Test method for evaluating anti-acid calcium locking effect of oral care product in vitro

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 anti-acid calcium locking effects of oral care products, which comprises the following specific steps: step 1, preparing an acidic solution, weighing a proper amount of phytic acid solution, adding into deionized water, adjusting the pH value to 4.5, fixing the volume to 1000mL, and shaking uniformly for later use; 2, sample configuration (pretreatment); the method comprises the following steps: by taking in-vitro bovine tooth enamel as a model, pretreating by using a sample, performing primary demineralization for 1 hour by using a phytic acid solution with the pH value of 4.5, measuring the calcium ion concentration of demineralization liquid, calculating the calcium locking multiple / proportion, and performing statistical test to judge whether the toothpaste has the acid-resisting and calcium-locking effects or not. According to the method, the experimental period is greatly shortened, only about 4 h is needed from sampling to quantitative result obtaining, and the time is shortened compared with that of a traditional pH circulation model; the cost is low, and expensive animal experiments or complex buffer systems are not needed; the method is good in reproducibility, adopts one-time phytic acid demineralization, is fixed in condition and high in stability, and reduces batch difference.

Description

Technical Field

[0001] This invention belongs to the field of oral care product efficacy evaluation technology, specifically a test method for evaluating the anti-acid and calcium-locking effects of oral care products in vitro. Background Technology

[0002] Teeth are prone to calcium and phosphorus loss (demineralization) in acidic environments, leading to thinning of tooth enamel and causing problems such as tooth decay and tooth sensitivity. Fluoride in toothpaste can promote mineral redeposition, repair early demineralized areas, and form a protective layer to reduce calcium loss. Therefore, accurately evaluating the anti-acid and calcium-locking effects of toothpaste is a key aspect of product development and quality testing.

[0003] In existing technologies, the evaluation of the anti-acid / anti-caries efficacy of oral care products on tooth enamel mainly adopts the following three types of schemes: 1. In vivo human or animal tests: long cycle, high cost, complex ethical approval, unsuitable for early formula screening; 2. Simple hardness or surface morphology tests (such as microhardness testers, SEM): only reflect morphological changes, cannot quantify calcium ion loss, and are highly dependent on operator experience; 3. Conventional pH cycling model: requires the preparation of multiple buffer solutions, cumbersome steps, many cycles (5-14 days), and reproducibility is significantly affected by batch differences in the buffer system. None of the above three schemes can quickly and quantitatively compare the differences in "calcium retention" of different toothpaste formulas with "calcium loss" as a direct evaluation indicator in a short period of time. Therefore, the industry urgently needs an in vitro evaluation experimental method that is short-cycle, low-cost, highly reproducible, and can directly quantify calcium loss. Summary of the Invention

[0004] The purpose of this invention is to provide an in vitro test method for evaluating the anti-acid and calcium-locking effects of oral care products, so as to solve the problems mentioned in the background art.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a test method for evaluating the anti-acid and calcium-locking effects of oral care products in vitro, the specific steps of which are as follows: Step 1: Preparation of acidic solution Weigh an appropriate amount of phytic acid solution, add it to deionized water to adjust the pH to 4.5, and make up to 1000 mL. Shake well and set aside. Step 2: Sample Preparation (Pretreatment) Toothpaste: Mix with deionized water at a mass ratio of 1:3 to form a uniform paste; Non-toothpaste: Use directly according to the instructions or the specified multiple. Blank control: Deionized water; Step 3: Grouping The purchased 5mm×5mm intact enamel-coated dental prostheses were randomly divided into two groups for use: a sample group and a blank control group. Each group had at least three tooth specimens in parallel. Step 4: Sample Processing of Dental Specimens The isolated bovine tooth specimens were immersed in the sample group and blank control group solutions, respectively, and soaked at 37°C for 1 hour. They were then rinsed with deionized water and stored. Step 5: Acid Etching Treatment The treated isolated bovine tooth specimens were placed in the phytic acid solution from step one and demineralized for 1 hour at a time. Step Six: Determination of Calcium Content After acid etching, the calcium content in the phytic acid solution of the sample group and the blank control group was determined by flame atomic absorption spectrometry. Step 7: Data Processing and Statistical Analysis Data processing: Calcium retention ratio of the sample group: blank control group / sample group; Calcium retention ratio between the sample and control groups: (blank control group - sample group) / sample group 100%; Statistical analysis: The t-test was used to determine whether there was a significant difference between the two groups of data, with a significance level of α=0.05; Step 8: Data Result Judgment If the calcium content in the sample group is lower than that in the blank control group, and the t-test comparison of the three parallel data of the sample group and the blank control group shows a statistical difference of P < 0.05, then the product can be determined to have the effect of resisting acid and locking calcium.

[0006] As a preferred technical solution of the present invention, when the sample preparation (pretreatment) in step two is required, a negative control is set when a control sample is needed for comparison: a slurry with the same base material as the sample but without fluorine active material.

[0007] As a preferred technical solution of the present invention, the specific method of rinsing and storing the sample with deionized water in step four is as follows: after taking out the soaked isolated bovine tooth specimen, rinse it with deionized water 3 times, 1 minute each time, and then place the rinsed isolated bovine tooth specimen in sterile water for temporary storage.

[0008] As a preferred technical solution of the present invention, the specific method of demineralization for 1 hour in step five is: constant temperature soaking at 37°C for 1 hour.

[0009] As a preferred technical solution of the present invention, the specific content of the flame atomic absorption spectrometry in step six is ​​as follows: after the sample is digested, a lanthanum solution is added as a releasing agent, and the absorbance is measured at 422.7 nm; the absorbance is linearly related to the calcium concentration within the standard curve range, and the calcium content in the sample can be quantitatively calculated through the calcium carbonate standard curve.

[0010] As a preferred technical solution of the present invention, the t-test described in steps seven and eight refers to a statistical method used to compare whether there is a significant difference between the means of two samples.

[0011] As a preferred embodiment of the present invention, the test method also requires quality control during use: Each batch of tests should include a sodium fluoride positive control (0.05% NaF) with a calcium retention ratio of ≥30%; otherwise, the batch is invalid. Glaze blocks should be randomly coded and tested blinded. The CV of three parallel data points within the experimental group should be ≤10%.

[0012] As a preferred technical solution of the present invention, the CV is the coefficient of variation, which is used to statistically analyze the dispersion of sample data. The formula for calculating the CV value is the standard deviation (SD) divided by the mean (Mean), and the result is in the form of a percentage.

[0013] As a preferred technical solution of the present invention, P mentioned in step eight is the probability of making an error that considers the observation result to be valid, i.e., representative of the whole, and a P value of 0.05 represents the acceptable error boundary level.

[0014] The beneficial effects of this invention are as follows: This invention uses isolated bovine tooth enamel as a model. The sample is first pretreated, then demineralized for 1 hour with a pH 4.5 phytic acid solution. The calcium ion concentration in the demineralized solution is measured, and the calcium retention ratio is calculated. Statistical analysis allows for the determination of whether the toothpaste has anti-acid and calcium-locking effects. This method significantly shortens the experimental cycle, requiring only about 4 hours from sampling to obtaining quantitative results, which is shorter than the traditional pH cycle model. It is low-cost, requiring no expensive animal experiments or complex buffer systems. The method has good reproducibility, using a single-stage phytic acid demineralization process with fixed conditions, resulting in high stability and reduced batch-to-batch variation. The evaluation is direct and quantitative, using calcium ion release as the core indicator, providing objective and accurate results. The operation is convenient and has high throughput, making it suitable for companies to quickly screen formulations and verify efficacy, providing efficient and reliable technical support for product development and patent claims. Attached Figure Description

[0015] Figure 1 This is a flowchart of the present invention. Detailed Implementation

[0016] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0017] like Figure 1As shown in the figure, this invention provides an in vitro test method for evaluating the anti-acid and calcium-locking effects of oral care products. The specific steps are as follows: Step 1: Preparation of acidic solution Weigh an appropriate amount of phytic acid solution, add it to deionized water to adjust the pH to 4.5, and make up to 1000 mL. Shake well and set aside. Step 2: Sample Preparation (Pretreatment) Toothpaste: Mix with deionized water at a mass ratio of 1:3 to form a uniform paste; Non-toothpaste: Use directly according to the instructions or the specified multiple. Blank control: Deionized water; Step 3: Grouping The purchased 5mm×5mm intact enamel-coated dental prostheses were randomly divided into two groups for use: a sample group and a blank control group. Each group had at least three tooth specimens in parallel. Step 4: Sample Processing of Dental Specimens The isolated bovine tooth specimens were immersed in the sample group and blank control group solutions, respectively, and soaked at 37°C for 1 hour. They were then rinsed with deionized water and stored. Step 5: Acid Etching Treatment The treated isolated bovine tooth specimens were placed in the phytic acid solution from step one and demineralized for 1 hour at a time. Step Six: Determination of Calcium Content After acid etching, the calcium content in the phytic acid solution of the sample group and the blank control group was determined by flame atomic absorption spectrometry. Step 7: Data Processing and Statistical Analysis Data processing: Calcium retention ratio of the sample group: blank control group / sample group; Calcium retention ratio between the sample and control groups: (blank control group - sample group) / sample group 100%; Statistical analysis: The t-test was used to determine whether there was a significant difference between the two groups of data, with a significance level of α=0.05; Step 8: Data Result Judgment If the calcium content in the sample group is lower than that in the blank control group, and the t-test comparison of the three parallel data of the sample group and the blank control group shows a statistical difference of P < 0.05, then the product can be determined to have the effect of resisting acid and locking calcium.

[0018] This experimental method replaces multi-step pH cycling with a one-step phytic acid demineralization: starting from pH 4.5, 0.1 mol L... -1Phytic acid solution completes the entire acid challenge in a single 1-hour demineralization process, eliminating the need for the traditional 5-14 day, multiple solution changes, and pH cycling steps. Calcium ion release is the sole direct-reading quantitative indicator: no hardness measurement or electron microscopy is required; the calcium ion concentration in the demineralization solution is directly determined using flame atomic absorption spectrometry (GB5009.92-2016), with the calcium retention multiple or ratio as the endpoint. The sample slurry adopts a reverse sequence design of "pretreatment + immediate demineralization": the active ingredients (fluorides, bioactive glass, etc.) are first allowed to fully react with the glaze surface for 1 hour before a single acid challenge, instead of the traditional "demineralization-treatment-evaluation" process. Phytic acid is used as a pioneering demineralizing agent: abandoning commonly used acetic acid, lactic acid, or citric acid, phytic acid is selected as the demineralization challenge agent.

[0019] In step two, when preparing the sample (pretreatment), if a control sample is required for comparison, a negative control is set: a slurry with the same base material as the sample but without fluorine active substances.

[0020] The samples can be oral care products such as toothpaste, mouthwash, and oral films. A slurry with the same base material as the sample but without fluoride active ingredients is set up as a negative control. On the one hand, it can clearly distinguish whether the experimental results are caused by fluoride active ingredients or by the characteristics of the sample base material itself, eliminating base material interference and ensuring the accuracy of experimental results. On the other hand, by comparing with the negative control, it is possible to intuitively judge whether fluoride active ingredients play a role and their effect, providing a reliable reference for subsequent analysis and enhancing the scientific nature and persuasiveness of the experiment.

[0021] The specific procedure for rinsing and storing the isolated bovine tooth specimen in step four is as follows: after taking out the soaked specimen, rinse it with deionized water three times for one minute each time, and then temporarily store the rinsed specimen in sterile water.

[0022] Rinsing the specimen three times with deionized water for one minute each time can effectively remove residual soaking reagents from the surface of the extracted bovine tooth specimen, avoiding interference with subsequent experiments and ensuring the accuracy of experimental results. Temporarily storing the specimen in sterile water can keep the specimen moist, while the sterile environment can reduce microbial contamination, ensuring that the specimen is in good condition before use in subsequent experiments, thus improving the reliability and effectiveness of the experiments.

[0023] The specific procedure for the one-hour demineralization in step five is as follows: soaking at a constant temperature of 37℃ for 1 hour.

[0024] This approach simulates the relatively stable temperature environment inside the oral cavity, making the demineralization process more realistic, ensuring consistency of experimental conditions, and improving the accuracy and reliability of the results.

[0025] The specific content of the flame atomic absorption spectrometry in step six is ​​as follows: after the sample is digested, a lanthanum solution is added as a releasing agent, and the absorbance is measured at 422.7 nm; the absorbance is linearly related to the calcium concentration within the standard curve range, and the calcium content in the sample can be quantitatively calculated through the calcium carbonate standard curve.

[0026] Flame atomic absorption spectrometry is an analytical technique that uses a chemical flame to atomize elements and quantitatively determine calcium content by utilizing the absorption of ground-state atoms by light of a specific wavelength. It can detect trace amounts of calcium (concentrations as low as ppm), meeting the needs for accurate evaluation of the anti-acid and calcium-locking effects of oral care products. Moreover, the equipment is mature, the analysis speed is fast, and it is suitable for batch determination of calcium content in sample groups and blank control groups.

[0027] Among them, the t-test in steps seven and eight refers to a statistical method used to compare whether there is a significant difference between the means of two samples.

[0028] The t-test is suitable for situations where the sample size is small and the population standard deviation is unknown. In this experiment, each group had at least 3 tooth specimens in parallel, resulting in a small sample size. The t-test calculates the ratio of the difference between the means of the two groups to the degree of data variation to obtain the t-value. Then, the corresponding p-value is obtained by looking up the t-distribution table based on the degrees of freedom. When p < 0.05, it indicates that there is a significant difference between the means of the two samples, thus determining whether the product has an anti-acid and calcium-locking effect.

[0029] The test methods also require quality control during use: Each batch of tests should include a sodium fluoride positive control (0.05% NaF) with a calcium retention ratio of ≥30%; otherwise, the batch is invalid. Glaze blocks should be randomly coded and tested blinded. The CV of three parallel data points within the experimental group should be ≤10%.

[0030] Setting up a sodium fluoride positive control and specifying a calcium-locking ratio of ≥30% verifies the effectiveness of the experimental system and ensures reliable test results. Random coding of enamel blocks and blind testing avoid subjective interference and ensure objective results. Limiting the CV of three parallel data points within the experimental group to ≤10% ensures data repeatability and stability and reduces errors. These quality control measures ensure the scientific rigor of the experiment from multiple aspects, making the evaluation of the acid-resistant calcium-locking effect of oral care products more accurate and reliable.

[0031] CV stands for Coefficient of Variation, which is used to statistically analyze the dispersion of sample data. The CV value is calculated by dividing the standard deviation (SD) by the mean (Mean), and the result is expressed as a percentage.

[0032] The smaller the CV value, the more concentrated the data distribution; conversely, the larger the CV value, the more dispersed the data. Under certain experimental conditions, the CV value is an extremely important indicator and is usually used to measure the stability and accuracy of experimental data.

[0033] In step eight, P is the probability of making an error that considers the observation to be valid, i.e., representative of the whole population. A P value of 0.05 represents the acceptable error boundary level.

[0034] The P-value is a key indicator for judging whether a product has the effect of resisting acid and locking calcium. When P < 0.05, it means that under the premise of assuming that the product has no effect, the probability of obtaining the current observation result or a more extreme result is less than 5%, that is, the probability of making a mistake is low, and the product can be considered to have the effect of resisting acid and locking calcium. This boundary level is scientific and reasonable.

[0035] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0036] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A test method for evaluating the anti-acid and calcium-locking effects of oral care products in vitro, characterized in that, The specific steps are as follows: Step 1: Preparation of acidic solution Weigh an appropriate amount of phytic acid solution, add it to deionized water to adjust the pH to 4.5, and make up to 1000 mL. Shake well and set aside. Step 2: Sample Preparation (Pretreatment) Toothpaste: Mix with deionized water at a mass ratio of 1:3 to form a uniform paste; Non-toothpaste: Use directly according to the instructions or the specified multiple. Blank control: Deionized water; Step 3: Grouping The purchased 5mm×5mm intact enamel-coated dental prostheses were randomly divided into two groups for use: a sample group and a blank control group. Each group had at least three tooth specimens in parallel. Step 4: Sample Processing of Dental Specimens The isolated bovine tooth specimens were immersed in the sample group and blank control group solutions, respectively, and soaked at 37°C for 1 hour. They were then rinsed with deionized water and stored. Step 5: Acid Etching Treatment The treated isolated bovine tooth specimens were placed in the phytic acid solution from step one and demineralized for 1 hour at a time. Step Six: Determination of Calcium Content After acid etching, the calcium content in the phytic acid solution of the sample group and the blank control group was determined by flame atomic absorption spectrometry. Step 7: Data Processing and Statistical Analysis Data processing: Calcium retention ratio of the sample group: blank control group / sample group; Calcium retention ratio between the sample and control groups: (blank control group - sample group) / sample group 100%; Statistical analysis: The t-test was used to determine whether there was a significant difference between the two groups of data, with a significance level of α=0.05; Step 8: Data Result Judgment If the calcium content in the sample group is lower than that in the blank control group, and the t-test comparison of the three parallel data of the sample group and the blank control group shows a statistical difference of P < 0.05, then the product can be determined to have the effect of resisting acid and locking calcium.

2. The test method for evaluating the acid-resistant and calcium-locking effects of oral care products in vitro according to claim 1, characterized in that: When preparing the sample (pretreatment) as described in step two, if a control sample is required for comparison, a negative control should be set up: a slurry with the same base material as the sample but without fluorine active substances.

3. The test method for evaluating the acid-resistant and calcium-locking effects of oral care products in vitro according to claim 1, characterized in that: The specific procedure for rinsing and storing the extracted bovine tooth specimens in step four is as follows: After removing the soaked specimens, rinse them three times with deionized water for one minute each time, and then temporarily store the rinsed specimens in sterile water.

4. The test method for evaluating the acid-resistant and calcium-locking effects of oral care products in vitro according to claim 1, characterized in that: The specific procedure for the one-hour demineralization process described in step five is as follows: soaking at a constant temperature of 37℃ for 1 hour.

5. The test method for evaluating the acid-resistant and calcium-locking effects of oral care products in vitro according to claim 1, characterized in that: The specific content of the flame atomic absorption spectrometry described in step six is ​​as follows: After the sample is digested, a lanthanum solution is added as a releasing agent, and the absorbance is measured at 422.7 nm; the absorbance is linearly related to the calcium concentration within the standard curve range, and the calcium content in the sample can be quantitatively calculated through the calcium carbonate standard curve.

6. The test method for evaluating the acid-resistant and calcium-locking effects of oral care products in vitro according to claim 1, characterized in that: The t-test mentioned in steps seven and eight refers to a statistical method used to compare whether there is a significant difference between the means of two samples.

7. The test method for evaluating the acid-resistant and calcium-locking effects of oral care products in vitro according to claim 1, characterized in that: The aforementioned test method also requires quality control during its use. Each batch of tests should include a sodium fluoride positive control (0.05% NaF) with a calcium retention ratio of ≥30%; otherwise, the batch is invalid. Glaze blocks should be randomly coded and tested blinded. The CV of three parallel data points within the experimental group should be ≤10%.

8. The test method for evaluating the acid-resistant and calcium-locking effects of oral care products in vitro according to claim 7, characterized in that: The CV stands for Coefficient of Variation, which is used to statistically analyze the dispersion of sample data. The CV value is calculated by dividing the standard deviation (SD) by the mean (Mean), and the result is expressed as a percentage.

9. The test method for evaluating the acid-resistant and calcium-locking effects of oral care products in vitro according to claim 1, characterized in that: The P mentioned in step eight is the probability of making an error when the observation is considered valid, i.e., representative of the whole population. A P value of 0.05 represents the acceptable error boundary level.

Images