Method for detecting nitzschia sp in cosmetics

Abstract

The application relates to the technical field of cosmetic detection, and specifically discloses a detection method for nannochloropsis sp. polysaccharide in cosmetics. The detection method is characterized in that: first, a cosmetic sample is mixed with a demulsification solvent, then elution is carried out, and then enzymolysis is carried out by using an enzymolysis agent; after boiling water bath, CTAB solution is added, and after ice bath, centrifugation is carried out; the precipitate is collected, redissolved, and constant volume is carried out to obtain a sample liquid. The sample liquid is mixed with sulfuric acid solution, boiling water bath is carried out, cooling is carried out, and pH is adjusted to 7.0 to obtain a pretreated sample liquid. The pretreated sample liquid is sequentially added with a chromogenic solution, an oxidizing agent and sodium thiosulfate to obtain a chromogenic solution. Then, double-wavelength detection is carried out on the chromogenic solution, the concentration of glucuronidase in the chromogenic solution is measured, and the higher the concentration of glucuronidase in the chromogenic solution, the higher the content of nannochloropsis sp. polysaccharide in the cosmetic sample. The method can improve the detection efficiency and anti-interference performance of the detection method for nannochloropsis sp. polysaccharide in cosmetics.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of cosmetic detection, and in particular to a detection method of nostoc polysaccharide in cosmetics. BACKGROUND

[0002] Nostoc polysaccharide contains multiple hydroxyl groups, which can form hydrogen bonds with water molecules, thereby firmly binding water molecules, which are the main "functional groups" for maintaining water. In addition, the complex spatial configuration of polysaccharide can also adsorb a certain amount of water molecules. Therefore, the application of nostoc polysaccharide in cosmetics can improve the moisturizing, anti-photoaging, absorption promoting and repairing effects of cosmetics. However, excessive content of nostoc polysaccharide in cosmetics can cause adverse reactions such as itching, erythema or pore blockage of the skin, and therefore it is necessary to control the content of nostoc polysaccharide in cosmetics.

[0003] In related technologies, phenol-sulfuric acid method or chromatography method is generally used to detect nostoc polysaccharide in cosmetics. However, phenol-sulfuric acid method detects total polysaccharide rather than target acidic polysaccharide, which is easily interfered by coexisting polysaccharides such as hyaluronic acid and sodium alginate. Chromatography method requires complex pretreatment and purification. Moreover, cosmetics contain complex components such as oil, emulsifier, pigment, preservative, thickening agent and surfactant, which are extremely easy to cause interference and misjudgment.

[0004] Therefore, it is necessary to develop a detection method of nostoc polysaccharide in cosmetics with high detection efficiency and strong anti-interference performance. SUMMARY

[0005] In order to improve the detection efficiency and anti-interference performance of the detection method of nostoc polysaccharide in cosmetics, the present application provides a detection method of nostoc polysaccharide in cosmetics.

[0006] The detection method of nostoc polysaccharide in cosmetics provided by the present application adopts the following technical solution:

[0007] The detection method of nostoc polysaccharide in cosmetics comprises the following steps:

[0008] S1, mixing a cosmetic sample and a demulsification solvent, centrifuging, removing an upper oil phase, collecting a lower water phase, passing the lower water phase through a SPE column, eluting the SPE column with an eluent, collecting the eluted eluent, centrifuging, and collecting a supernatant;

[0009] S2, adding an enzymatic agent to the supernatant, oscillating at 42-48℃ for 30-40min, boiling in a water bath for 4-6min, adding a CTAB solution, ice-bathing for 20-25min, centrifuging, washing the precipitate with ethanol, and then resolubilizing in ultrapure water to obtain a sample solution, wherein the enzymatic agent comprises beta-glucosidase and lysozyme;

[0010] S3, add the sample liquid into the sulfuric acid solution, boil in water bath for 40-50 min, cool to room temperature, adjust pH to 7.0, to obtain a pretreated sample liquid; add the pretreated sample liquid into the enzyme label plate hole of the 96-hole plate, add the chromogenic liquid and oxidant in turn, shake and mix, stand at room temperature in dark, then add sodium thiosulfate to obtain a chromogenic solution;

[0011] S4, carry out double-wavelength detection on the chromogenic solution, the main detection wavelength is 620 nm, and the reference wavelength is 450 nm, read the absorbance value A 620 and the absorbance value A 450 at 450 nm of each hole at 620 nm, calculate the difference between the absorbance value A 620 and the absorbance value A 450 , to obtain the corrected absorbance, judge the concentration of glucuronide in the chromogenic solution corresponding to the corrected absorbance according to the glucuronide standard curve equation, the higher the concentration of glucuronide in the chromogenic solution, the higher the content of the nostoc polysaccharide in the cosmetic sample.

[0012] By adopting the above technical scheme, the demulsification solvent in the method can destroy the emulsification structure, release the wrapped nostoc polysaccharide, and eliminate the interference caused by the wrapping of silicon oil and mineral oil on the polysaccharide. The C18 bonded phase in the SPE column can adsorb organic pigments and fragrances. The diatomite can intercept particulate matter, and simultaneously remove main interference sources such as pigments, titanium dioxide and carbomer colloids. In the S1 step, the supernatant can be collected by centrifugation, which can remove most of the solid impurities and prevent the subsequent enzymatic reaction from being blocked. In the S2 step, the β-glucosidase can hydrolyze non-target or interfering small molecular glucan or oligosaccharide into glucose monosaccharide, and through terminal modification, the nostoc polysaccharide is more easily precipitated and enriched by CTAB. The CTAB solution is electrostatically combined with the nostoc polysaccharide to form a precipitate, thereby selectively enriching the nostoc polysaccharide. The ice bath can reduce the solubility of the CTAB-polysaccharide complex and enhance the precipitation efficiency. The precipitate is washed with ethanol, which can remove the liposoluble impurities combined on the precipitate. In the S3 step, the 2M sulfuric acid solution can completely cleave the nostoc polysaccharide in the sample liquid into monosaccharide, eliminating the influence of the difference in polysaccharide polymerization degree. Adjusting the pH to 7.0 can prevent over-acidification from causing the decomposition of the chromogenic product. The chromogenic liquid is condensed with the aldehyde group of the uronic acid to generate blue hydrazone compounds, the oxidizing agent oxidizes the condensation product to generate stable blue dyes, and sodium thiosulfate terminates the reaction and blocks the absorbance drift caused by excessive oxidation. Then, the double-wavelength detection method is used for detection, 620 nm captures the target signal, and 450 nm deducts the background scattering, which can eliminate the interference of residual emulsion particles, pigments and colloidal refraction. Therefore, the method has high detection efficiency and strong anti-interference performance on impurities in cosmetics, which can improve the detection efficiency and anti-interference performance of the detection method of the nostoc polysaccharide in cosmetics.

[0013] In one specific embodiment, the demulsification solvent comprises a demulsifier and a non-polar solvent in a volume ratio of 1:(2-3).

[0014] By adopting the above technical solution, the demulsifier can destroy the emulsification structure and release the encapsulated gonyaulax polysaccharide. The non-polar solvent can extract liposoluble components such as silicone oil, mineral oil and synthetic esters, and eliminate the interference of oil and fat on subsequent detection.

[0015] In one specific embodiment, the demulsification solvent further comprises tetramethyl ethylenediamine, and the volume ratio of the demulsifier to tetramethyl ethylenediamine is 1:(1-1.2).

[0016] By adopting the above technical solution, the emulsion layer stability of cosmetics usually depends on the 'ionic bridge' effect of metal ions at the oil-water interface, and the demulsifier usually has insufficient effect on such emulsion layer. Tetramethyl ethylenediamine can chelate metal ions, directly destroy the ionic crosslinking bonds in the emulsion layer, make the emulsion layer structure collapse, and then the demulsifier penetrates and collapses the oil-water interface film, and the non-polar solvent dissolves the free oil, so that the three work together to realize the collapse of the emulsion layer, and the oil / water is completely separated. Therefore, the above S1 step can further reduce the interference of the complex matrix of cosmetics and improve the anti-interference performance of the method.

[0017] In one specific embodiment, the eluent comprises the following raw materials in parts by weight: 0.1-0.3 parts of formic acid, 38-44 parts of methanol, 38-44 parts of acetonitrile, and the balance is deionized water, with a total amount of 100 parts.

[0018] By adopting the above technical solution, the present application uses low-concentration formic acid and high-concentration methanol and acetonitrile. The high concentration of methanol and acetonitrile greatly reduces the polarity of the solution, which can greatly weaken the hydrophobic interaction force, and helps to effectively elute the gonyaulax polysaccharide adsorbed on the SPE column. Moreover, the above-mentioned proportion of methanol and acetonitrile combines the strong elution force and low viscosity of acetonitrile and the solubility of methanol, and the elution effect is better than that of using one of them alone.

[0019] In one specific embodiment, the preparation method of the chromogenic solution comprises the following steps: uniformly mixing 3,3',5,5'-tetramethylbenzidine with anhydrous N,N-dimethylformamide to obtain a 3,3',5,5'-tetramethylbenzidine stock solution, uniformly mixing horseradish peroxidase stock solution with glycerol to obtain a horseradish peroxidase working solution, and uniformly mixing the 3,3',5,5'-tetramethylbenzidine stock solution, the horseradish peroxidase working solution and the citrate buffer to obtain the chromogenic solution.

[0020] By adopting the technical scheme, 3,3',5,5'-tetramethylbenzidine is dissolved in N,N-dimethylformamide, and a blue product is generated after oxidation. Horseradish peroxidase catalyzes the oxidation of 3,3',5,5'-tetramethylbenzidine, which can amplify the detection signal. Citrate buffer provides the optimal pH environment for the oxidation of 3,3',5,5'-tetramethylbenzidine by horseradish peroxidase to form a blue product. The three cooperate to improve the sensitivity of the detection method.

[0021] In a specific implementable embodiment, in the S3 step, the pretreated sample liquid is mixed uniformly with the graphene quantum dot solution, and then added into the enzyme-labeled plate hole of the 96-hole plate. The color developing liquid and the oxidizing agent are sequentially added, mixed uniformly, and placed in the dark at room temperature. Then, sodium thiosulfate is added to obtain a color developing solution.

[0022] By adopting the technical scheme, graphene quantum dots can enhance the color developing reaction of 3,3',5,5'-tetramethylbenzidine. Even if the color developing reaction is weak, a stronger absorbance signal can be generated under the assistance of quantum dots, so that lower content of botryosphaeran can be detected.

[0023] In a specific implementable embodiment, the preparation method of the oxidizing agent comprises the following steps: dissolving ferric chloride and potassium ferricyanide in ultrapure water, and mixing uniformly to obtain the oxidizing agent. The amount of ferric chloride and potassium ferricyanide is 2.5 g / L.

[0024] By adopting the technical scheme, potassium ferricyanide itself is a strong oxidizing agent and can directly oxidize reducing substances. Ferric chloride can enhance the oxidation capacity or stability of the system. This chemical oxidizing agent does not depend on specific enzymes, which helps to complete the color developing reaction.

[0025] In a specific implementable embodiment, the enzymatic agent further comprises tetrabutylammonium bromide.

[0026] By adopting the technical scheme, tetrabutylammonium bromide can dissolve or loosen lipid and membrane structures, and enhance the contact and effect of lysozyme and beta-glucosidase on cell wall components.

[0027] In a specific implementable embodiment, in the S4 step: glucose uronic acid is dissolved in ultrapure water to obtain several glucose uronic acid standard samples with different concentrations of glucose uronic acid. A microplate reader is used to detect the glucose uronic acid standard samples at double wavelengths, the main detection wavelength is 620 nm, the reference wavelength is 450 nm, ultrapure water is used for zero setting, and the absorbance value A 620 at 620 nm and the absorbance value A 450 at 450 nm of each hole are read, and the absorbance value A 620 and the absorbance value A 450The difference between the two is obtained, and the corrected absorbance of the glucuronic acid standard is obtained. The concentration of the glucuronic acid standard is used as the horizontal coordinate, and the corrected absorbance of the glucuronic acid standard is used as the vertical coordinate. A linear equation y=ax+b is fitted. The corrected absorbance of the cosmetic sample to be tested and the control group is substituted into the glucuronic acid standard curve equation to obtain the concentration of glucuronic acid in the chromogenic solution. The higher the concentration of glucuronic acid in the chromogenic solution, the higher the content of the nostoc polysaccharide in the cosmetic sample.

[0028] In a specific implementation, the concentration of glucuronic acid in the chromogenic solution is denoted as C, the content of nostoc polysaccharide = (C x Vt) ÷ W x K, Vt is the volume of the sample in step S2, W is the initial mass of the cosmetic sample, and K is the inverse of the proportion of glucuronic acid in nostoc polysaccharide.

[0029] By using the above technical solution and the specific test and judgment method, the content of nostoc polysaccharide in the cosmetic can be accurately judged.

[0030] In summary, the present application has the following beneficial effects:

[0031] 1. The method of the present application has high detection efficiency, and strong anti-interference performance to impurities in the cosmetic, which can improve the detection efficiency and anti-interference performance of the detection method of nostoc polysaccharide in the cosmetic.

[0032] 2. The demulsifying solvent, chromogenic solution and oxidizing agent in the present application are preferably prepared by using specific raw materials, which can further improve the detection efficiency and anti-interference performance of the detection method of nostoc polysaccharide in the cosmetic. DETAILED DESCRIPTION

[0033] Unless otherwise specified, the reagents used in the present application are commercially available. Among them, the SPE column is Discovery DSC-MCAX SPE solid phase extraction tube. The β-glucosidase is purchased from Guangdong Wengjiang Chemical Reagent Co., Ltd., with a purity of AR grade. The lysozyme model number is AS90021, and the brand is AMEKO. The CTAB solution is purchased from Shanghai Shangbao Biological Technology Co., Ltd. The CAS number of 3-methyl-2-benzothiazolinone oxime is 38894-11-0, and the purity is AR grade. The potassium ferricyanide model number is P3289, and the brand is SIGMA. The ferric chloride is PWX4038 ferric chloride purchased from Langfang Jinnuo Biological Technology Co., Ltd. The tetramethyl ethylenediamine is N,N,N',N'-tetramethyl ethylenediamine purchased from Nanjing Huikang Biological Technology Co., Ltd. The tetrabutylammonium bromide is purchased from Guangdong Wengjiang Chemical Reagent Co., Ltd., with a purity of AR grade. The graphene quantum dot solution is model BKMK427, purchased from Beikona Nano. 3,3',5,5'-tetramethylbenzidine is purchased from Guangdong Fangxin Biological Technology Co., Ltd., with a purity of AR grade. N,N-dimethylformamide is purchased from Nanjing Chemical Reagent Co., Ltd. The horseradish peroxidase stock solution is purchased from Hubei Ruibaoxin Chemical Co., Ltd. Glucuronic acid is purchased from Chengdu Desite Biological Technology Co., Ltd. The porphyridium polysaccharide is purchased from Shaanxi Senyuan Biological Technology Co., Ltd. Gyeonggi polysaccharide.

[0034] The present application is further described in detail in combination with the following examples and comparative examples.

[0035] Example

[0036] Example 1

[0037] The present example provides a method for detecting porphyridium polysaccharide in cosmetics, which adopts the following steps:

[0038] S1, mix 1.0 g of cosmetic sample, 1.0 mL of demulsifier (Triton X-114) and 2.0 mL of n-hexane, ultrasonically oscillate uniformly, then centrifuge at 3000 rpm for 10 min, remove the upper oil phase, and collect the lower water phase. Then pass the lower water phase through the SPE column, elute the SPE column with eluent, collect the eluted eluent, centrifuge at 3000 rpm for 3 min, and take the supernatant.

[0039] S2, add 100 μL of β-glucosidase and 50 μL of lysozyme to the supernatant, oscillate at 45°C for 35 min, then boil in water bath for 5 min to complete the enzyme inactivation. Then add 200 μL of CTAB solution with a mass concentration of 1%, ice bath for 23 min, then centrifuge at 12000 rpm for 15 min, collect the precipitate, wash the precipitate with ethanol, then redissolve in ultrapure water, and dilute to 1 mL with ultrapure water to obtain the sample solution.

[0040] S3. Add 0.5 mL of sample solution to 0.5 mL of 2 M sulfuric acid solution, boil in a water bath for 45 min, cool to room temperature, then add 10 M sodium hydroxide to adjust the pH to 7.0 to obtain the pretreated sample solution. Add 50 μL of the pretreated sample solution to the wells of a 96-well microplate, then add 50 μL of the colorimetric solution, shake with a plate shaker for 10 s, let stand at room temperature in the dark for 15 min, then add 50 μL of oxidant, shake to mix, let stand at room temperature in the dark for 15 min, then add 20 μL of 10% sodium thiosulfate to terminate the reaction, thus obtaining the colorimetric solution.

[0041] S4. Use an ELISA reader to perform dual-wavelength detection on the chromogenic solution. The main detection wavelength is 620 nm, and the reference wavelength is 450 nm. Zero the instrument with ultrapure water and read the absorbance value A of each well at 620 nm. 620 and absorbance value A at 450 nm 450 Calculate the absorbance value A 620 With absorbance value A 450 The difference is used to obtain the corrected absorbance. The concentration of glucuronic acid in the colorimetric solution corresponding to the corrected absorbance is determined according to the glucuronic acid standard curve equation. Based on the glucuronic acid concentration, the content of Nostoc commune polysaccharide in the cosmetic sample is determined.

[0042] The eluent was prepared from the following ingredients: 0.2 g formic acid, 40 g methanol, 40 g acetonitrile, and 19.8 g deionized water. The formic acid, methanol, acetonitrile, and deionized water were mixed thoroughly to obtain the eluent.

[0043] The colorimetric solution was prepared as follows: 5g of 3-methyl-2-benzothiazolinone oxime was dissolved in 1L of 0.1M hydrochloric acid and stirred until completely dissolved to obtain the colorimetric solution.

[0044] The oxidant is prepared as follows: 2.5g of ferric chloride and 2.5g of potassium ferricyanide are dissolved in 1L of ultrapure water, shaken and mixed to obtain the oxidant.

[0045] Example 2

[0046] The only difference between this embodiment and Example 1 is that step S2 is as follows: 100 μL of β-glucosidase and 50 μL of lysozyme are added to the supernatant. After shaking at 42°C for 40 min, the mixture is then boiled in a water bath for 6 min to inactivate the enzymes. Next, 200 μL of 1% CTAB solution is added, and the mixture is incubated on ice for 25 min. Then, it is centrifuged at 12000 rpm for 15 min, the precipitate is collected, washed with ethanol, reconstituted in ultrapure water, and brought to a final volume of 1 mL with ultrapure water to obtain the sample solution.

[0047] Example 3

[0048] The difference between this embodiment and embodiment 1 is only that the S2 step is as follows: 100 μL of β-glucosidase and 50 μL of lysozyme are added to the supernatant, and after oscillation reaction at 48°C for 30 min, enzyme inactivation is completed by boiling in a water bath for 4 min. Then 200 μL of 1% CTAB solution is added, and after ice bath for 20 min, centrifugation is performed at 12000 rpm for 15 min, and the precipitate is collected, washed with ethanol, and then redissolved in ultrapure water, and then diluted with ultrapure water to 1 mL to obtain a sample solution.

[0049] Embodiment 4

[0050] The difference between this embodiment and embodiment 1 is only that the S3 step is as follows: 0.5 mL of sample solution is added to 0.5 mL of 2M sulfuric acid solution, and after boiling in a water bath for 40 min, the solution is cooled to room temperature, and then 10M sodium hydroxide is added to adjust the pH to 7.0 to obtain a pretreated sample solution. Then 50 μL of the pretreated sample solution is added to the enzyme plate well of a 96-well plate, and then 50 μL of color developing solution is added, and oscillation is performed for 10 s with a plate oscillator, and after standing at room temperature in the dark for 20 min, 50 μL of oxidizing agent is added, and then oscillation is performed for mixing, and after standing at room temperature in the dark for 20 min, 20 μL of 10% sodium thiosulfate is added to terminate the reaction, and a color developing solution is obtained.

[0051] Embodiment 5

[0052] The difference between this embodiment and embodiment 1 is only that the S3 step is as follows: 0.5 mL of sample solution is added to 0.5 mL of 2M sulfuric acid solution, and after boiling in a water bath for 50 min, the solution is cooled to room temperature, and then 10M sodium hydroxide is added to adjust the pH to 7.0 to obtain a pretreated sample solution. Then 50 μL of the pretreated sample solution is added to the enzyme plate well of a 96-well plate, and then 50 μL of color developing solution is added, and oscillation is performed for 10 s with a plate oscillator, and after standing at room temperature in the dark for 12 min, 50 μL of oxidizing agent is added, and then oscillation is performed for mixing, and after standing at room temperature in the dark for 12 min, 20 μL of 10% sodium thiosulfate is added to terminate the reaction, and a color developing solution is obtained.

[0053] Embodiment 6

[0054] The difference between this embodiment and embodiment 1 is only that the eluent is prepared by using the following raw materials: 0.1 g of formic acid, 38 g of methanol, 38 g of acetonitrile, and 23.9 g of deionized water.

[0055] Embodiment 7

[0056] The difference between this embodiment and embodiment 1 is only that the eluent is prepared by using the following raw materials: 0.3 g of formic acid, 44 g of methanol, 44 g of acetonitrile, and 11.7 g of deionized water.

[0057] Example 8

[0058] The difference between this example and Example 1 is only that the eluent is prepared using the following raw materials: 0.1 g formic acid, 80 g methanol, 19.9 g deionized water.

[0059] Example 9

[0060] The difference between this example and Example 1 is only that the eluent is prepared using the following raw materials: 80 g methanol, 20 g deionized water.

[0061] Example 10

[0062] The difference between this example and Example 1 is only that in the S1 step: replace n-hexane with an equal amount of dichloromethane.

[0063] Example 11

[0064] The difference between this example and Example 1 is only that in the S1 step: replace n-hexane with an equal amount of diethyl ether.

[0065] Example 12

[0066] The difference between this example and Example 1 is only that the S1 step is as follows: mix 1.0 g of the cosmetic sample, 1.0 mL of the demulsifier (Triton X-114), and 2.5 mL of n-hexane, uniformly ultrasonically oscillate, centrifuge at 3000 rpm for 10 min, remove the upper oil phase, collect the lower aqueous phase. Then pass the lower aqueous phase through the SPE column, elute the SPE column with the eluent, collect the eluted eluent, centrifuge at 3000 rpm for 3 min, and take the supernatant.

[0067] Example 13

[0068] The difference between this example and Example 1 is only that the S1 step is as follows: mix 1.0 g of the cosmetic sample, 1.0 mL of the demulsifier (Triton X-114), and 3.0 mL of n-hexane, uniformly ultrasonically oscillate, centrifuge at 3000 rpm for 10 min, remove the upper oil phase, collect the lower aqueous phase. Then pass the lower aqueous phase through the SPE column, elute the SPE column with the eluent, collect the eluted eluent, centrifuge at 3000 rpm for 3 min, and take the supernatant.

[0069] Example 14

[0070] The difference between this embodiment and embodiment 1 is only that the S1 step is as follows: 1.0 g of the cosmetic sample, 1.0 mL of demulsifier (Triton X-114), 1.0 mL of tetramethyl ethylenediamine and 2.0 mL of n-hexane are mixed, and after ultrasonic oscillation, centrifugation is performed at 3000 rpm for 10 min, the upper oil phase is removed, and the lower aqueous phase is collected. The lower aqueous phase is then passed through a SPE column, the SPE column is eluted with an eluent, the eluted eluent is collected, centrifugation is performed at 3000 rpm for 3 min, and the supernatant is taken.

[0071] Embodiment 15

[0072] The difference between this embodiment and embodiment 1 is only that the S1 step is as follows: 1.0 g of the cosmetic sample, 1.0 mL of demulsifier (Triton X-114), 1.2 mL of tetramethyl ethylenediamine and 2.0 mL of n-hexane are mixed, and after ultrasonic oscillation, centrifugation is performed at 3000 rpm for 10 min, the upper oil phase is removed, and the lower aqueous phase is collected. The lower aqueous phase is then passed through a SPE column, the SPE column is eluted with an eluent, the eluted eluent is collected, centrifugation is performed at 3000 rpm for 3 min, and the supernatant is taken.

[0073] Embodiment 16

[0074] The difference between this embodiment and embodiment 1 is only that in the S2 step: 100 μL of β-glucosidase, 50 μL of lysozyme and 20 μL of tetrabutylammonium bromide are added to the supernatant, and after oscillation at 45°C for 35 min, boiling water bath for 5 min, the enzyme is inactivated.

[0075] Embodiment 17

[0076] The difference between this embodiment and embodiment 1 is only that in the S3 step: the pretreated sample solution is mixed with 10 μL of graphene quantum dot solution, and then added to the enzyme-labeled plate hole of the 96-hole plate, 50 μL of color developing solution is added, and oscillation is performed with a plate oscillator for 10 s. After standing at room temperature for 15 min in the dark, 50 μL of oxidizing agent is added, and oscillation is performed for mixing, and then standing at room temperature for 15 min in the dark, and then 20 μL of 10% sodium thiosulfate is added, and the reaction is terminated, and a color developing solution is obtained.

[0077] Embodiment 18

[0078] The difference between this embodiment and embodiment 1 is only that in the S2 step: 100 μL of β-glucosidase, 50 μL of lysozyme and 20 μL of tetrabutylammonium bromide are added to the supernatant, and after oscillation at 45°C for 35 min, boiling water bath for 5 min, the enzyme is inactivated; in the S3 step: the pretreated sample solution is mixed with 10 μL of graphene quantum dot solution, and then added to the enzyme-labeled plate hole of the 96-well plate, and then 50 μL of color developing solution is added, oscillated with a plate oscillator for 10 s, and then placed at room temperature for 15 min after avoiding light. 50 μL of oxidizing agent is added, and then oscillated and mixed, and then placed at room temperature for 15 min after avoiding light, and then 20 μL of 10% sodium thiosulfate is added to terminate the reaction, and then the color developing solution is obtained.

[0079] Example 19

[0080] The difference between this embodiment and embodiment 1 is only that the color developing solution is prepared as follows: 21.6 mg of 3,3',5,5'-tetramethylbenzidine is mixed with 10 mL of anhydrous N,N-dimethylformamide to obtain a 3,3',5,5'-tetramethylbenzidine stock solution with a concentration of 2.16 mg / mL, 10 mL of raw horseradish peroxidase solution with a concentration of 10,000 U / mL is mixed with 19 mL of glycerol with a mass concentration of 50% to obtain a horseradish peroxidase working solution, and 1 part of the 3,3',5,5'-tetramethylbenzidine stock solution, 0.2 parts of the horseradish peroxidase working solution and 5 parts of 0.1 M citrate buffer solution are mixed to obtain the color developing solution.

[0081] Example 20

[0082] The difference between this embodiment and embodiment 1 is only that the oxidizing agent is prepared as follows: 2.5 g of ferric chloride is dissolved in 1 L of ultrapure water and oscillated and mixed to obtain the oxidizing agent.

[0083] Example 21

[0084] The difference between this embodiment and embodiment 1 is only that the oxidizing agent is prepared as follows: 2.5 g of potassium ferricyanide is dissolved in 1 L of ultrapure water and oscillated and mixed to obtain the oxidizing agent.

[0085] Comparative Example

[0086] Comparative Example 1

[0087] The difference between this comparative example and embodiment 1 is only that the S1 step is as follows: 1.0 g of the cosmetic sample is mixed with 3.0 mL of n-hexane, and after ultrasonic oscillation, centrifugation is performed at 3000 rpm for 10 min, the upper oil phase is removed, and the lower aqueous phase is collected. The lower aqueous phase is passed through a SPE column, the SPE column is eluted with an eluent, and the eluted eluent is collected and centrifuged at 3000 rpm for 3 min, and the supernatant is collected.

[0088] Comparative Example 2

[0089] The difference between this comparative example and Example 1 is only that the S1 step is as follows: 1.0 g of the cosmetic sample, 3.0 mL of deionized water are mixed, and after ultrasonic oscillation, centrifugation is performed at 3000 rpm for 10 min, the upper oil phase is removed, and the lower aqueous phase is collected. The lower aqueous phase is then passed through a SPE column, the SPE column is eluted with an eluent, and the eluted eluent is collected, centrifuged at 3000 rpm for 3 min, and the supernatant is taken.

[0090] Comparative Example 3

[0091] The difference between this comparative example and Example 1 is only that the S1 step is as follows: 1.0 g of the cosmetic sample, 1.0 mL of demulsifier (Triton X-114) and 2.0 mL of n-hexane are mixed, and after ultrasonic oscillation, centrifugation is performed at 3000 rpm for 10 min, the upper oil phase is removed, and the lower aqueous phase is collected. The lower aqueous phase is then passed through a C18 solid phase extraction column, the C18 solid phase extraction column is eluted with an eluent, and the eluted eluent is collected, centrifuged at 3000 rpm for 3 min, and the supernatant is taken.

[0092] Comparative Example 4

[0093] The difference between this comparative example and Example 1 is only that the S2 step is as follows: 200 μL of a 1% mass concentration CTAB solution is added to the supernatant, ice-bath for 23 min, then centrifuged at 12000 rpm for 15 min, the precipitate is washed with ethanol, then redissolved in ultrapure water, and diluted with ultrapure water to 1 mL to obtain a sample solution.

[0094] Comparative Example 5

[0095] The difference between this comparative example and Example 1 is only that the S3 step is as follows: 50 μL of the sample solution is added to the enzyme-labeled plate well of the 96-well plate, 50 μL of the color developing solution is added, and the plate is shaken for 10 s with a plate shaker, then 50 μL of the oxidizing agent is added after standing at room temperature in the dark for 15 min, and the mixture is mixed well, then 20 μL of a 10% mass concentration sodium thiosulfate solution is added to terminate the reaction, and a color developing solution is obtained.

[0096] Comparative Example 6

[0097] The difference between the present comparative example and Example 1 is only that the S3 step is as follows: 0.5 mL of the sample solution is added to 0.5 mL of a sulfuric acid solution with a concentration of 2 M, a boiling water bath is used for 45 min, and then the solution is cooled to room temperature, and then 10 M sodium hydroxide is added to adjust the pH to 7.0 to obtain a pretreated sample solution. Then 50 μL of the pretreated sample solution is added to the enzyme-labeled plate well of a 96-well plate, and 50 μL of a chromogenic solution is added, and the plate is shaken for 10 s using a plate shaker, and then the chromogenic solution is obtained after the plate is left to stand at room temperature in the dark for 15 min.

[0098] Comparative Example 7

[0099] The difference between the present comparative example and Example 1 is only that the S4 step is as follows: the chromogenic solution is detected using a spectrophotometer, the detection wavelength is 620 nm, and the absorbance is obtained, the concentration of glucuronolactone in the chromogenic solution corresponding to the corrected absorbance is determined according to the standard curve equation of glucuronolactone, and the content of botryoid polysaccharide in the cosmetic sample is determined according to the concentration of glucuronolactone.

[0100] Performance detection test

[0101] For Examples 1-21 and Comparative Examples 1-7, the following performance detection is performed:

[0102] Glucuronolactone 10 mg is dissolved in 10 mL of ultrapure water to prepare a 1 mg / mL stock solution, which is then gradiently diluted to 0, 0.1, 0.5, 1.0, 5.0, 10, 50, and 100 μg / mL of glucuronolactone standard. The enzyme-labeled instrument is used to detect the glucuronolactone standard at a double wavelength, the main detection wavelength is 620 nm, the reference wavelength is 450 nm, ultrapure water is used for zero adjustment, and the absorbance values A 620 and A 450 at 450 nm of each well at 620 nm are read, the difference between the absorbance values A 620 and A 450 is calculated to obtain the corrected absorbance of the glucuronolactone standard. As shown in Table 1, the linear equation y = ax + b is fitted with the concentration of the glucuronolactone standard as the abscissa and the corrected absorbance of the glucuronolactone standard as the ordinate. The calculated glucuronolactone standard curve equation is y = 0.2286x + 0.005, and the correlation coefficient R 2 = 0.9998. The detection limit LOD = 0.03 μg / mL.

[0103] Preparation of the cosmetic sample to be tested: 14 g of stearic acid, 1.0 g of monoglyceride, 1 g of 1618 alcohol, 2 g of white oil, 8 g of glycerol, 0.5 g of potassium hydroxide and 73.5 g of deionized water were mixed uniformly to obtain a cosmetic base. Then the cosmetic base and the batyl alcohol were mixed uniformly to obtain the cosmetic samples to be tested with the contents of 0.1 mg / kg, 1 mg / kg, 10 mg / kg, 100 mg / kg and 1000 mg / kg of batyl alcohol.

[0104] Testing of the cosmetic samples to be tested with different contents of batyl alcohol by the testing method of Example 1: The cosmetic samples to be tested with different contents were tested by the testing method of Example 1, and the cosmetic base was used as a control group. The corrected absorbance of the cosmetic samples to be tested and the control group was recorded, as shown in Table 2.

[0105] The corrected absorbance of the cosmetic samples to be tested and the control group was substituted into the standard curve equation of glucuronic acid to obtain the concentration of glucuronic acid in the chromogenic solution, denoted as C. According to the content of batyl alcohol (μg / g) = (C x Vt) ÷ W x K, the content of batyl alcohol (μg / g) in the cosmetic samples to be tested and the cosmetic base was calculated, as shown in Table 2. Wherein Vt is the constant volume in step S2 of Example 1, i.e. 1 mL. W is the initial mass of the cosmetic sample to be tested. K is the conversion factor. The proportion of glucuronic acid in the batyl alcohol used in the present application is approximately 18%, so K = 1 ÷ 0.18 = 5.56.

[0106] According to the above testing method, the testing method of Examples 1-21 and Comparative Examples 1-7 was used to test the cosmetic sample to be tested with the content of 10 mg / kg of batyl alcohol. The corrected absorbance of the cosmetic sample to be tested and the content of batyl alcohol (μg / g) in the cosmetic sample to be tested were determined, as shown in Table 3.

[0107] Table 1

[0108]

[0109]

[0110] Table 2

[0111]

[0112] Table 3

[0113]

[0114]

[0115] It can be seen from the combination of Example 1 and Table 2 that the determination results of each of the S. polyrhiza polysaccharide content of the cosmetic samples to be tested in Example 1 are relatively close to the actual S. polyrhiza polysaccharide content of the cosmetic samples to be tested, and the determination values of the cosmetic samples to be tested with a S. polyrhiza polysaccharide content of ≥1 μg / g are less than 2% different from the actual values. This shows that the detection method of Example 1 of the present application has high accuracy, the complex components in the cosmetic have little interference on the detection results, and the entire detection process is time-saving and efficient.

[0116] It can be seen from the combination of Example 1, Comparative Examples 1-7 and Table 3 that the S. polyrhiza polysaccharide content determined by Comparative Examples 1-7 is significantly different from the actual S. polyrhiza polysaccharide content in the cosmetic samples to be tested, and the determination values of the cosmetic samples to be tested with a S. polyrhiza polysaccharide content of ≥1 μg / g are all more than 15% different from the actual values. This shows that the detection steps and reagent usage of Example 1 of the present application can reduce the interference of complex components in the cosmetic on the detection results, thereby improving the accuracy of the detection method.

[0117] It can be seen from the combination of Examples 1-21 and Table 3 that the determination results of Examples 1-21 are relatively accurate. However, the determination values of Examples 8-9 and Examples 20-21 deviate from the actual values by a larger margin, and the determination values of Examples 14-19 are less different from the actual values. This shows that the raw material ratio of Examples 14-19 helps to further reduce the interference of complex components in the cosmetic on the detection results, thereby improving the accuracy of the detection method.

[0118] The specific embodiments are merely an explanation of the present application, and are not a limitation of the present application. Those skilled in the art can make modifications to the embodiments without creative contribution after reading the present specification, as long as the modifications are within the scope of the claims of the present application.

Claims

1. A method for detecting Nostoc polysaccharide in a cosmetic product, characterized by, The method comprises the following steps: S1, mixing the cosmetic sample and a demulsification solvent, centrifuging, removing the upper oil phase, collecting the lower water phase, passing the lower water phase through a SPE column, eluting the SPE column with an eluent, collecting the eluted eluent, centrifuging, and collecting the supernatant; S2, adding an enzymatic agent to the supernatant, oscillating at 42-48℃ for 30-40min, boiling in a water bath for 4-6min, adding a CTAB solution, ice-bathing for 20-25min, centrifuging, washing the precipitate with ethanol, and then re-dissolving in ultrapure water to obtain a sample solution, wherein the enzymatic agent comprises β-glucosidase and lysozyme; S3, adding the sample solution into a sulfuric acid solution, boiling in a water bath for 40-50min, cooling to room temperature, and adjusting the pH to 7.0 to obtain a pretreated sample solution; adding the pretreated sample solution into an enzyme-labeled plate hole of a 96-well plate, sequentially adding a color developing solution and an oxidizing agent, oscillating and mixing, standing at room temperature in the dark, and then adding sodium thiosulfate to obtain a color developing solution; S4, double-wavelength detection is performed on the chromogenic solution, the main detection wavelength is 620 nm, the reference wavelength is 450 nm, and the absorbance value A of each well at 620 nm is read 620 and the absorbance value A at 450 nm 450 The difference between the absorbance value A 620 and the absorbance value A 450 is calculated to obtain the corrected absorbance, the concentration of glucuronide in the chromogenic solution corresponding to the corrected absorbance is determined according to the standard curve equation of glucuronide, and the higher the concentration of glucuronide in the chromogenic solution, the higher the content of the nostoc polysaccharide in the cosmetic sample is determined; The preparation method of the color developing solution comprises the following steps: uniformly mixing 3,3',5,5'-tetramethylbenzidine and anhydrous N,N-dimethylformamide to obtain a 3,3',5,5'-tetramethylbenzidine stock solution, uniformly mixing horseradish peroxidase stock solution and glycerol to obtain a horseradish peroxidase working solution, and uniformly mixing the 3,3',5,5'-tetramethylbenzidine stock solution, the horseradish peroxidase working solution and a citrate buffer to obtain the color developing solution.

2. The method for detecting the nitzschia polysaccharide in the cosmetic according to claim 1, characterized in that, The demulsification solvent comprises a demulsifier and a non-polar solvent in a volume ratio of 1:(2-3).

3. The method for detecting the nitzschia polysaccharide in the cosmetic according to claim 2, characterized in that, The demulsification solvent further comprises tetramethyl ethylenediamine, and the volume ratio of the demulsifier to the tetramethyl ethylenediamine is 1:(1-1.2).

4. The method for detecting the nitzschia polysaccharide in the cosmetic according to claim 1, characterized in that, The eluent comprises the following raw materials in parts by weight: 0.1-0.3 parts of formic acid, 38-44 parts of methanol, 38-44 parts of acetonitrile, and the balance of deionized water, with a total amount of 100 parts.

5. The method for detecting the nitzschia polysaccharide in the cosmetic according to claim 1, characterized in that, In the step S3, the pretreated sample solution is uniformly mixed with a graphene quantum dot solution, and then added into an enzyme-labeled plate hole of a 96-well plate, sequentially adding a color developing solution and an oxidizing agent, oscillating and mixing, standing at room temperature in the dark, and then adding sodium thiosulfate to obtain a color developing solution.

6. The method for detecting the nitzschia polysaccharide in the cosmetic according to claim 1, characterized in that, The preparation method of the oxidizing agent comprises the following steps: dissolving ferric chloride and potassium ferricyanide in ultrapure water, and oscillating and mixing to obtain the oxidizing agent, wherein the amount of the ferric chloride and the potassium ferricyanide is 2.5g / L.

7. The method for detecting the nitzschia polysaccharide in the cosmetic according to claim 1, characterized in that, The enzymatic agent further comprises tetrabutylammonium bromide.

8. The method for detecting the nitzschia polysaccharide in the cosmetic according to claim 1, characterized in that, In S4 step: glucose uronic acid is dissolved in ultrapure water to obtain several glucose uronic acid standard samples with different concentrations. The glucose uronic acid standard samples are detected by a microplate reader at a main detection wavelength of 620 nm and a reference wavelength of 450 nm, and the ultrapure water is used for zero adjustment. The absorbance values A of each well at 620 nm are read 620 and the absorbance values A at 450 nm are read 450 . The difference between the absorbance values A 620 and the absorbance values A 450 is calculated to obtain the corrected absorbance of the glucose uronic acid standard sample. The concentration of the glucose uronic acid standard sample is used as the abscissa, and the corrected absorbance of the glucose uronic acid standard sample is used as the ordinate to fit a linear equation y = ax + b. The corrected absorbance of the test cosmetic sample and the control group is substituted into the glucose uronic acid standard curve equation to obtain the concentration of glucose uronic acid in the chromogenic solution. The higher the concentration of glucose uronic acid in the chromogenic solution, the higher the content of the botryosphaerio polysaccharide in the cosmetic sample.

9. The method for detecting the nitzschia polysaccharide in the cosmetic according to claim 8, characterized in that, The concentration of glucuronic acid in the color developing solution is denoted as C, and the content of the nostoc polysaccharide is (C×Vt)÷W×K, wherein Vt is the volume of the sample solution in the step S2, W is the initial mass of the cosmetic sample, and K is the reciprocal of the proportion of glucuronic acid in the nostoc polysaccharide.

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