A hyaluronic acid substance indicator composition and its application in rapid detection of hyaluronic acid substances

Abstract

The present invention discloses a hyaluronic acid indicator composition and its use in the rapid detection of hyaluronic acid substances. The composition contains zerovalent iodine, has an ionic strength greater than 2000µs / cm, and has a pH of 2.0 to 9.0. The hyaluronic acid indicator composition of the present invention can be used for rapid qualitative detection of hyaluronic acid substances. The detection method is simple, low-cost, and requires no pretreatment of the product. A visible color change can be produced within a specified time, resulting in a short detection time, thereby enabling rapid detection of hyaluronic acid substances.

Description

Technical Field

[0001] The present invention relates to a hyaluronic acid substance indicator composition, a preparation method thereof and application in rapid detection of hyaluronic acid substances, belonging to the technical field of chemical detection. Background Art

[0002] Hyaluronic acid (HA), also known as hyaluronic acid, is a high molecular weight straight-chain mucopolysaccharide composed of (1-3)-2-acetylamino-2-deoxy-D-glucose (1-4)-DD-glucuronic acid disaccharide repeating units. Hyaluronic acid has good skin care properties and has been added to various skin-friendly daily necessities in recent years, such as paper towels, wet wipes, non-woven products and fabric products, etc. However, for products with HA added, if you want to detect HA, conventional detection methods need to go through the series operation of "extraction-enzyme hydrolysis-HPLC", which is not only time-consuming, but also limited by the equipment model and cannot quickly detect HA in the product. For example, patent CN109298112B provides a method for measuring the content of hyaluronic acid, which is suitable for the determination of hyaluronic acid content in health foods, medicines, medical devices, cosmetics, hair care products, oral care products, and paper products. It is necessary to first use hyaluronidase to enzymolyze the sample to be tested containing hyaluronic acid and then use high performance liquid chromatography to measure the HA content. In the prior art, no method has been found that can quickly detect HA in products. Summary of the Invention

[0003] In response to the shortcomings of the existing technology, the inventors, after extensive experiments and research, surprisingly discovered a hyaluronic acid indicator composition, which contains zero-valent iodine. When the composition is mixed with hyaluronic acid, it can undergo a color change, so that the presence of hyaluronic acid can be determined by the naked eye, thereby realizing rapid qualitative detection of hyaluronic acid substances.

[0004] The specific technical solutions of the present invention are as follows:

[0005] The present invention provides a hyaluronic acid indicator composition, which contains zerovalent iodine, and the ionic strength of the hyaluronic acid indicator composition is greater than 2000 μs / cm and the pH value is 2.0 to 9.0.

[0006] The zerovalent iodine includes one or more of elemental iodine and a mixture of elemental iodine and a polymer.

[0007] The hyaluronic acid indicator composition includes one or more of an inorganic acid, an inorganic base, and a buffer solution as a pH regulator.

[0008] The hyaluronic acid indicator composition optionally contains an ionic strength regulator, and the ionic strength regulator includes one or more inorganic salts.

[0009] The hyaluronic acid indicator composition may further include at least one of a water-soluble dye and a solvent.

[0010] The present invention also provides the use of the above-mentioned hyaluronic acid indicator composition in detecting hyaluronic acid substances in products, wherein the hyaluronic acid substances include at least one of hyaluronic acid, hyaluronate, cross-linked hyaluronic acid, hyaluronic acid derivatives, and derivatives of cross-linked hyaluronic acid.

[0011] The products include paper products, nonwoven products, and / or fabric products.

[0012] The present invention also provides a method for qualitatively detecting hyaluronic acid substances in a product, wherein the hyaluronic acid substance indicator composition is directly brought into contact with the product to observe the color change of the product.

[0013] The hyaluronic acid substances referred to in the present invention include hyaluronic acid, hyaluronates, cross-linked hyaluronic acid, cross-linked hyaluronates, hyaluronic acid derivatives, cross-linked hyaluronic acid derivatives, and the like. Hyaluronates include sodium hyaluronate, potassium hyaluronate, zinc hyaluronate, magnesium hyaluronate, calcium hyaluronate, cobalt hyaluronate, and gold hyaluronate. Hyaluronic acid derivatives include acetylated hyaluronic acid and thiolated hyaluronic acid, and cross-linked hyaluronic acid derivatives include acetylated cross-linked hyaluronic acid and thiolated cross-linked hyaluronic acid.

[0014] Zero-valent iodine reacts rapidly with hyaluronic acid substances. As long as iodine and hyaluronic acid substances exist in the system, they will react with each other. Therefore, the content of zero-valent iodine can be greater than 0%. However, the iodine content should not be too high. Excessive iodine makes it difficult to observe color changes. Therefore, the amount of zero-valent iodine added is preferably less than or equal to the theoretical molar amount of zero-valent iodine reacting with hyaluronic acid substances.

[0015] In some embodiments, the content of zerovalent iodine is 0.005 to 0.09 wt %, preferably 0.007 to 0.07 wt %, such as 0.008 wt %, 0.009 wt %, 0.01 wt %, 0.02 wt %, 0.03 wt %, 0.04 wt %, 0.05 wt %, 0.06 wt %, 0.07 wt %, and preferably 0.01-0.03 wt %. This content ensures good color change time and color differentiation, facilitating observation of color changes.

[0016] The zerovalent iodine includes one or more of elemental iodine and a compound of elemental iodine and a polymer. The compound of elemental iodine and a polymer is not particularly limited, for example, povidone iodine, etc. The zerovalent iodine is preferably elemental iodine.

[0017] Since zerovalent iodine is yellow, the color of the hyaluronic acid indicator composition itself is yellow. During use, when iodine reacts with the hyaluronic acid, the zerovalent iodine disappears and the hyaluronic acid indicator composition becomes light-colored or colorless.

[0018] In some embodiments, when the zerovalent iodine is elemental iodine, potassium iodide, sodium iodide, etc. can be added to the hyaluronic acid indicator composition to assist in the dissolution of elemental iodine.

[0019] The hyaluronic acid indicator composition of the present invention can be directly applied to a product for detection of hyaluronic acid, without dissolving the product in a solvent to form a solution or pre-extracting the product. The hyaluronic acid indicator composition of the present invention can specifically detect hyaluronic acid in a product by utilizing the interaction of pH, ionic strength, and zerovalent iodine.

[0020] In some embodiments, the hyaluronic acid indicator composition of the present invention includes one or more of an inorganic acid, an inorganic base, and a buffer as a pH regulator. The inorganic acid is not particularly limited, for example, hydrochloric acid, sulfuric acid, nitric acid, etc., the inorganic base is not particularly limited, for example, sodium hydroxide, potassium hydroxide, etc., and the buffer is not particularly limited, for example, a phosphate buffer, a citrate buffer, etc., wherein the phosphate buffer can be a sodium hydrogen phosphate / sodium phosphate buffer, etc., and the citrate buffer can be a citric acid / sodium citrate buffer.

[0021] When the pH value of the hyaluronic acid substance indicator of the present invention is in the range of 2 to 9, the color change of the measured object corresponds obviously to the hyaluronic acid substance, and the indicator effect is significant. The pH value can be specifically 2, 3, 4, 5, 6, 7, 8, 9, preferably in the range of 5 to 9.

[0022] The ionic strength of the hyaluronic acid indicator composition of the present invention is above 2000 μs / cm, for example, 2200 μs / cm, 2500 μs / cm, 2800 μs / cm, 3000 μs / cm, 3300 μs / cm, 3500 μs / cm, 3900 μs / cm, 4500 μs / cm, 4800 μs / cm, 5300 μs / cm, 5500 μs / cm, 5800 μs / cm, 6500 μs / cm, 7000 μs / cm, 7500 μs / cm, 8000 μs / cm, 8500 μs / cm, 9000 μs / cm, etc. In some embodiments, the ionic strength is 2000-9000 μs / cm, preferably 2200-9000 μs / cm, and more preferably 2200-3000 μs / cm. The ionic strength of the hyaluronic acid indicator cooperates with the pH value of the above-mentioned hyaluronic acid indicator to control the color change time and color difference, allowing the indicator to directly contact the product to detect hyaluronic acid substances without pre-treatment of the product.

[0023] The ionic strength of the hyaluronic acid indicator composition of the present invention is determined by all components of the hyaluronic acid indicator composition. For example, zerovalent iodine, pH adjusters, and other dyes can all contribute to ionic strength. When the components of the composition are capable of achieving an ionic strength of 2000 µS / cm or greater, no additional components are required to adjust the ionic strength. However, when the total ionic strength of the components of the composition is less than 2000 µS / cm, an ionic strength-enhancing component can be added to achieve the desired ionic strength. In addition to the aforementioned components, the ionic strength-enhancing component can also be an ionic strength modifier.

[0024] In some embodiments, the ionic strength regulator is not particularly limited, for example, it is one or more of inorganic salts, the cations of the inorganic salts include alkali metal ions or alkaline earth metal ions, for example, potassium ions, sodium ions, calcium ions, magnesium ions, etc., and the anions include chloride ions, sulfate ions, nitrate ions, etc.; the ionic strength regulator of the present invention includes, for example, sodium chloride or potassium chloride.

[0025] In some embodiments, a solvent is optionally added to the hyaluronic acid indicator composition of the present invention, and the solvent may be water. It should be noted that when the pH adjuster contained in the hyaluronic acid indicator composition of the present invention is a buffer solution, since the buffer solution itself can be used as a solvent, there is no need to add another solvent.

[0026] In some embodiments, in order to impart a variety of color changes to the hyaluronic acid indicator composition of the present invention, a water-soluble dye may be added. The added water-soluble dye may be selected from water-soluble dyes that differ significantly in color from iodine, such as CI 45430, CI 16255, CI 15510, CI 45410, CI 16035, CI 42090, and the like. For example, when CI 45430 red dye is added, the original color of the hyaluronic acid indicator composition of the present invention changes to orange. When used to detect hyaluronic acid, as the iodine disappears, the final color changes to red (only the red dye remains). The amount of water-soluble dye used can be adjusted according to actual conditions to meet the pH and ionic strength requirements of the hyaluronic acid indicator composition of the present invention, and to form a new color that is easily observed by the naked eye.

[0027] The present invention also provides a method for preparing the above-mentioned hyaluronic acid indicator composition. The preparation method is simple and can be obtained by uniformly mixing the components. For example, zero-valent iodine, a pH regulator, an ionic strength regulator and water are mixed to obtain the hyaluronic acid indicator composition. There is no special requirement for the order of adding the raw materials.

[0028] The present invention also provides the application of the above-mentioned hyaluronic acid indicator composition in the hyaluronic acid substance in the detection product, and the definition of hyaluronic acid substance is consistent with that shown above. The hyaluronic acid substance in the described product is not particularly limited, for example, the hyaluronic acid substance in paper products, nonwoven products, fabric products, etc. When detecting, directly the hyaluronic acid indicator composition is contacted with the product. When the composition is used as the indicator of hyaluronic acid substance, a color change visible to the naked eye can occur within a prescribed time, and the time of this regulation, for example, can be less than 300 s, and therefore, it is short to consume time, and it is possible to achieve the rapid qualitative detection of hyaluronic acid substance. Through experimental verification, the hyaluronic acid indicator composition of the present invention can detect hyaluronic acid substances of about 10 ppm, and detection sensitivity is high.

[0029] The present invention also provides a method for qualitatively detecting hyaluronic acid substances in a product. The definition of hyaluronic acid substances is consistent with that shown above. The method is: a hyaluronic acid substance indicator composition of the present invention is directly contacted with a product, and then the color change on the surface of the product is observed. After the hyaluronic acid substance indicator composition of the present invention is contacted with the product, a color change visible to the naked eye will occur on the surface of the product within a prescribed time. If the color changes significantly, it is indicated that hyaluronic acid substances exist. If the color does not change significantly, it is indicated that no hyaluronic acid substances exist or the hyaluronic acid substance amount is very low. Wherein, the shorter the time required for the color change, the more hyaluronic acid substance content is in the sample.

[0030] The above products are not particularly limited, for example, paper products, non-woven products, fabric products, etc., more specifically, such as paper towels, wet wipes, masks, clothes, etc.

[0031] In some embodiments, the above-mentioned method for qualitatively detecting hyaluronic acid substances in products can be used to analyze the hyaluronic acid content between different products. For example, the same type of products produced by different manufacturers containing hyaluronic acid substances can be tested for hyaluronic acid substances using this method, and the speed of color change can be used to evaluate which product has a higher hyaluronic acid content.

[0032] In some embodiments, the above-described method for qualitatively detecting hyaluronic acid in products can also be used for quality control during the production process. For example, in the production of paper products containing hyaluronic acid, the hyaluronic acid is applied to the paper by spraying. If the spraying equipment becomes clogged, the hyaluronic acid may not be applied. This qualitative method can be used to quickly detect the presence of hyaluronic acid as the product comes off the production line, thereby controlling product quality.

[0033] In some embodiments, the above-described method for qualitatively detecting hyaluronic acid in products can also be used to quickly distinguish genuine and counterfeit goods in the market. This method is simple to operate, intuitive, time-efficient, highly efficient, requires no additional equipment, and is low-cost, thus possessing high application value.

[0034] In some embodiments, the above-mentioned method for qualitatively detecting hyaluronic acid substances in products can also be used for experimental demonstration, allowing consumers to intuitively see that hyaluronic acid substances are added to the product.

[0035] The present invention has the following beneficial effects:

[0036] 1. The qualitative detection limit of the hyaluronic acid indicator composition of the present invention is as low as 10 ppm of hyaluronic acid.

[0037] 2. The hyaluronic acid indicator composition of the present invention has simple ingredients, low cost, and can undergo a color change visible to the naked eye within a specified time. The color change time is appropriate, and can achieve rapid detection of hyaluronic acid substances.

[0038] 3. The hyaluronic acid indicator composition of the present invention is effectively suitable for the detection of hyaluronic acid in paper products, non-woven products, textile products, etc. (which are not easy to detect using traditional HPLC methods), and has good application prospects.

[0039] 4. The method for detecting hyaluronic acid substances of the present invention can be applied to a variety of different scenario requirements, and can realize rapid qualitative detection of hyaluronic acid substances. It has the advantages of simple operation, intuitive phenomenon, short time consumption, high efficiency, no need for other equipment assistance, and low cost. DETAILED DESCRIPTION

[0040] The present invention will be further described below through specific examples. The following description is only exemplary and does not limit its content.

[0041] In the following examples, all reagents used are commercially available products, and some of the reagents are shown in Table 1 below:

[0042] Table 1

[0043]

[0044] Example 1

[0045] Hyaluronic acid solution: Weigh hyaluronic acid and dissolve it in purified water to prepare HA solution;

[0046] Zero-valent iodine solution: Weigh zero-valent iodine and dissolve it in purified water to prepare a zero-valent iodine solution;

[0047] Sodium alginate solution: Weigh sodium alginate and dissolve it in purified water to prepare a sodium alginate solution;

[0048] Chitosan solution: Weigh chitosan and dissolve it in purified water to prepare chitosan solution;

[0049] The above-mentioned hyaluronic acid solution, sodium alginate solution or chitosan solution were mixed with the zerovalent iodine solution in proportion, and the components in the mixture after mixing were shown in Table 2 below.

[0050] Table 2

[0051]

[0052] Color change experiment

[0053] The time after the hyaluronic acid solution, sodium alginate solution or chitosan solution and the zerovalent iodine solution are mixed is defined as the initial time. Observe whether the color of the solution changes. If so, record the time required for the solution color to become colorless from the initial time, which is the color change time.

[0054] The color change of each mixture solution in Table 2 is shown in Table 3 below:

[0055] Table 3

[0056]

[0057] The above experiments show that sodium hyaluronate, cross-linked sodium hyaluronate and sodium hyaluronate derivatives of different concentrations and molecular weights all change color after mixing with zero-valent iodine, while polyhydroxy polysaccharides with a similar structure to hyaluronic acid, such as chitosan and sodium alginate, do not change color when mixed with zero-valent iodine. This shows that the color reaction between zero-valent iodine and hyaluronic acid substances has a certain specificity, and zero-valent iodine can specifically indicate the presence of hyaluronic acid substances.

[0058] Example 2

[0059] From Example 1, zerovalent iodine and hyaluronic acid substances react rapidly under solution environment, and color change reaction can occur quickly. Considering that this reaction time is short, it is unfavorable for the color observation in actual use process, and moreover simple zerovalent iodine has a good indication effect to hyaluronic acid substances under solution environment, but when being directly used in paper products, fabrics, non-woven products etc., because hyaluronic acid substances are fixed in products by hydrogen bonds etc., it is necessary to carry out the pre-extraction of these products to hyaluronic acid substances, increases the inconvenience of operation. Therefore, in order to simplify the operation step, the pretreatment of product can be omitted, hyaluronic acid substance indicator composition is screened.

[0060] 1. Selection of formula of hyaluronic acid indicator composition:

[0061] Table 4 shows 15 groups of hyaluronic acid indicator compositions, wherein the zerovalent iodine is elemental iodine or povidone iodine, the ionic strength regulator is sodium chloride or potassium chloride, and the pH regulator is dilute hydrochloric acid, sodium hydroxide aqueous solution, citric acid / sodium citrate buffer or disodium hydrogen phosphate / sodium phosphate buffer.

[0062] Table 4

[0063]

[0064]

[0065] Note: 1. The iodine content in the composition is detected using the direct titration method of GB / T13025.7-2012.

[0066] 2. The pH of the composition shall be tested in accordance with the instructions for pH determination in GB / T 13531.1 General test methods for cosmetics.

[0067] 3. The ionic strength of the composition is tested in accordance with the test instructions for conductivity in GB / T 6908-2005.

[0068] 2. Preparation of hyaluronic acid indicator composition:

[0069] Each hyaluronic acid indicator composition was prepared by physically mixing the ingredients according to the formula in Table 4 above.

[0070] 3. The 15 groups of hyaluronic acid indicator compositions prepared above were used to detect hyaluronic acid in paper products and non-woven products. The method is as follows:

[0071] 3.1 Samples to be tested:

[0072] Sample 1: Tuxiaomiao brand hyaluronic acid moisturizing facial tissue (hyaluronic acid content of 0.1% to 0.5% by weight);

[0073] Sample 2: Bio-MESO hyaluronic acid soft protective mask skin-friendly layer non-woven fabric (hyaluronic acid content by weight is 0.1% to 0.5%);

[0074] Blank control: Moisturizing paper facial tissue (does not contain hyaluronic acid, other ingredients are the same as Tuxiaomiao brand hyaluronic acid moisturizing paper facial tissue).

[0075] Fabric sample containing 10 ppm hyaluronic acid: Cut a 100 gsm cotton base fabric into 10 x 10 cm pieces, weighing 1.0 g. Weigh 0.1 g of sodium hyaluronate (Bloomage Biosciences Co., Ltd., batch number 19021401) and dissolve it in purified water to a volume of 1000 ml to prepare a 0.01% solution. Use a manual spray bottle to evenly spray 0.1 g of this solution onto the cut cotton base fabric piece and air-dry it to obtain a fabric sample containing 10 ppm hyaluronic acid.

[0076] Hyaluronic acid solution: 0.01 wt% sodium hyaluronate aqueous solution (sodium hyaluronate: Bloomage Biotech Co., Ltd., batch number 19021401).

[0077] 3.2 Hyaluronic acid indicator: 15 groups of hyaluronic acid indicator compositions prepared above;

[0078] 3.3 Detection method:

[0079] Spread test sample 1, test sample 2, a fabric sample containing 10 ppm hyaluronic acid, and a blank control substance flat on a horizontal table. Use a pipette to accurately weigh 50ul of each hyaluronic acid indicator composition. Evenly apply each hyaluronic acid indicator composition to the sample surface in a direction parallel to the long axis of the sample. The application length should be approximately 10±2cm. After application, place the sample in the air at room temperature. The time after application is completed is recorded as 0s. Observe the color change. The time required for the color to change and remain unchanged is the color change time.

[0080] Hyaluronic acid solution detection method: Take 10ml of 0.01% sodium hyaluronate aqueous solution, accurately weigh 50ul of hyaluronic acid indicator composition into the above solution using a pipette, and mark the time after addition as 0s. Observe the color change. The time required for the color to change and remain unchanged is the color change time.

[0081] 3.4 Experimental Results

[0082] The color development results of the hyaluronic acid indicator in each group are shown in Table 5 below:

[0083] Table 5

[0084]

[0085]

[0086] The above experiments show that when the zerovalent iodine solution directly contacts the untreated paper product, no color change occurs. When the zerovalent iodine content in the composition is within the range of 0.005-0.09wt%, the ionic strength is greater than 2000 µs / cm, and the pH is within the range of 2.0 to 9.0, the hyaluronic acid indicator composition contacts the product and exhibits a visible color change, which facilitates observation. It has the advantages of short detection time, convenient operation, and rapid color development, enabling rapid detection of hyaluronic acid substances.

Claims

1. A hyaluronic acid indicator composition, characterized in that: The hyaluronic acid indicator composition contains zerovalent iodine, and has a pH value of 2.0 to 9.0 and an ionic strength of greater than 2000 μs / cm.

2. The hyaluronic acid indicator composition according to claim 1, wherein: The zerovalent iodine includes one or more of elemental iodine and a mixture of elemental iodine and a polymer.

3. The hyaluronic acid indicator composition according to claim 1 or 2, characterized in that: It also includes one or more of inorganic acids, inorganic bases, and buffer solutions as pH regulators.

4. The hyaluronic acid indicator composition according to claim 1 or 2, characterized in that: Also included is an ionic strength adjuster, which includes one or more inorganic salts.

5. The hyaluronic acid indicator composition according to claim 3, characterized in that: Also included is an ionic strength adjuster, which includes one or more inorganic salts.

6. The hyaluronic acid indicator composition according to claim 1 or 2, characterized in that: Also included is at least one of a water-soluble dye and a solvent.

7. The hyaluronic acid indicator composition according to claim 3, characterized in that: Also included is at least one of a water-soluble dye and a solvent.

8. The hyaluronic acid indicator composition according to claim 4, characterized in that: Also included is at least one of a water-soluble dye and a solvent.

9. The hyaluronic acid indicator composition according to claim 5, characterized in that: Also included is at least one of a water-soluble dye and a solvent.

10. Use of the hyaluronic acid indicator composition according to any one of claims 1 to 9 in detecting hyaluronic acid substances in products, wherein the hyaluronic acid substances comprise at least one of hyaluronic acid, hyaluronate, cross-linked hyaluronic acid, hyaluronic acid derivatives, and derivatives of cross-linked hyaluronic acid.

11. The use according to claim 10, characterized in that: The product includes at least one of a paper product, a non-woven product, and a fabric product.

12. A method for qualitatively detecting hyaluronic acid substances in a product, characterized by: The hyaluronic acid indicator composition according to any one of claims 1 to 9 is directly brought into contact with a product to observe the color change of the product.

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