Method for detecting nostoc polysaccharide in cosmetics

Abstract

The invention relates to the technical field of cosmetic detection, and particularly discloses a method for detecting nostoc polysaccharide in cosmetics. The detection method comprises the following steps: mixing a cosmetic sample with a demulsification solvent, eluting, carrying out enzymolysis by using an enzymolysis agent, carrying out boiling water bath, adding a CTAB solution, carrying out ice bath, centrifuging, collecting precipitates, redissolving, and fixing the volume to obtain a sample solution. Mixing the sample solution with a sulfuric acid solution, carrying out boiling water bath, cooling, and adjusting the pH value to 7.0 to obtain a pretreated sample solution; sequentially adding a color developing solution, an oxidizing agent and sodium thiosulfate into the pretreated sample solution to obtain a color developing solution. Then carrying out dual-wavelength detection on the color developing solution, detecting the concentration of glucuronic acid in the color developing solution, and judging that the content of the nostoc polysaccharide in the cosmetic sample is higher when the concentration of the glucuronic acid in the color developing solution is higher. According to the method, the detection efficiency and the anti-interference performance of the method for detecting the nostoc polysaccharide in the cosmetics can be improved.

Description

Technical Field

[0001] The present invention relates to the technical field of cosmetics detection, in particular to a method for detecting Nostoc polysaccharide in cosmetics. Background Art

[0002] Nostoc polysaccharides contain multiple hydroxyl groups, which can form hydrogen bonds with water molecules, thereby firmly binding them. These groups are the primary "functional groups" for retaining moisture. Furthermore, the polysaccharide's complex spatial configuration can also adsorb a certain amount of water molecules. Therefore, when used in cosmetics, Nostoc polysaccharides can improve their moisturizing, anti-photoaging, absorption, and repair properties. However, excessive levels of Nostoc polysaccharides in cosmetics can cause adverse reactions such as itching, erythema, or clogged pores. Therefore, the content of Nostoc polysaccharides in cosmetics needs to be controlled.

[0003] In related technologies, the phenol-sulfuric acid method or chromatography is generally used to detect Nostoc polysaccharides in cosmetics. However, the phenol-sulfuric acid method detects total polysaccharides rather than the target acidic polysaccharides, and is susceptible to interference from coexisting polysaccharides such as hyaluronic acid and sodium alginate. Chromatographic methods require complex pretreatment and purification. Furthermore, cosmetics are rich in complex ingredients such as oils, emulsifiers, pigments, preservatives, thickeners, and surfactants, which can easily cause interference and misidentification.

[0004] Therefore, it is necessary to develop a detection method for Nostoc polysaccharides in cosmetics with high detection efficiency and strong anti-interference performance. Summary of the Invention

[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] This application provides a method for detecting Nostoc polysaccharides in cosmetics, which adopts the following technical solution:

[0007] A method for detecting Nostoc polysaccharide in cosmetics comprises the following steps:

[0008] S1. Mix the cosmetic sample and the demulsifying solvent, centrifuge, remove the upper oil phase, collect the lower aqueous phase, pass the lower aqueous phase through an SPE column, elute the SPE column with an eluent, collect the eluted eluate, centrifuge, and collect the supernatant;

[0009] S2. Add an enzymatic hydrolyzing agent to the supernatant, shake and react at 42-48° C. for 30-40 minutes, then boil in water for 4-6 minutes, add CTAB solution, ice bath for 20-25 minutes, centrifuge, collect the precipitate, wash the precipitate with ethanol, and redissolve in ultrapure water to obtain a sample solution, wherein the enzymatic hydrolyzing agent includes β-glucosidase and lysozyme;

[0010] S3, adding the sample solution to a sulfuric acid solution, boiling water bath for 40-50 min, cooling to room temperature, adjusting the pH to 7.0, and obtaining a pretreated sample solution; adding the pretreated sample solution to the wells of a 96-well ELISA plate, adding a color developing solution and an oxidant in sequence, shaking and mixing, and standing at room temperature in the dark, and then adding sodium thiosulfate to obtain a color developing solution;

[0011] S4. Perform dual wavelength detection on the colorimetric solution, with the main detection wavelength at 620 nm and the reference wavelength at 450 nm, and read the absorbance value A of each well at 620 nm. 620 and the absorbance value A at 450 nm 450 Calculate the absorbance value A 620 and absorbance value A 450 The difference between the two values ​​is used to obtain the corrected absorbance, and the concentration of glucuronic acid in the color developing solution corresponding to the corrected absorbance is determined according to the glucuronic acid standard curve equation. The higher the concentration of glucuronic acid in the color developing solution, the higher the content of Nostoc polysaccharide in the cosmetic sample.

[0012] By employing the above-mentioned technical solution, the demulsifying solvent in this method disrupts the emulsion structure, releasing the encapsulated Nostoc polysaccharides and eliminating interference caused by silicone and mineral oil encapsulation. The C18 bonded phase in the SPE column adsorbs organic pigments and flavors. Diatomaceous earth traps particulate matter, simultaneously removing major sources of interference such as pigments, titanium dioxide, and carbomer colloid. In step S1, centrifugation and collection of the supernatant removes most solid impurities, preventing subsequent enzymatic hydrolysis from being blocked. In step S2, β-glucosidase hydrolyzes non-target or interfering small glucans or oligosaccharides into glucose monosaccharides. Terminal modification makes the Nostoc polysaccharides more susceptible to CTAB precipitation and enrichment. The CTAB solution electrostatically binds to the Nostoc polysaccharides, forming a precipitate, thereby selectively enriching the Nostoc polysaccharides. Ice bathing reduces the solubility of the CTAB-polysaccharide complex, enhancing precipitation efficiency. Washing the precipitate with ethanol removes fat-soluble impurities bound to the precipitate. In step S3, 2M sulfuric acid solution can completely break down the Nostoc polysaccharide in the sample solution into monosaccharides, eliminating the influence of the difference in the degree of polymerization of polysaccharides. Adjusting to a pH of 7.0 can prevent the decomposition of the color-developed product caused by excessive acid. The color-developing solution condenses with the uronic acid aldehyde group to generate a blue hydrazone compound, and the oxidant oxidizes the condensation product to generate a stable blue dye. Sodium thiosulfate terminates the reaction and blocks the absorbance drift caused by excessive oxidation. Then, a dual-wavelength detection method is used for detection, with 620nm capturing the target signal and 450nm subtracting background scattering to eliminate interferences such as residual opacified particles, pigments and colloid refraction. Therefore, this method has a fast detection efficiency and strong anti-interference performance against impurities in cosmetics, which can improve the detection efficiency and anti-interference performance of the detection method for Nostoc polysaccharide in cosmetics.

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

[0014] By employing this technical solution, the demulsifier can disrupt the emulsion structure and release the encapsulated Nostoc polysaccharide. The non-polar solvent can extract fat-soluble components such as silicone oil, mineral oil, and synthetic esters, eliminating any interference from oils and fats in subsequent testing.

[0015] In a specific embodiment, the demulsifier solvent further includes tetramethylethylenediamine, and the volume ratio of the demulsifier to tetramethylethylenediamine is 1:(1-1.2).

[0016] By adopting the above technical solution, the stability of the cosmetic emulsion layer often relies on the "ionic bridge" effect of metal ions at the oil-water interface, and demulsifiers are generally ineffective against such emulsion layers. Tetramethylethylenediamine can chelate metal ions and directly destroy the ionic crosslinks in the emulsion layer, causing the emulsion layer structure to collapse. The demulsifier then penetrates and disintegrates the oil-water interface membrane, and the non-polar solvent dissolves the free oil. The three work together to achieve the disintegration of the emulsion layer and complete oil / water separation. Therefore, the above step S1 can further reduce interference from the complex matrix of the cosmetic and improve the anti-interference performance of this method.

[0017] In a 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 concentrations of methanol and acetonitrile greatly reduce the polarity of the solution, significantly weakening the hydrophobic interaction force, which helps to effectively elute the Nostoc polysaccharide adsorbed on the SPE column. Moreover, the above ratio of methanol and acetonitrile combines the strong elution power and low viscosity of acetonitrile with the solubility of methanol, resulting in a better elution effect than using either one alone.

[0019] In a specific embodiment, the method for preparing 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 a 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 a color developing solution.

[0020] Using this technical solution, 3,3',5,5'-tetramethylbenzidine is dissolved in N,N-dimethylformamide and oxidized to produce a blue product. Horseradish peroxidase catalyzes the oxidation of 3,3',5,5'-tetramethylbenzidine, amplifying the detection signal. Citrate buffer provides the optimal pH environment for horseradish peroxidase to oxidize 3,3',5,5'-tetramethylbenzidine to form a blue product. The combination of these three factors helps improve the sensitivity of the detection method.

[0021] In a specific implementation scheme, in step S3, the pretreated sample liquid and the graphene quantum dot solution are mixed evenly, and then added to the 96-well ELISA plate wells, and the color developing solution and the oxidant are added in sequence, shaken and mixed, and allowed to stand in the dark at room temperature, and then sodium thiosulfate is added to obtain a color developing solution.

[0022] By adopting the above technical solution, graphene quantum dots can enhance the color reaction of 3,3',5,5'-tetramethylbenzidine. Even a weak color reaction can produce a stronger absorbance signal with the assistance of quantum dots, thereby detecting lower levels of Nostoc polysaccharides.

[0023] In a specific embodiment, the preparation method of the oxidant includes the following steps: dissolving ferric chloride and potassium ferrocyanide in ultrapure water, shaking and mixing to obtain the oxidant, wherein the amount of ferric chloride and potassium ferrocyanide is 2.5 g / L.

[0024] By adopting the above technical solution, potassium ferrocyanide is a strong oxidant that can directly oxidize reducing substances, and ferric chloride can enhance the oxidizing ability or stability of the system. This chemical oxidant does not rely on specific enzymes and helps to complete the color development reaction.

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

[0026] By adopting the above technical solution, tetrabutylammonium bromide can dissolve or loosen lipids and membrane structures, thereby enhancing the contact and action effects of lysozyme and beta-glucosidase on cell wall components.

[0027] In a specific embodiment, in step S4: glucuronic acid is dissolved in ultrapure water to obtain glucuronic acid standards with several glucuronic acid concentrations, and the glucuronic acid standards are detected by dual wavelength using an enzyme reader, with the main detection wavelength being 620 nm and the reference wavelength being 450 nm. The instrument is zeroed with ultrapure water, and the absorbance value A of each well at 620 nm is read. 620 and the absorbance value A at 450 nm 450 Calculate the absorbance value A 620 and absorbance value A 450The difference between the values ​​of 0.05 and 0.06 was used to obtain the corrected absorbance of the glucuronic acid standard. The concentration of the glucuronic acid standard was used as the horizontal coordinate and the corrected absorbance of the glucuronic acid standard was used as the vertical coordinate. The linear equation was fitted: y = ax + b. The corrected absorbance of the cosmetic sample to be tested and the control group was substituted into the glucuronic acid standard curve equation to obtain the glucuronic acid concentration in the color developing solution. The higher the glucuronic acid concentration in the color developing solution, the higher the content of Nostoc polysaccharide in the cosmetic sample.

[0028] In a specific embodiment, the glucuronic acid concentration in the color development solution is recorded as C, and the content of Nostoc polysaccharide = (C×Vt) ÷ W×K, where Vt is the volume of the sample solution in step S2, W is the initial mass of the cosmetic sample, and K is the reciprocal of the proportion of glucuronic acid in Nostoc polysaccharide.

[0029] By adopting the above technical solution and the above specific testing and judgment methods, the content of Nostoc polysaccharide in cosmetics can be accurately determined.

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

[0031] 1. The method of the present application has a fast detection efficiency and strong anti-interference performance against impurities in cosmetics, which can improve the detection efficiency and anti-interference performance of the detection method of Nostoc polysaccharide in cosmetics.

[0032] 2. In this application, the demulsification solvent, color developing solution and oxidant with a specific raw material ratio are preferably used, which helps to further improve the detection efficiency and anti-interference performance of the detection method of Nostoc polysaccharide in cosmetics. DETAILED DESCRIPTION

[0033] Unless otherwise specified, the reagents used in this application are all commercially available. Among them, the SPE column is Discovery DSC-MCAX SPE solid phase extraction tube. β-glucosidase was purchased from Guangdong Wengjiang Chemical Reagent Co., Ltd. with a purity of AR grade. The lysozyme model is AS90021 and the brand is AMEKO. CTAB solution was purchased from Shanghai Shangbao Biotechnology Co., Ltd. The CAS number of 3-methyl-2-benzothiazolinone oxime is 38894-11-0 and the purity is AR grade. The model of potassium ferrocyanide is P3289 and the brand is SIGMA. Ferric chloride is PWX4038 ferric chloride purchased from Langfang Jinnuo Biotechnology Co., Ltd. Tetramethylethylenediamine is N,N,N',N'-tetramethylethylenediamine purchased from Nanjing Huikang Biotechnology Co., Ltd. Tetrabutylammonium bromide was purchased from Guangdong Wengjiang Chemical Reagent Co., Ltd. with a purity of AR grade. The model of graphene quantum dot solution is BKMK427, purchased from Beike Nano. 3,3',5,5'-Tetramethylbenzidine was purchased from Guangdong Fangxin Biotechnology Co., Ltd. with AR grade purity. N,N-Dimethylformamide was purchased from Nanjing Chemical Reagent Co., Ltd. Horseradish peroxidase stock solution was purchased from Hubei Ruiboxin Chemical Co., Ltd. Glucuronic acid was purchased from Chengdu Desite Biotechnology Co., Ltd. Nostoc polysaccharide was purchased from Shaanxi Senyuan Biotechnology Co., Ltd.

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

[0035] Example

[0036] Example 1

[0037] This embodiment provides a method for detecting Nostoc polysaccharide in cosmetics, which comprises 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. After ultrasonic oscillation, centrifuge at 3000 rpm for 10 min. Remove the upper oil phase and collect the lower aqueous phase. Pass the lower aqueous phase through an SPE column and elute the SPE column with eluent. Collect the eluate and centrifuge at 3000 rpm for 3 min. The supernatant is collected.

[0039] S2. Add 100 μL of β-glucosidase and 50 μL of lysozyme to the supernatant. Incubate the mixture at 45°C with shaking for 35 minutes, then incubate in a boiling water bath for 5 minutes to inactivate the enzymes. Add 200 μL of 1% CTAB solution, incubate on ice for 23 minutes, and then centrifuge at 12,000 rpm for 15 minutes. Collect the precipitate, wash it with ethanol, redissolve it in ultrapure water, and dilute to 1 mL with ultrapure water to obtain the sample solution.

[0040] S3. Add 0.5 mL of the sample solution to 0.5 mL of a 2 M sulfuric acid solution, boil in a boiling water bath for 45 min, cool to room temperature, then add 10 M sodium hydroxide and adjust the pH to 7.0 to obtain a pretreated sample solution. 50 μL of the pretreated sample solution is then added to a 96-well ELISA plate, followed by 50 μL of the color development solution. Oscillate the solution on a plate shaker for 10 s. After standing at room temperature in the dark for 15 min, add 50 μL of the oxidant and oscillate to mix. Stand at room temperature in the dark for 15 min, then add 20 μL of 10% sodium thiosulfate to terminate the reaction to obtain a color development solution.

[0041] S4. Use a microplate reader to perform dual-wavelength detection on the color solution, with the main detection wavelength at 620nm and the reference wavelength at 450nm. Adjust the zero value with ultrapure water and read the absorbance value A of each well at 620nm. 620 and the absorbance value A at 450 nm 450 Calculate the absorbance value A 620 and absorbance value A 450 The difference between the two values ​​is used to obtain the corrected absorbance. The concentration of glucuronic acid in the color developing solution corresponding to the corrected absorbance is determined according to the glucuronic acid standard curve equation. The content of Nostoc polysaccharide in the cosmetic sample is determined according to the glucuronic acid concentration.

[0042] The eluent is prepared by mixing the formic acid, methanol, acetonitrile and deionized water to obtain a washing solution.

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

[0044] The oxidant was prepared as follows: 2.5 g of ferric chloride and 2.5 g of potassium ferrocyanide were dissolved in 1 L of ultrapure water, and the mixture was shaken to obtain the oxidant.

[0045] Example 2

[0046] This example differs from Example 1 only in that step S2 is as follows: 100 μL of β-glucosidase and 50 μL of lysozyme are added to the supernatant, and the reaction is shaken at 42°C for 40 minutes, followed by a boiling water bath for 6 minutes to inactivate the enzymes. Then, 200 μL of a 1% CTAB solution is added, the reaction is infused on ice for 25 minutes, and then centrifuged at 12,000 rpm for 15 minutes. The precipitate is collected, washed with ethanol, redissolved in ultrapure water, and diluted to 1 mL with ultrapure water to obtain a sample solution.

[0047] Example 3

[0048] This example differs from Example 1 only in that step S2 is as follows: 100 μL of β-glucosidase and 50 μL of lysozyme are added to the supernatant, and the reaction is shaken at 48°C for 30 minutes, followed by a boiling water bath for 4 minutes to inactivate the enzymes. Then, 200 μL of a 1% CTAB solution is added, the reaction is ice-bathed for 20 minutes, and then centrifuged at 12,000 rpm for 15 minutes. The precipitate is collected, washed with ethanol, redissolved in ultrapure water, and diluted to 1 mL with ultrapure water to obtain a sample solution.

[0049] Example 4

[0050] The only difference between this embodiment and embodiment 1 is that step S3 is as follows: 0.5 mL of sample solution is added to 0.5 mL of 2 M sulfuric acid solution, boiled in a water bath for 40 min, cooled to room temperature, and then sodium hydroxide with a concentration of 10 M is added to adjust the pH to 7.0 to obtain a pre-treated sample solution. Then 50 μL of the pre-treated sample solution is added to the ELISA plate well of a 96-well plate, and then 50 μL of the color developing solution is added, and the solution is shaken on a flatbed shaker for 10 s and allowed to stand at room temperature in the dark for 20 min. 50 μL of an oxidant is added, and the mixture is shaken and mixed, and allowed to stand at room temperature in the dark for 20 min, and then 20 μL of 10% sodium thiosulfate is added to terminate the reaction to obtain a color developing solution.

[0051] Example 5

[0052] The only difference between this embodiment and embodiment 1 is that step S3 is as follows: 0.5 mL of sample solution is added to 0.5 mL of 2 M sulfuric acid solution, boiled in a water bath for 50 min, cooled to room temperature, and then sodium hydroxide with a concentration of 10 M is added to adjust the pH to 7.0 to obtain a pre-treated sample solution. Then 50 μL of pre-treated sample solution is added to the ELISA plate well of a 96-well plate, and then 50 μL of color development solution is added, shaken on a flat plate shaker for 10 s, and allowed to stand in the dark for 12 min at room temperature. 50 μL of oxidant is added, shaken to mix, allowed to stand in the dark for 12 min at room temperature, and then 20 μL of 10% sodium thiosulfate is added to terminate the reaction to obtain a color development solution.

[0053] Example 6

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

[0055] Example 7

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

[0057] Example 8

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

[0059] Example 9

[0060] The only difference between this embodiment and embodiment 1 is that the eluent uses the following raw materials: 80 g methanol and 20 g deionized water.

[0061] Example 10

[0062] The only difference between this embodiment and embodiment 1 is that in step S1, an equal amount of dichloromethane is used to replace n-hexane.

[0063] Example 11

[0064] The only difference between this embodiment and embodiment 1 is that, in step S1, n-hexane is replaced by an equal amount of diethyl ether.

[0065] Example 12

[0066] This example differs from Example 1 only in that step S1 is as follows: 1.0 g of cosmetic sample, 1.0 mL of demulsifier (Triton X-114), and 2.5 mL of n-hexane are mixed and homogenized by ultrasonication. The mixture is then centrifuged at 3000 rpm for 10 minutes, the upper oil phase is removed, and the lower aqueous phase is collected. The lower aqueous phase is then passed through an SPE column and eluted with an eluent. The eluate is collected and centrifuged at 3000 rpm for 3 minutes, and the supernatant is collected.

[0067] Example 13

[0068] This Example differs from Example 1 only in that step S1 is as follows: 1.0 g of cosmetic sample, 1.0 mL of demulsifier (Triton X-114), and 3.0 mL of n-hexane are mixed and homogenized by ultrasonication. The mixture is then centrifuged at 3000 rpm for 10 minutes, the upper oil phase is removed, and the lower aqueous phase is collected. The lower aqueous phase is then passed through an SPE column and eluted with an eluent. The eluate is collected and centrifuged at 3000 rpm for 3 minutes to obtain the supernatant.

[0069] Example 14

[0070] This Example differs from Example 1 only in that step S1 is as follows: 1.0 g of cosmetic sample, 1.0 mL of demulsifier (Triton X-114), 1.0 mL of tetramethylethylenediamine, and 2.0 mL of n-hexane are mixed and homogenized by ultrasonication. The mixture is then centrifuged at 3000 rpm for 10 minutes, the upper oil phase is removed, and the lower aqueous phase is collected. The lower aqueous phase is then passed through an SPE column and eluted with an eluent. The eluate is collected and centrifuged at 3000 rpm for 3 minutes to obtain the supernatant.

[0071] Example 15

[0072] This Example differs from Example 1 only in that step S1 is as follows: 1.0 g of cosmetic sample, 1.0 mL of demulsifier (Triton X-114), 1.2 mL of tetramethylethylenediamine, and 2.0 mL of n-hexane are mixed and homogenized by ultrasonication. The mixture is then centrifuged at 3000 rpm for 10 minutes, the upper oil phase is removed, and the lower aqueous phase is collected. The lower aqueous phase is then passed through an SPE column and eluted with an eluent. The eluate is collected and centrifuged at 3000 rpm for 3 minutes to obtain the supernatant.

[0073] Example 16

[0074] The only difference between this embodiment and embodiment 1 is that, in step S2, 100 μL of β-glucosidase, 50 μL of lysozyme, and 20 μL of tetrabutylammonium bromide were added to the supernatant, and the mixture was shaken at 45° C. for 35 min, followed by boiling in water for 5 min to inactivate the enzyme.

[0075] Example 17

[0076] The only difference between this embodiment and embodiment 1 is that, in step S3, the pretreated sample solution and 10 μL of the graphene quantum dot solution are mixed evenly, and then added to the wells of a 96-well ELISA plate. 50 μL of the color development solution is then added, and the mixture is shaken on a plate shaker for 10 seconds. The mixture is then allowed to stand at room temperature in the dark for 15 minutes. 50 μL of the oxidant is then added, and the mixture is shaken and mixed evenly. The mixture is allowed to stand at room temperature in the dark for 15 minutes. Then, 20 μL of 10% sodium thiosulfate is added to terminate the reaction, thereby obtaining a color development solution.

[0077] Example 18

[0078] The only difference between this embodiment and embodiment 1 is that, in step S2: 100 μL of β-glucosidase, 50 μL of lysozyme and 20 μL of tetrabutylammonium bromide are added to the supernatant, and the reaction is shaken at 45 ° C for 35 minutes, and then the mixture is boiled in water for 5 minutes to complete the enzyme inactivation; in step S3: the pretreated sample solution is mixed evenly with 10 μL of graphene quantum dot solution, and then added to the ELISA plate well of a 96-well plate, and then 50 μL of color development solution is added, and the mixture is shaken on a plate shaker for 10 seconds, and then allowed to stand at room temperature in the dark for 15 minutes. 50 μL of oxidant is added, and then shaken to mix, and allowed to stand at room temperature in the dark for 15 minutes, and then 20 μL of 10% sodium thiosulfate is added to terminate the reaction to obtain a color development solution.

[0079] Example 19

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

[0081] Example 20

[0082] The only difference between this embodiment and embodiment 1 is that the oxidant is prepared according to the following steps: 2.5 g of ferric chloride is dissolved in 1 L of ultrapure water, and the mixture is shaken to obtain the oxidant.

[0083] Example 21

[0084] The only difference between this embodiment and embodiment 1 is that the oxidant is prepared according to the following steps: 2.5 g of potassium ferrocyanide is dissolved in 1 L of ultrapure water, and the mixture is shaken to obtain the oxidant.

[0085] Comparative Example

[0086] Comparative Example 1

[0087] This comparative example differs from Example 1 only in that step S1 is as follows: 1.0 g of the cosmetic sample was mixed with 3.0 mL of n-hexane, ultrasonically shaken for uniformity, and then centrifuged at 3000 rpm for 10 minutes. The upper oil phase was removed, and the lower aqueous phase was collected. The lower aqueous phase was then passed through an SPE column and eluted with an eluent. The eluate was collected and centrifuged at 3000 rpm for 3 minutes, and the supernatant was collected.

[0088] Comparative Example 2

[0089] This comparative example differs from Example 1 only in that step S1 is as follows: 1.0 g of the cosmetic sample and 3.0 mL of deionized water are mixed, ultrasonically shaken for uniformity, and then centrifuged at 3000 rpm for 10 minutes. The upper oil phase is removed, and the lower aqueous phase is collected. The lower aqueous phase is then passed through an SPE column and eluted with an eluent. The eluate is collected and centrifuged at 3000 rpm for 3 minutes to obtain the supernatant.

[0090] Comparative Example 3

[0091] This comparative example differs from Example 1 only in that step S1 is as follows: 1.0 g of the cosmetic sample, 1.0 mL of a demulsifier (Triton X-114), and 2.0 mL of n-hexane are mixed and homogenized by ultrasonication. The mixture is then centrifuged at 3000 rpm for 10 minutes, the upper oil phase removed, and the lower aqueous phase collected. The lower aqueous phase is then passed through a C18 solid-phase extraction column and eluted with an eluent. The eluate is collected and centrifuged at 3000 rpm for 3 minutes to obtain the supernatant.

[0092] Comparative Example 4

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

[0094] Comparative Example 5

[0095] The only difference between this comparative example and Example 1 is that step S3 is as follows: 50 μL of sample solution is added to the ELISA plate wells of a 96-well plate, and then 50 μL of color development solution is added, and the mixture is shaken on a plate shaker for 10 s. After standing in the dark at room temperature for 15 min, 50 μL of oxidant is added, and the mixture is shaken to mix, and the mixture is stood in the dark at room temperature for 15 min. Then, 20 μL of 10% sodium thiosulfate is added to terminate the reaction to obtain a color development solution.

[0096] Comparative Example 6

[0097] The only difference between this comparative example and Example 1 is that step S3 is as follows: 0.5 mL of the sample solution is added to 0.5 mL of a 2 M sulfuric acid solution, the solution is in a boiling water bath for 45 minutes, and then cooled to room temperature. 10 M sodium hydroxide is then added to adjust the pH to 7.0 to obtain a pretreated sample solution. 50 μL of the pretreated sample solution is then added to a well of a 96-well ELISA plate, followed by 50 μL of a color development solution. The solution is shaken on a plate shaker for 10 seconds and allowed to stand in the dark at room temperature for 15 minutes to obtain a color development solution.

[0098] Comparative Example 7

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

[0100] Performance testing

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

[0102] Weigh 10 mg of glucuronic acid and dissolve it in 10 mL of ultrapure water to prepare a 1 mg / mL stock solution. Then, dilute it to 0, 0.1, 0.5, 1.0, 5.0, 10, 50, and 100 μg / mL of glucuronic acid standard. Use a microplate reader to perform dual-wavelength detection on the glucuronic acid standard, with the main detection wavelength at 620 nm and the reference wavelength at 450 nm. Adjust the zero value with ultrapure water and read the absorbance value A of each well at 620 nm. 620 and the absorbance value A at 450 nm 450 Calculate the absorbance value A 620 and absorbance value A 450 The difference between the two values ​​was used to obtain the corrected absorbance of the glucuronic acid standard. As shown in Table 1. With the concentration of the glucuronic acid standard as the horizontal axis and the corrected absorbance of the glucuronic acid standard as the vertical axis, the linear equation was fitted: y = ax + b. After calculation, the equation of the glucuronic acid standard curve was y = 0.2286x + 0.005, and the correlation coefficient R 2 =0.9998. Limit of detection LOD =0.03 μg / mL.

[0103] Prepare the cosmetic samples to be tested: Mix 14g of stearic acid, 1.0g of monoglyceride, 1g of 1618 alcohol, 2g of white oil, 8g of glycerin, 0.5g of potassium hydroxide, and 73.5g of deionized water to obtain a cosmetic base. Then, mix the cosmetic base with Nostoc polysaccharide to obtain cosmetic samples with Nostoc polysaccharide contents of 0.1mg / kg, 1mg / kg, 10mg / kg, 100mg / kg, and 1000mg / kg, respectively.

[0104] Cosmetic samples containing varying levels of Nostoc polysaccharides were tested using the detection method described in Example 1. The method described in Example 1 was used to test cosmetic samples containing varying levels of Nostoc polysaccharides, with the cosmetic matrix serving as a control group. The corrected absorbances of the test cosmetic samples and the control group were recorded, as shown in Table 2.

[0105] Substitute the corrected absorbance of the cosmetic sample to be tested and the control group into the glucuronic acid standard curve equation to obtain the glucuronic acid concentration in the color development solution, recorded as C, and calculate the Nostoc polysaccharide content (μg / g) in the cosmetic sample to be tested and the cosmetic matrix according to the Nostoc polysaccharide content (μg / g) = (C×Vt) ÷ W×K, 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 glucuronic acid content in the Nostoc polysaccharide used in this application is ≈18%, so K=1÷0.18=5.56.

[0106] According to the above detection method, the detection methods of Examples 1-21 and Comparative Examples 1-7 were respectively used to detect the cosmetic samples to be tested with a polysaccharide content of 10 mg / kg of Nostoc. The measured corrected absorbance of the cosmetic samples to be tested and the polysaccharide content of Nostoc in the cosmetic samples to be tested (μg / g) are shown in Table 3.

[0107] Table 1

[0108]

[0109]

[0110] Table 2

[0111]

[0112] Table 3

[0113]

[0114]

[0115] In conjunction with Example 1 and Table 2, it can be seen that the results of Example 1 for the determination of the polysaccharide content of each cosmetic sample tested are relatively close to the actual polysaccharide content of Nostoc algae in the tested cosmetic sample. Moreover, the measured value of the cosmetic sample tested with a polysaccharide content of ≥1 μg / g differs from the actual value by less than 2%. This demonstrates that the detection method of Example 1 of the present application has high accuracy, with minimal interference from complex ingredients in the cosmetics on the test results. Furthermore, the entire detection process is short and efficient.

[0116] Combining Example 1, Comparative Examples 1-7, and Table 3, it can be seen that compared to Example 1, the Nostoc polysaccharide content measured in Comparative Examples 1-7 differed significantly from the actual Nostoc polysaccharide content in the tested cosmetic samples. For the tested cosmetic samples with a Nostoc polysaccharide content of ≥1 μg / g, the measured values ​​differed from the actual values ​​by more than 15%. This demonstrates that Example 1 of the present application, by coordinating the detection steps and reagent dosages, can reduce the interference of complex ingredients in cosmetics on the test results, thereby improving the accuracy of the detection method.

[0117] Combining Examples 1-21 with Table 3 shows that the measurement results of Examples 1-21 are all relatively accurate. However, the measured values ​​of Examples 8-9 and Examples 20-21 deviate significantly from the true values, while the measured values ​​of Examples 14-19 have a smaller difference from the true values. This indicates that adopting the raw material ratios of Examples 14-19 helps further reduce the interference of complex ingredients in cosmetics on the test results, thereby improving the accuracy of the detection method.

[0118] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed, but as long as they are within the scope of the claims of the present application, they are protected by the patent law.

Claims

1. A method for detecting Nostoc polysaccharide in cosmetics, characterized in that: The steps include: S1. Mix the cosmetic sample and the demulsifying solvent, centrifuge, remove the upper oil phase, collect the lower aqueous phase, pass the lower aqueous phase through an SPE column, elute the SPE column with an eluent, collect the eluted eluate, centrifuge, and collect the supernatant; S2. Add an enzymatic hydrolyzing agent to the supernatant, shake and react at 42-48° C. for 30-40 minutes, then boil in water for 4-6 minutes, add CTAB solution, ice bath for 20-25 minutes, centrifuge, collect the precipitate, wash the precipitate with ethanol, and redissolve in ultrapure water to obtain a sample solution, wherein the enzymatic hydrolyzing agent includes β-glucosidase and lysozyme; S3, adding the sample solution to a sulfuric acid solution, boiling in a water bath for 40-50 min, cooling to room temperature, and adjusting the pH to 7.0 to obtain a pretreated sample solution; The pretreated sample solution was added to the wells of the ELISA plate of a 96-well plate, and the color developing solution and the oxidant were added in sequence, shaken and mixed, and allowed to stand at room temperature in the dark, and then sodium thiosulfate was added to obtain a color developing solution; S4. Perform dual wavelength detection on the colorimetric solution, with the main detection wavelength at 620 nm and the reference wavelength at 450 nm, and read the absorbance value A of each well at 620 nm. 620 and the absorbance value A at 450 nm 450 Calculate the absorbance value A 620 and absorbance value A 450 The difference between the two values ​​is used to obtain the corrected absorbance, and the concentration of glucuronic acid in the color developing solution corresponding to the corrected absorbance is determined according to the glucuronic acid standard curve equation. The higher the concentration of glucuronic acid in the color developing solution, the higher the content of Nostoc polysaccharide in the cosmetic sample.

2. The method for detecting Nostoc polysaccharide in cosmetics according to claim 1, characterized in that: The demulsifying solvent includes a demulsifier and a non-polar solvent in a volume ratio of 1:(2-3).

3. The method for detecting Nostoc polysaccharide in cosmetics according to claim 2, characterized in that: The demulsifier solvent further comprises tetramethylethylenediamine, and the volume ratio of the demulsifier to tetramethylethylenediamine is 1:(1-1.2).

4. The method for detecting Nostoc polysaccharide in cosmetics 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 is deionized water, with a total amount of 100 parts.

5. The method for detecting Nostoc polysaccharide in cosmetics according to claim 1, characterized in that: 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 a 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 a color developing solution.

6. The method for detecting Nostoc polysaccharide in cosmetics according to claim 5, characterized in that: In step S3, the pretreated sample solution and the graphene quantum dot solution are mixed evenly, and then added to the 96-well ELISA plate wells, and the color developing solution and the oxidant are added in sequence, shaken and mixed, and allowed to stand in the dark at room temperature, and then sodium thiosulfate is added to obtain a color developing solution.

7. The method for detecting Nostoc polysaccharide in cosmetics according to claim 1, characterized in that: The preparation method of the oxidant comprises the following steps: dissolving ferric chloride and potassium ferrocyanide in ultrapure water, and oscillating and mixing to obtain the oxidant, wherein the dosage of the ferric chloride and potassium ferrocyanide is 2.5 g / L.

8. The method for detecting Nostoc polysaccharide in cosmetics according to claim 1, characterized in that: The enzymatic agent also includes tetrabutylammonium bromide.

9. The method for detecting Nostoc polysaccharide in cosmetics according to claim 1, characterized in that: In step S4: dissolve glucuronic acid in ultrapure water to obtain glucuronic acid standards with different glucuronic acid concentrations. Use a microplate reader to perform dual-wavelength detection on the glucuronic acid standards, with the main detection wavelength at 620 nm and the reference wavelength at 450 nm. Zero the detection with ultrapure water and read the absorbance value A of each well at 620 nm. 620 and the absorbance value A at 450 nm 450 Calculate the absorbance value A 620 and absorbance value A 450 The difference between the values ​​of 0.05 and 0.06 was used to obtain the corrected absorbance of the glucuronic acid standard. The concentration of the glucuronic acid standard was used as the horizontal coordinate and the corrected absorbance of the glucuronic acid standard was used as the vertical coordinate. The linear equation was fitted: y = ax + b. The corrected absorbance of the cosmetic sample to be tested and the control group was substituted into the glucuronic acid standard curve equation to obtain the glucuronic acid concentration in the color developing solution. The higher the glucuronic acid concentration in the color developing solution, the higher the content of Nostoc polysaccharide in the cosmetic sample.

10. The method for detecting Nostoc polysaccharide in cosmetics according to claim 9, characterized in that: The concentration of glucuronic acid in the color development solution is recorded as C, and the content of Nostoc polysaccharide = (C×Vt)÷W×K, where Vt is the volume of the sample solution in step S2, W is the initial mass of the cosmetic sample, and K is the reciprocal of the proportion of glucuronic acid in Nostoc polysaccharide.

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