportion is more than or equal to 30%, otherwise, the batch is invalidated, the glaze is randomly coded and tested by a blind method, and the CV of 3 parallel data in the test group is less than or equal to 10%.
  8. 8. The method according to claim 7, wherein CV is a coefficient of variation for calculating the degree of dispersion of the sample data, and CV is calculated as Standard Deviation (SD) divided by Mean (Mean) in percent.
  9. 9. The method of claim 1, wherein P in the step eight is a probability of making an error that the observation is considered valid, i.e., is generally representative, and a P value of 0.05 indicates a boundary level of acceptable error.

Description

Test method for in-vitro evaluation of acid-resistant calcium locking effect of oral care product Technical Field The invention belongs to the technical field of efficacy evaluation of oral care products, and particularly relates to a test method for in-vitro evaluation of acid and calcium locking effects of oral care products. Background The tooth is easy to lose calcium and phosphorus (demineralize) in an acidic environment, so that enamel becomes thin, further problems of dental caries, tooth sensitivity and the like are caused, fluorine in the toothpaste can promote mineral redeposition, repair an early demineralized area and form a protective layer to reduce calcium loss, and therefore accurate evaluation of the acid and calcium locking effect of the toothpaste is a key link of product research and development and quality detection. In the prior art, the oral care product mainly adopts three types of schemes of long period, high cost and complex ethical approval for in vivo experiments of human or animals, which are not suitable for early formulation screening, two types of simple hardness or surface morphology tests (such as microhardness meters and SEM) which only reflect morphological changes and cannot quantify calcium ion loss and have great dependence on operator experience, three types of conventional pH circulating models (pH-circulating) which are required to prepare various buffers, have complicated steps and multiple circulating times (5-14 days), the reproducibility is obviously influenced by batch differences of a buffer system, and the three types of schemes cannot take the calcium loss as a direct evaluation index in a short time and rapidly and quantitatively compare the calcium locking differences of different toothpaste formulations, so that the industry rapidly needs an in vitro evaluation experimental method which has short period, low cost and high reproducibility and can directly quantify the calcium loss. Disclosure of Invention The invention aims to provide a test method for in-vitro evaluation of the acid and calcium locking resistance effect of an oral care product, so as to solve the problems in the background art. In order to achieve the aim, the invention provides the following technical scheme that the test method for evaluating the acid and calcium locking effect of the oral care product in vitro comprises the following specific steps: step one, preparing an acidic solution Weighing a proper amount of phytic acid solution, adding the phytic acid solution into deionized water, adjusting the pH to 4.5, fixing the volume to 1000mL, and shaking uniformly for later use; Step two, sample configuration (pretreatment) The toothpaste is prepared into uniform slurry according to the mass ratio of 1:3 (toothpaste: deionized water), and the non-toothpaste is directly used according to the original or specified times of the instruction; Blank control, deionized water; Step three, grouping Randomly dividing the purchased 5mm multiplied by 5mm intact insert module teeth into 2 groups for standby, wherein at least 3 tooth samples in each group are parallel to each other; step four, sample processing tooth specimen Immersing the isolated bovine tooth specimen in the solutions of the sample group and the blank control group respectively, soaking for 1 hour at the constant temperature of 37 ℃, and then washing with deionized water and storing; step five, acid etching treatment Placing the treated isolated bovine tooth specimen into the phytic acid solution in the first step, and carrying out primary ore removal for 1 hour; Step six, measuring the content of the calcium element After the acid etching is finished, measuring the calcium content in the phytic acid solution of the sample group and the blank group by adopting a flame atomic absorption spectrometry; seventh, data processing and statistical analysis Data processing, namely, calcium locking multiple of a sample group, namely, a blank control group/a sample group, and calcium locking proportion of the sample and the control group, namely, the blank control group, the sample group/the sample group100%; Statistical analysis, namely adopting a data statistical method t-test to judge whether the two groups of data have significance difference, wherein the significance level alpha=0.05; step eight, judging the data result When the calcium content of the sample group is smaller than that of the blank control group, and meanwhile, the t-test comparison is carried out on the parallel data of the sample group and 3 groups of the blank control group, and the P is smaller than 0.05, the statistical difference exists, and the product can be judged to have the effect of resisting acid and locking calcium. As a preferred embodiment of the present invention, when the sample is prepared (pretreated) in the second step, and when a comparison is made with a control sample, a negative control is provided, which is a slurry of the same base material as