A method for testing anti-glycation efficacy based on Maillard reaction

Abstract

The present invention provides an anti-glycation efficacy testing method, comprising: defining several areas on the skin surface as control areas and test sample areas, respectively, collecting initial skin color parameters; applying a control substance and a test sample to the control area and the test sample area, respectively; applying a test composition containing reducing sugars to the control area and the test sample area; collecting skin color parameters again at a specified time point; respectively calculating the change rate of the skin color parameters in the test sample area and the change rate of the skin color parameters in the control area, and comparing them. The testing method of the present invention can accurately evaluate the anti-glycation effect of cosmetics in vivo without trauma and intervention, eliminating the deviation caused by factors such as age, skin quality, and ethnicity in the deduction from in vitro experiments to actual use effects.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology detection, and specifically relates to an anti-glycation efficacy testing method based on the Maillard reaction. Background Art

[0002] Skin aging is the result of the overlap of endogenous aging (individual, genetic) and exogenous aging (depending on external factors such as ultraviolet rays, pollution and lifestyle). One of the causes of skin aging is the appearance of advanced glycation end products (AGEs) in our life process (see non-patent literature 1). AGEs are the end products produced by the Maillard reaction between the aldehyde group of reducing sugar and the amino group of protein, lipid or nucleic acid. AGEs gradually accumulate in the epidermis and dermis of the skin, and have a negative impact on the color and texture of the skin, such as yellowing and loss of elasticity (see non-patent literature 2). In addition, AGEs have the property of forming crosslinks by binding to structural proteins present in their vicinity. The crosslinking between AGEs and the collagen that constitutes the dermis reduces the elasticity of the skin and also causes dull skin.

[0003] Preventing the formation of glycation products in the skin through the use of skin care products has practical significance for delaying skin aging. In recent years, with the rise of the concept of anti-glycation, people have begun to pursue the use of anti-glycation skin care products to fight skin aging and dullness. At present, there are anti-glycation (anti-aging) cosmetics on the market that can reduce the accumulation of advanced glycation end products (AGEs) in the skin. For example, La Roche-Posay's anti-glycation antioxidant essence mainly contains carnosine, anthocyanins, and tea polyphenols as anti-glycation ingredients, as well as brands such as POLA, SkinCeuticals, and Mepa.

[0004] Recently, researchers have upgraded skin imaging equipment and used the fluorescence characteristics of AGEs to test and analyze the skin glycation of subjects, greatly enriching the evaluation methods of skin anti-glycation efficacy in the cosmetics field. CN115568822A discloses a device for detecting skin autofluorescence and a method for evaluating anti-glycation efficacy. The method comprises: (a) marking the skin area to be tested; (b) using a fluorescence detection device to determine the basic value of the skin fluorescence intensity and content in the marked area; (c) using a sample to be tested; (d) using a fluorescence detection device to determine the skin fluorescence intensity and content in the marked area after using the sample; (e) comparing the result of step (d) with the result of step (b) to evaluate the anti-glycation efficacy.

[0005] Based on the current "Evaluation Standards for Cosmetic Efficacy Claims", anti-glycation is a new efficacy claim, which requires scientific and reasonable analysis and the establishment of a reliable efficacy evaluation method. At present, the evaluation methods for anti-glycation efficacy are mostly in vitro experiments. AGEs have fluorescent properties. The principle that glucose and bovine serum albumin undergo glycation reactions in vitro to generate AGEs can be used for modeling. After adding anti-glycation products, the level of inhibition of AGEs is evaluated by fluorescence quantitative methods to reflect the anti-glycation efficacy. Among the evaluation methods for anti-glycation efficacy, human trials are relatively rare, mainly due to the limitations of human experimental detection methods. However, the anti-glycation ingredients and dosages of the same ingredients will produce different anti-glycation effects depending on factors such as age, skin quality, and race. Therefore, the reliability of extrapolating in vitro experimental results to humans is limited, and its accuracy needs to be further verified. At present, a detection method that can directly detect the effect of anti-glycation ingredients on human skin is needed to accurately reflect the true effect of anti-glycation ingredients in cosmetics and topical creams.

[0006] Non-patent document 1: Pageon, H. Reaction of Glycation and Human Skin: The Effectson the Skin and Its Components, Reconstructed Skin as a Model. Pathol. Biol. 2010, 58(3), 226–231.

[0007] https: / / doi.org / 10.1016 / j.patbio.2009.09.009.

[0008] Non-patent literature 2: Yokota, M.; Tokudome, Y. The Effect of Glycation on EpidermalLipid Content, Its Metabolism and Change in Barrier Function. SkinPharmacol. Physiol. 2016, 29(5), 231–242.

[0009] https: / / doi.org / 10.1159 / 000448121. Summary of the invention

[0010] Technical Problems to be Solved by the Invention

[0011] As mentioned above, the reliability of extrapolating the results of in vitro anti-glycation experiments to humans is limited, and a detection method that can directly detect the effect of anti-glycation ingredients on human skin is needed to accurately reflect the true effect of anti-glycation ingredients in commercial products such as cosmetics or topical lotions. The present invention aims to establish a new evaluation method that can directly and quantitatively evaluate the anti-glycation efficacy of topical skin products.

[0012] The method of the present invention utilizes reducing sugar to react with keratin containing amino groups in the stratum corneum of the skin to produce glycosylation end products, thereby causing the skin color to turn yellow and dark. A reagent containing reducing sugar and a functional skin care product are simultaneously applied to the surface of human skin, and the anti-glycation efficacy of the skin care product is detected by detecting the change in skin color.

[0013] Technical solutions to technical problems

[0014] The present invention includes the following technical solutions:

[0015] The present invention provides an anti-glycation efficacy testing method, comprising:

[0016] Several areas are defined on the skin surface as control areas and sample areas to be tested, and initial skin color parameters are collected;

[0017] Applying a control substance and a sample to be tested to the control area and the sample to be tested area, respectively;

[0018] applying a test composition containing a reducing sugar to the control area and the test sample area;

[0019] Collect skin color parameters again at a specified time point;

[0020] The change rate of the skin color parameter of the sample area to be tested and the change rate of the skin color parameter of the control area are calculated respectively and compared.

[0021] The change rate of the skin color parameter refers to the percentage of the difference between the initial skin color parameter value and the skin color parameter value collected a period of time after the start of the test (ie, the specified time point) relative to the initial skin color parameter value.

[0022] In the anti-glycation efficacy testing method of the present invention, the skin color parameter is selected from one or more of L value, ITA° value, A value or B value.

[0023] In the anti-glycation efficacy testing method of the present invention, the rate of change of the parameter is the rate of decrease of ITA°, the rate of decrease of L value, the rate of increase of A value or the rate of increase of B value.

[0024] In the anti-glycation efficacy testing method of the present invention, the reducing sugar is preferably dihydroxyacetone and / or erythrulose, and the content of the reducing sugar is preferably in the range of 0.1-10% by weight.

[0025] In the anti-glycation efficacy testing method of the present invention, the test composition further contains one or more of a thickener, a surfactant or an emulsifier.

[0026] In the anti-glycation efficacy testing method of the present invention, the surfactant is selected from an ionic surfactant or a non-ionic surfactant, preferably a polyether surfactant.

[0027] In the anti-glycation efficacy testing method of the present invention, the thickener is selected from one or more of hyaluronic acid, cellulose, starch, acrylic acid (ester) polymers, polyethylene glycols, xanthan gum, guar gum, sclerotium gum, gum arabic, and gellan gum.

[0028] In the anti-glycation efficacy testing method of the present invention, the specified time point is 4 hours later.

[0029] In the anti-glycation efficacy testing method of the present invention, the test composition containing reducing sugar is in the form of a gel or an emulsion.

[0030] The anti-glycation efficacy testing method of the present invention collects the skin color parameters by means of a skin imaging device or a colorimeter.

[0031] Beneficial technical effects

[0032] The present invention can accurately evaluate the anti-glycation effect of the active ingredients in cosmetics on the user in a non-invasive and non-invasive manner in vivo, eliminating the deviation caused by factors such as age, skin quality, and ethnicity in the extrapolation from in vitro experiments to actual use effects, and provides an evaluation method for anti-glycation efficacy that is directly related to human use.

[0033] The evaluation method of the present invention has the following advantages:

[0034] Convenience: The instruments and modeling reagents used in this evaluation method are highly available. The instruments used are skin imaging instruments (Vplus\Visia) or colorimeter (Colorimeter CL400) widely used by existing human efficacy evaluation institutions. The reagents used, dihydroxyacetone and erythrulose, are reducing sugar tanning agents and are general cosmetic raw materials.

[0035] Low cost: No need to upgrade the testing equipment, and the reagents used are low cost.

[0036] Fast: Since reducing sugar tanning agents can produce perceptible skin color changes in a relatively short period of time, the inhibitory effect of anti-glycation ingredients on reducing sugar tanning agents can be evaluated in a relatively short period of time.

[0037] Intuitive and visual: Compared with in vitro biochemical experiments and anti-glycation animal models, this method is based on in vivo skin testing and evaluates the anti-glycation efficacy by changes in skin color, so it is more intuitive and visual. DETAILED DESCRIPTION

[0038] The present invention is described in detail below with reference to the following examples. The examples provide detailed implementation methods and processes to fully disclose and demonstrate that the invention can be implemented. The examples do not represent limitations on the invention, and the protection scope of the present invention is not limited to the following examples.

[0039] Experimental methods without specific conditions in the examples of the specification are usually carried out according to conventional conditions in the art or conditions recommended by the manufacturer. Instruments, reagents and drugs without sources in the examples of the specification can be commercially available.

[0040] In the present invention, unless otherwise specified, "above", "below", "within" means including the number itself. When "comprising", "including", "containing" are used to describe an embodiment herein, other similar embodiments described as "consisting of" and / or "consisting essentially of" are also provided. When "consisting essentially of" is used to describe an embodiment herein, other similar embodiments described as "consisting of" are also provided.

[0041] The term "and / or" as used herein in phrases such as "A and / or B" is intended to include both A and B; A or B; A (alone); and B (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to include each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0042] In this article, "non-enzymatic glycosylation" and "glycation" are different names for the same concept with the same meaning. Therefore, "non-enzymatic glycosylation reaction" and "glycation reaction" are sometimes used interchangeably, and "non-enzymatic glycosylation end products" and "glycation products" are sometimes used interchangeably.

[0043] In the present invention, unless otherwise specified, % refers to weight percentage.

[0044] Detection principle

[0045] The method of the present invention utilizes a reducing sugar tanning agent to react with keratin containing amino groups in the stratum corneum of the skin to produce glycosylation end products, thereby causing the skin color to turn yellow and dark. A detection composition containing a reducing sugar tanning agent and a skin care product with anti-glycation efficacy are simultaneously applied to the surface of human skin, and by detecting the change in skin color, whether the skin care product has an anti-glycation efficacy is detected.

[0046] In the test composition of the present invention, as an essential component, a reducing sugar tanning agent and a solvent acceptable to cosmetics are contained. The content range of the reducing sugar tanning agent is 0.1-10% by weight. Available reducing sugars are, for example, dihydroxyacetone (DHA) and / or erythrulose. Dihydroxyacetone is a reducing sugar with a simple molecular structure, which is widely used in tanning cosmetic formulas as a tanning ingredient. According to the Chinese cosmetic raw material used catalog and SCCS, the upper limit of its use in products as a skin tanning ingredient is 10%. Accordingly, dihydroxyacetone is a safe reagent. Erythrulose is a natural 4-carbon ketose, which is the ketose form of erythrose. In the field of biochemistry and molecular biology, erythrulose belongs to a carbohydrate compound. Erythrulose is usually used in conjunction with dihydroxyacetone (DHA) to enhance the color of DHA and make its distribution more uniform. Erythrulose is used as a tanning agent in cosmetics and has been included in the "Catalogue of Used Cosmetic Raw Materials (2021 Edition)". It is not a prohibited substance in the "Cosmetic Safety Technical Specifications (2015 Edition)". In sunless tanning products, erythrulose has a more natural color and potential benefits for sensitive and dry skin compared to dihydroxyacetone.

[0047] Acceptable solvents in cosmetics can be selected by referring to the "Catalogue of Used Cosmetic Raw Materials" or the "Technical Specifications for Safety of Cosmetics", including: water, propylene glycol, glycerin, butylene glycol, etc.

[0048] The test composition of the present invention preferably further contains one or more of a thickener, a surfactant and / or an emulsifier as required.

[0049] The main function of the thickener is to increase the consistency and leveling. The content of the thickener is preferably 0-1% by weight. The thickener that can be used is not particularly limited, and can be, for example, one or more of hyaluronic acid, cellulose, starch, acrylic acid (ester) polymer, polyethylene glycol, xanthan gum, guar gum, sclerotium gum, gum arabic, and gellan gum.

[0050] The test composition of the present invention may further contain a surfactant or an emulsifier as required. The surfactant / emulsifier that can be used is not particularly limited, as long as it can play an emulsifying role in the composition or help the test composition to spread better on the skin, for example, it can be an ionic surfactant or a nonionic surfactant, preferably a nonionic surfactant, for example, preferably a polyether surfactant. The content of the surfactant and / or emulsifier is preferably 0-5% by weight.

[0051] In the test composition of the present invention, as required, an emollient may also be contained. Available emollients are not particularly limited, and the emollients commonly used in cosmetics may be used, for example, one or more of caprylic / capric triglyceride, C12-15 alcohol benzoate, isononyl isononanoate, isopropyl palmitate, pentaerythritol tetra(ethylhexanoate), squalane, isohexadecane, polydimethylsiloxane, mineral oil, avocado tree fat, jojoba seed oil, sweet almond oil, etc. The content of the emollient is preferably 0-5% by weight.

[0052] The test composition of the present invention may further contain an acid-base regulator as required. For example, when the sample to be tested is an acidic substance (such as vitamin C), in order to reduce irritation to the skin, an appropriate amount of sodium hydroxide solution is added to the sample to reduce the acidity, and accordingly, an appropriate amount of sodium hydroxide solution is also added to the matrix reference substance and the test composition.

[0053] Experimental methods

[0054] The anti-glycation efficacy testing method of the present invention comprises the following steps:

[0055] (1) defining several areas on the skin surface as control areas and sample areas, respectively, and collecting initial skin color parameters;

[0056] (2) applying equal amounts of matrix reference substance and test sample to the control area and the test sample area, respectively, and waiting for full absorption;

[0057] (3) applying a test composition containing reducing sugar to the control area and the test sample area respectively, and waiting for sufficient absorption;

[0058] (4) collecting skin color parameters again at a specified time point;

[0059] (5) Calculate the change rate of the skin color parameters of the sample area to be tested and the change rate of the skin color parameters of the control area respectively, and compare them. If the change rate of the skin color parameters of the sample test area (such as the decrease rate of ITA°, L value or the increase rate of B value) is significantly lower than the change rate of the skin color parameters of the control area, it means that the test sample has the effect of inhibiting the Maillard reaction between reducing sugar and the skin surface, which further indicates that the product has anti-glycation effect.

[0060] The skin color parameters refer to parameters used to describe and classify human skin color. Common skin color parameters include CIELAB color space parameters and ITA° (International Standard Skin Color Measurement).

[0061] CIELAB color space parameters include L value: (Lightness, brightness, the higher the L value, the brighter the skin color);

[0062] A value: the red-green axis, representing the red or green component of the color; and

[0063] B value: yellow-blue degree. The larger the B value, the yellower the skin color.

[0064] ITA° (Individual typology angle) is a measurement method based on skin color depth, ranging from 0° (very white) to 60° (very dark). The larger the ITA° value, the darker the skin color.

[0065] Example

[0066] Example 1 Testing the anti-glycation efficacy of vitamin C

[0067] 1. Sample Preparation

[0068] Dihydroxyacetone gel, matrix and vitamin C samples were prepared as shown in Table 1.

[0069] Table 1:

[0070]

[0071]

[0072] 2. Detection steps

[0073] After cleaning the subject's arm, place it in a constant temperature and humidity environment (e.g., test temperature 25℃±1℃, test humidity 50%±10%) and sit and rest for 30 minutes. Keep sitting still and mark two areas of 2x2cm on the skin, one as the control area and the other as the sample area;

[0074] Vplus skin imaging equipment (purchased from Shanghai Fuhuan Technology Co., Ltd.) was used to take basic photos of two demarcated areas of the skin and collect initial ITA°, B, and L values;

[0075] Apply an equal amount of matrix control substance to the control area delineated on the arm, and an equal amount of test sample vitamin C to the sample area, and keep it there for 30 minutes to allow the sample to be fully absorbed;

[0076] A gel containing 5 wt% dihydroxyacetone was applied to the control area and sample area of ​​the arm at a dosage of 10 mg / cm 2 .

[0077] After the first day and the second day, the control area and sample area of ​​the arm were photographed and the ITA°, B and L values ​​were collected, and the change rates of the ITA°, B and L values ​​of the control area and the sample area were compared.

[0078] The change rate refers to the percentage of the difference between the initial skin color parameter value and the skin color parameter value collected a period of time after the start of the experiment (after the first day, after the second day) relative to the initial skin color parameter value.

[0079] If the decrease rate of ITA and L value in the sample area or the increase rate of B value in the sample area is significantly lower than that in the control area, it means that the test sample has the effect of inhibiting the Maillard reaction between dihydroxyacetone and the skin surface, thereby reflecting the anti-glycation effect of the product.

[0080] 3. Test results:

[0081] According to the L value, ITA° value, and B value of the photographed area of ​​the two subjects, the change rates of the L value, ITA° value, and B value were calculated, respectively, as shown in Table 2.

[0082] Table 2:

[0083]

[0084]

[0085] It can be seen from the above table that the absolute change rates of L value, ITA° value and B value in the experimental area where the vitamin C sample was applied were significantly lower than those in the control area, indicating that the experimental sample vitamin C has anti-glycation effect.

[0086] Example 2 Testing the Anti-glycation Efficacy of Ergothioneine

[0087] 1. Sample Preparation

[0088] As shown in Table 3, dihydroxyacetone gel, matrix and ergothioneine samples were prepared respectively.

[0089] Table 3:

[0090]

[0091] 2. Detection steps

[0092] Except that the test sample vitamin C is replaced with ergothioneine, the same steps as in Example 1 are followed.

[0093] 3. Test results:

[0094] According to the L value, ITA° value, and B value of the photographed area of ​​the two subjects, the change rates of the L value, ITA° value, and B value were calculated, respectively, as shown in Table 4.

[0095] Table 4:

[0096]

[0097]

[0098] As can be seen from the above table, the absolute rates of change of L value, ITA ° value and B value in the experimental area where the thioneine sample was applied were significantly lower than those in the control area, indicating that the experimental sample thioneine has anti-glycation effect.

[0099] Example 3 Testing Collrepair TM Anti-glycation effect

[0100] 1. Sample Preparation

[0101] According to Table 5, dihydroxyacetone gel, matrix and Collrepair were prepared respectively. TM Sample (purchased from BASF).

[0102] Table 5:

[0103]

[0104] 2. Detection steps

[0105] In addition to replacing the test sample vitamin C with Collrepair TM Except for the sample, the detection was carried out according to the same steps as in Example 1.

[0106] 3. Test results:

[0107] According to the L value, ITA° value, and B value of the photographed area of ​​the two subjects, the change rates of the L value, ITA° value, and B value were calculated, respectively, as shown in Table 6.

[0108] Table 6:

[0109]

[0110]

[0111] As can be seen from the table above, the application of Collrepair TM The absolute change rates of L value, ITA° value and B value in the experimental area of ​​the sample are significantly lower than those in the control area, indicating that the experimental sample Collrepair TM Has anti-glycation effect.

[0112] Example 4

[0113] 1. Sample preparation:

[0114] Dihydroxyacetone gel: Prepare dihydroxyacetone gel as shown in Table 7

[0115] Table 7:

[0116] Reagents Dihydroxyacetone gel Pure water 94.58％ Acrylates / C10-30 Alkyl Acrylate Crosspolymer 0.2％ Xanthan gum 0.1％ 10% sodium hydroxide solution 0.12％ Dihydroxyacetone 5％

[0117] Reference substance: pure water

[0118] Sample: Commercially available olive extract

[0119] 2. Detection steps

[0120] The test was performed in the same manner as in Example 1, except that the test sample vitamin C was replaced with a commercially available olive extract sample.

[0121] 3. Test results:

[0122] According to the L value, ITA° value, and B value of the photographed area of ​​the subject, the change rates of the L value, ITA° value, and B value were calculated, respectively, as shown in Table 8.

[0123] Table 8:

[0124]

[0125] As can be seen from the above table, the absolute change rates of L value, ITA° value and B value in the experimental area where the commercially available olive extract sample was applied were significantly lower than those in the control area, indicating that the commercially available olive extract has anti-glycation effect.

[0126] Example 5

[0127] 1. Sample preparation:

[0128] As shown in Table 9, erythrulose gel, matrix and vitamin C samples were prepared respectively.

[0129] Table 9:

[0130]

[0131] 2. Detection steps

[0132] The detection was carried out according to the same procedures as in Example 1, except that the dihydroxyacetone gel was replaced by erythrulose gel.

[0133] 3. Test results:

[0134] According to the L value, ITA° value, and B value of the photographed area of ​​the subject, the change rates of the L value, ITA° value, and B value were calculated respectively. The results are shown in Table 10.

[0135] Table 10:

[0136]

[0137]

[0138] It can be seen from the above table that the absolute change rates of L value, ITA° value and B value in the experimental area where the vitamin C sample was applied were significantly lower than those in the control area, indicating that the experimental sample vitamin C has anti-glycation effect.

[0139] It should be noted that the present invention is not limited to the above implementation modes and examples. The above implementation modes and examples are only examples, and implementation modes that have substantially the same structure as the technical idea and exert the same effect within the scope of the technical solution of the present application are all included in the technical scope of the present application. In addition, within the scope of the subject matter of the present application, various modifications and substitutions of the implementation modes / examples that can be thought of by those skilled in the art, and other methods formed by combining some of the constituent elements in the implementation modes are also included in the scope of the present application, and the scope of protection of the present invention is defined by the attached claims.

Claims

1. A method for testing anti-glycation efficacy, comprising: Several areas are defined on the skin surface as control areas and sample areas to be tested, and initial skin color parameters are collected; Applying a control substance and a sample to be tested to the control area and the sample to be tested area, respectively; applying a test composition containing a reducing sugar to the control area and the test sample area; Collect skin color parameters again at a specified time point; The change rate of the skin color parameter of the sample area to be tested and the change rate of the skin color parameter of the control area are calculated respectively and compared.

2. The anti-glycation efficacy testing method according to claim 1, wherein the skin color parameter comprises one or more of an L value, an ITA° value, an A value or a B value.

3. The anti-glycation efficacy testing method according to claim 1, wherein the rate of change of the skin color parameter is a decrease rate of ITA°, a decrease rate of L value, an increase rate of A value or an increase rate of B value.

4. The anti-glycation efficacy testing method according to claim 1, wherein the reducing sugar is dihydroxyacetone and / or erythrulose, and the reducing sugar content ranges from 0.1 to 10% by weight.

5. The anti-glycation efficacy testing method according to claim 1, wherein the test composition further comprises one or more of a thickener, a surfactant or an emulsifier.

6. The anti-glycation efficacy testing method according to claim 5, wherein the surfactant is a nonionic surfactant.

7. The anti-glycation efficacy testing method according to claim 5, wherein the thickener is selected from one or more of hyaluronic acid, cellulose, starch, acrylic acid (ester) polymers, polyethylene glycols, xanthan gum, guar gum, sclerotium gum, gum arabic, and gellan gum.

8. The anti-glycation efficacy testing method according to claim 1, wherein the specified time point is 4 hours later.

9. The anti-glycation efficacy testing method according to claim 1, wherein the test composition containing reducing sugar is in the form of a gel or an emulsion.

10. The anti-glycation efficacy testing method according to claim 1, wherein the skin color parameters are collected by a skin imaging device or a colorimeter.

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