Method for detecting content of trehalose in dressing
By preparing DNS reagents and glucose standard solutions, a glucose standard curve was prepared, dressing samples were hydrolyzed and trehalose content was calculated, which solved the accuracy and reproducibility of trehalose content detection in the prior art, and achieved a fast and accurate detection effect.
Patent Information
- Application Number
- CN202510455413.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-11
- Publication Date
- 2025-06-27
AI Technical Summary
The prior art has problems such as poor accuracy and reproducibility, complex operation, weak anti-interference ability and long detection cycle when detecting trehalose content in dressings, especially when other sugars are present in the sample at the same time.
The preparation of DNS reagents and glucose standard solutions, the preparation of glucose standard curves, the hydrolysis treatment of dressing samples and the calculation steps of trehalose content, and the trehalose content was hydrolyzed by acid reagents, and the reduced sugar content was determined by DNS method to calculate the content of trehalose.
It realizes rapid and accurate detection of the trehalose content in the dressing, with simple operation and short detection cycles, and can accurately calculate the trehalose content when other sugars are present.
Smart Images
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of detecting the content of trehalose in dressings, and specifically provides a method for detecting the content of trehalose in dressings. Background Art
[0002] Trehalose is a natural and safe sugar. It is a non-reducing disaccharide composed of two glucose molecules and has very stable properties. Research shows that exogenous trehalose can also provide good non-specific protection for organisms and biological macromolecules. In recent years, more and more tests have shown that trehalose exhibits various effects in medical beauty products. Significant progress has been made in the research and application of trehalose in aspects such as moisturizing and locking water, wound repair, anti-radiation, and post-sun repair. It can improve symptoms such as increased dandruff, heat, and horny sclerosis caused by skin dryness by effectively protecting the epidermal cell membrane structure, activating cells, and enhancing the hydration function of cells.
[0003] With the continuous development of research and application technologies, it has become increasingly important to quickly and accurately detect the content of trehalose, especially when other sugars are present in the sample.
[0004] Existing methods for determining the content of saccharide substances have their own advantages and disadvantages. Currently, commonly used methods include paper chromatography separation and elution method, anthrone-sulfuric acid method, gas chromatography, high-performance liquid chromatography, ion chromatography, and enzyme-biosensor analyzer method. Among them, the paper chromatography separation and elution method and the anthrone-sulfuric acid method have cumbersome operation steps, are easily interfered with, and have poor accuracy and reproducibility. Although the gas chromatography method has high accuracy and reproducibility, it requires the use of derivatization reagents and has complex operations. The anti-interference abilities of ion chromatography and enzyme methods are relatively weak. Although high-performance liquid chromatography can accurately determine the content of trehalose in a polysaccharide mixture system, the determination period is long and it is difficult to achieve rapid detection.
[0005] In view of the disadvantages existing in the existing methods for determining the content of saccharide substances, the present invention provides a method for detecting the content of trehalose in dressings. Summary of the Invention
[0006] The purpose of the present invention is to provide a method for detecting the content of trehalose in dressings to solve the problems in the above background art.
[0007] To achieve the above purpose, the present invention provides the following technical solution: A method for detecting the content of trehalose in dressings, comprising the following steps:
[0008] Step (1), preparing DNS reagent and glucose standard solution;
[0009] Step (2), making a glucose standard curve;
[0010] Step (3), performing hydrolysis treatment on the dressing sample;
[0011] Step (4), calculate the trehalose content.
[0012] Preferably, the specific steps for preparing the DNS reagent in step (1) are as follows:
[0013] Add 6.3 g of DNS and 262 mL of 2 mol / L NaOH solution to a hot aqueous solution of 500 mL containing 185 g of potassium sodium tartrate. Then add 5 g of crystalline phenol and 5 g of sodium sulfite, stir to dissolve, cool, and make up the volume to 1000 mL with distilled water. Store in a brown bottle for later use.
[0014] Preferably, the specific steps for preparing the glucose standard solution in step (1) are as follows:
[0015] Accurately weigh 50 mg of analytical pure glucose dried to constant weight at 80 °C, place it in a small beaker, dissolve it with a small amount of distilled water, transfer it to a 100 mL volumetric flask, make up the volume to 100 mL with distilled water, mix well, and store in a refrigerator at 4 °C for later use.
[0016] Preferably, the specific steps for making the glucose standard curve in step (2) are as follows:
[0017] Prepare glucose solutions with different concentrations, then add DNS reagent, heat in a boiling water bath for 5 minutes, then cool to room temperature, dilute with water, use the blank tube as a control, and use a spectrophotometer to measure the absorbance values of each other tube at a wavelength of 540 nm. Draw a glucose standard curve with the glucose content in the sample as the abscissa and the absorbance value of each tube at 540 nm as the ordinate.
[0018] Preferably, the specific steps for hydrolyzing the dressing sample in step (3) are as follows:
[0019] Add the dressing sample containing trehalose to a small beaker, add a certain amount of acid reagent, wait for the sample to completely dissolve, seal it, hydrolyze at high temperature for a certain time, adjust the pH of the solution to neutral after hydrolysis, and then make up the volume to 100 mL.
[0020] Preferably, the specific steps for calculating the trehalose content in step (4) are as follows:
[0021] Take the hydrolyzed sample, add DNS reagent, then heat in a boiling water bath for 5 minutes, then cool to room temperature, dilute with water and measure the absorbance value, calculate the concentration of reducing sugar according to the linear equation, and multiply by the dilution factor of the sample to obtain the reducing sugar content in the sample.
[0022] Preferably, the calculation formula for the trehalose content in the sample is as follows:
[0023] Trehalose content = Reducing sugar content after hydrolysis × 342 / 360.
[0024] The present invention has at least the following beneficial effects:
[0025] The detection method for the trehalose content in a dressing provided by the present invention utilizes the basic principle that an acid reagent can hydrolyze one molecule of trehalose into two molecules of glucose, and then uses the conventional method of the 3,5-dinitrosalicylic acid (DNS) method to determine the reducing sugar content, detects the reducing sugar content in the sample after acid hydrolysis, can accurately calculate the trehalose content in the dressing sample, is simple to operate, has a short detection period, and can achieve rapid detection. Specific embodiments
[0026] The technical solutions in the embodiments of the present invention are clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the scope of protection of the present invention.
[0027] In view of the deficiencies of the existing trehalose content detection technology, the present invention provides a detection method for the trehalose content in a facial mask dressing with high accuracy, low cost and simple operation.
[0028] The test principle is as follows: Utilize the basic principle that an acid reagent can hydrolyze one molecule of trehalose into two molecules of glucose. By adding a sufficient amount of hydrochloric acid or sulfuric acid to the sample to be tested and reacting under high-temperature conditions, it can be ensured that within a certain time, trehalose can be completely hydrolyzed into glucose; then use the conventional method of the 3,5-dinitrosalicylic acid (DNS) method to determine the reducing sugar content, detect the reducing sugar content in the sample after acid hydrolysis, and the trehalose content in the dressing sample can be accurately calculated.
[0029] Based on the above technical solutions, the present invention provides the following partial embodiments:
[0030] Example 1
[0031] Experimental method and process:
[0032] (1) Prepare DNS reagent and glucose standard solution
[0033] 3,5-dinitrosalicylic acid (DNS) reagent: Add 6.3 g of DNS and 262 mL of 2 mol / L NaOH solution to a 500 mL hot water solution containing 185 g of potassium sodium tartrate, then add 5 g of crystalline phenol and 5 g of sodium sulfite, stir and dissolve, and make up to 1000 mL with distilled water after cooling, and store in a brown bottle for later use.
[0034] 500 μg / mL Glucose Standard Solution: Accurately weigh 50 mg of analytical pure glucose dried to a constant weight at 80 °C, place it in a small beaker, dissolve it with a small amount of distilled water, transfer it to a 100 mL volumetric flask, make up the volume to 100 mL with distilled water, mix well, and store it in a refrigerator at 4 °C for later use.
[0035] (2) Preparation of Glucose Standard Curve
[0036] Prepare 6 test tubes as shown in the following table, add different amounts of glucose standard solution respectively, dilute to 1 mL with distilled water, and add DNS reagent.
[0037]
[0038] After shaking the above test tubes well, place them in a boiling water bath and heat for 5 min, cool to room temperature, and then make up the volume to 10 mL with water. Using the blank tube as a control, use a spectrophotometer to measure the absorbance values of the other tubes at a wavelength of 540 nm. Draw a glucose standard curve with the glucose content (mg / mL) in the sample as the abscissa and the absorbance value of each tube at 540 nm as the ordinate.
[0039] (3) Hydrolysis Treatment of Dressing Samples
[0040] Take a part of the dressing sample containing trehalose and add it to a small beaker, add 30 mL of 5% hydrochloric acid solution, wait for the sample to completely dissolve, seal it, hydrolyze at 100 °C for 4 h, adjust the pH of the solution to neutral after hydrolysis, and then make up the volume to 100 mL.
[0041] (4) Calculation of Trehalose Content:
[0042] Take 1 mL of the hydrolyzed sample, add 1.5 mL of DNS reagent, then heat in a boiling water bath for 5 minutes, then cool to room temperature, add 7.5 mL of distilled water, measure the absorbance value at 540 nm, calculate the concentration of reducing sugar according to the linear equation obtained in step (2), and multiply by the dilution factor of the sample to obtain the reducing sugar content in the sample. Since one H2O molecule is added during the hydrolysis of trehalose to glucose, the content of trehalose in the sample should be calculated according to the following formula:
[0043] Trehalose content = Reducing sugar content after hydrolysis × 342 / 360
[0044] The test results are as follows in the table:
[0045] Serial number Reducing sugar content in the sample after hydrolysis (mg / mL) Trehalose content in the sample (mg / mL) 1 5.2271 4.9658 2 5.2164 4.9556
[0046] Example 2
[0047] Experimental Method and Procedure:
[0048] (1) Preparation of DNS Reagent and Glucose Standard Solution
[0049] 3,5-Dinitrosalicylic acid (DNS) reagent: Dissolve 6.3 g of DNS and 262 mL of 2 mol / L NaOH solution in a hot aqueous solution of 500 mL containing 185 g of potassium sodium tartrate. Then add 5 g of crystalline phenol and 5 g of sodium sulfite, stir to dissolve, and after cooling, make up the volume to 1000 mL with distilled water. Store in a brown bottle for later use.
[0050] 500 μg / mL glucose standard solution: Accurately weigh 50 mg of analytical pure glucose dried to constant weight at 80 °C, place it in a small beaker, dissolve it with a small amount of distilled water, transfer it to a 100 mL volumetric flask, make up the volume to 100 mL with distilled water, mix well, and store in a refrigerator at 4 °C for later use.
[0051] (2) Preparation of glucose standard curve
[0052] Prepare 6 test tubes as shown in the following table, add different amounts of glucose standard solution respectively, dilute to 1 mL with distilled water, and add DNS reagent.
[0053]
[0054] After shaking the above test tubes well, place them in a boiling water bath and heat for 5 min, then cool to room temperature, and then make up the volume to 10 mL with water. Using the blank tube as a control, use a spectrophotometer to measure the absorbance values of the other tubes at a wavelength of 540 nm. Draw a glucose standard curve with the glucose content (mg / mL) in the sample as the abscissa and the absorbance value of each tube at 540 nm as the ordinate.
[0055] (3) Hydrolysis treatment of dressing samples
[0056] Take a part of the dressing sample containing trehalose and add it to a small beaker, add 20 mL of 10% hydrochloric acid solution, wait for the sample to dissolve completely, seal it, and hydrolyze at 100 °C for 5 h. After hydrolysis, adjust the pH of the solution to neutral, and then make up the volume to 100 mL.
[0057] (4) Calculation of trehalose content:
[0058] Take 1 mL of the hydrolyzed sample, add 1.5 mL of DNS reagent, then heat in a boiling water bath for 5 minutes, then cool to room temperature, add 7.5 mL of distilled water, measure the absorbance value at 540 nm, calculate the concentration of reducing sugar according to the linear equation obtained in step (2), and multiply by the dilution factor of the sample to obtain the reducing sugar content in the sample. Since one H2O molecule is added during the hydrolysis of trehalose to glucose, the content of trehalose in the sample should be calculated according to the following formula:
[0059] Trehalose content = Reducing sugar content after hydrolysis × 342 / 360
[0060] The test results are shown in the following table:
[0061] Serial number Reducing sugar content in the sample after hydrolysis (mg / mL) Trehalose content in the sample (mg / mL) 1 5.1398 4.8828 2 5.1366 4.8798
[0062] Example 3
[0063] Experimental method and process:
[0064] (1) Prepare DNS reagent and glucose standard solution
[0065] 3,5-Dinitrosalicylic acid (DNS) reagent: Add 6.3 g of DNS and 262 mL of 2 mol / L NaOH solution to a 500 mL hot aqueous solution containing 185 g of potassium sodium tartrate. Then add 5 g of crystalline phenol and 5 g of sodium sulfite, stir to dissolve, cool and make up the volume to 1000 mL with distilled water, and store in a brown bottle for later use.
[0066] 500 μg / mL glucose standard solution: Accurately weigh 50 mg of analytical pure glucose dried to constant weight at 80 °C, place it in a small beaker, dissolve it with a small amount of distilled water, transfer it to a 100 mL volumetric flask, make up the volume to 100 mL with distilled water, mix well, and store in a refrigerator at 4 °C for later use.
[0067] (2) Preparation of glucose standard curve
[0068] Prepare 6 test tubes as shown in the following table, add different amounts of glucose standard solution respectively, dilute to 1 mL with distilled water, and add DNS reagent.
[0069]
[0070] After shaking the above test tubes well, place them in a boiling water bath and heat for 5 min, cool to room temperature, and then make up the volume to 10 mL. Using the blank tube as a control, use a spectrophotometer to measure the absorbance values of the other tubes at a wavelength of 540 nm. Draw a glucose standard curve with the glucose content (mg / mL) in the sample as the abscissa and the absorbance value of each tube at 540 nm as the ordinate.
[0071] (3) Hydrolysis treatment of dressing sample
[0072] Take a part of the dressing sample containing trehalose and add it to a small beaker, add 10 mL of 15% hydrochloric acid solution, wait for the sample to dissolve completely, seal it, hydrolyze at 100 °C for 6 h, adjust the pH of the solution to neutral after hydrolysis, and then make up the volume to 100 mL.
[0073] (4) Calculation of trehalose content:
[0074] Take 1 mL of the hydrolyzed sample, add 1.5 mL of DNS reagent, then heat in a boiling water bath for 5 minutes, then cool to room temperature, add 7.5 mL of distilled water, measure the absorbance value at 540 nm, calculate the concentration of reducing sugar according to the linear equation obtained in step (2), and multiply by the dilution factor of the sample to obtain the content of reducing sugar in the sample. Since one H2O molecule is added during the hydrolysis of trehalose to glucose, the content of trehalose in the sample should be calculated according to the following formula:
[0075] Trehalose content = Reducing sugar content after hydrolysis × 342 / 360
[0076] The test results are as follows:
[0077] Serial number Reducing sugar content in the sample after hydrolysis (mg / mL) Trehalose content in the sample (mg / mL) 1 4.9745 4.7258 2 5.0313 4.7798
[0078] Example 4
[0079] Replace the hydrochloric acid solution for hydrolyzing trehalose in Example 1 with 30 mL of a 5% sulfuric acid solution, and the other operation steps are the same as in Example 1. The test results are as follows:
[0080] Serial number Reducing sugar content in the sample after hydrolysis (mg / mL) Trehalose content in the sample (mg / mL) 1 5.1739 4.9152 2 5.1106 4.8551
[0081] Example 5
[0082] Replace the hydrochloric acid solution for hydrolyzing trehalose in Example 2 with 20 mL of a 10% sulfuric acid solution, and the other operation steps are the same as in Example 2. The test results are as follows:
[0083] Serial number Reducing sugar content in the sample after hydrolysis (mg / mL) Trehalose content in the sample (mg / mL) 1 4.9637 4.7155 2 4.9702 4.7217
[0084] Example 6
[0085] Replace the hydrochloric acid solution for hydrolyzing trehalose in Example 3 with 10 mL of a 15% sulfuric acid solution, and the other operation steps are the same as in Example 3. The test results are as follows:
[0086] Serial number Reducing sugar content in the sample after hydrolysis (mg / mL) Trehalose content in the sample (mg / mL) 1 5.0369 4.7851 2 5.0755 4.8217
[0087] Comparative Example 1
[0088] Refer to the method in the national standard GB / T 23529-2009, and use a high-performance liquid chromatograph equipped with a differential detector to determine the content of trehalose in the dressing; use an acetonitrile-water solution with a volume ratio of 7:3 as the mobile phase for elution, and use an amino column (4.6 mm × 300 mm, 5 μm) for separation and detection of the content of trehalose in the dressing. Repeat the detection 3 times, and the average value of the trehalose content is 4.9856 mg / mL.
[0089] Combining the results obtained from the above experiments, it can be concluded that: compared with the detection results obtained by the traditional high-performance liquid chromatography method, the error of the hydrochloric acid hydrolysis detection method in Example 1 of the present invention is the lowest. Therefore, in step (3), hydrochloric acid with a concentration of 5% should be used and heated in a boiling water bath for 4 hours to obtain more accurate results.
[0090] The foregoing has shown and described the basic principles, main features and advantages of the present invention. For those skilled in the art, it is obvious that the present invention is not limited to the details of the above-described exemplary embodiments, and without departing from the spirit or basic characteristics of the present invention, the present invention can be implemented in other specific forms. Therefore, from any point of view, the embodiments should be regarded as exemplary and non-limiting. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be embraced by the present invention.
[0091] Although the embodiments of the present invention have been shown and described, for those of ordinary skill in the art, it can be understood that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principle and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.
Claims
1. A method for detecting the trehalose content in a dressing, characterized in that: The following steps are involved: Step (1), preparing DNS reagent and glucose standard solution; Step (2), preparing a glucose standard curve; Step (3), hydrolyzing the dressing sample; Step (4), calculate the trehalose content.
2. The method for detecting the trehalose content in a dressing according to claim 1, characterized in that: The specific steps of preparing the DNS reagent in step (1) are as follows: Add 6.3g DNS and 262mL 2mol / L NaOH solution to 500mL hot water solution containing 185g potassium sodium tartrate, then add 5g crystalline phenol and 5g sodium sulfite, stir to dissolve, cool, add distilled water to make up to 1000mL, and store in a brown bottle for later use.
3. The method for detecting the trehalose content in a dressing according to claim 1, characterized in that: The specific steps of step (1) of preparing the glucose standard solution are as follows: Accurately weigh 50 mg of analytical grade glucose dried at 80°C to constant weight, place it in a small beaker, add a small amount of distilled water to dissolve it, transfer it to a 100 mL volumetric flask, make up to 100 mL with distilled water, mix well, and store in a refrigerator at 4°C for later use.
4. The method for detecting the trehalose content in a dressing according to claim 1, characterized in that: The specific steps of preparing the glucose standard curve in step (2) are as follows: Prepare glucose solutions of different concentrations, then add DNS reagent, heat in a boiling water bath for 5 minutes, then cool to room temperature, dilute with water, use a blank tube as a control, and use a spectrophotometer to measure the absorbance values of other tubes at a wavelength of 540 nm. Draw a glucose standard curve with the glucose content in the sample as the horizontal axis and the absorbance value of each tube at 540 nm as the vertical axis.
5. The method for detecting the trehalose content in a dressing according to claim 1, characterized in that: The specific steps of step (3) of hydrolyzing the dressing sample are as follows: Add the dressing sample containing trehalose into a small beaker, add a certain amount of acid reagent, wait for the sample to be completely dissolved and then seal it, hydrolyze it at high temperature for a certain period of time, adjust the pH of the solution to neutral after the hydrolysis is completed, and then make up to 100 mL.
6. The method for detecting the trehalose content in a dressing according to claim 1, characterized in that: The specific steps of calculating the trehalose content in step (4) are as follows: Take the hydrolyzed sample, add DNS reagent, then heat it in a boiling water bath for 5 minutes, then cool it to room temperature, dilute it with water and measure the absorbance value. Calculate the concentration of reducing sugar according to the linear equation, and multiply it by the dilution factor of the sample to get the reducing sugar content in the sample.
7. The method for detecting the trehalose content in a dressing according to claim 6, characterized in that: The calculation formula of the trehalose content in the sample is as follows: Trehalose content = reducing sugar content after hydrolysis × 342 / 360.