Process for the preparation of modified hydroxytyrosol, applications and cosmetics

Abstract

This application relates to the field of cosmetic formulation technology, and particularly to a method for preparing modified hydroxytyrosol, its application, and cosmetics. The preparation method includes the following steps: hydroxytyrosol, collagen, alkaline protease, and arginine are reacted in an oxygen-containing environment in an aqueous solvent with a pH of 9-10 under stirring conditions to prepare a reaction product containing the modified hydroxytyrosol; wherein the pH of the aqueous solvent is adjusted by arginine. The modified hydroxytyrosol prepared by this method exhibits significantly enhanced free radical scavenging ability, can inhibit the expression of inflammatory factors TNF-α and IL-6, inhibit hyaluronidase activity, and thus possesses soothing and antioxidant effects. It is stable in an alkaline environment and also has moisturizing effects, making it suitable as an active ingredient in cosmetics, endowing cosmetics with at least one of the following effects: antioxidant, soothing, and moisturizing.

Description

Technical Field

[0001] This application relates to the field of cosmetic technology, and in particular to the preparation method, application and cosmetics of modified hydroxytyrosol. Background Technology

[0002] Hydroxytyrosol (HT) is a natural polyphenol compound whose natural sources include olive oil and olive plants. For example, it is found in extra virgin olive oil, olive fruit, and leaves.

[0003] HT is an amphoteric compound, also known as 3,4-dihydroxyphenylethanol, with the structural formula shown in formula (I). Its molecular weight is 154.16 g / mol. Pure hydroxytyrosol is a colorless to yellow oily substance. HT is mainly excreted from the body as HT-3-sulfate. HT has high safety, lacks genotoxicity and mutagenicity, and no adverse reactions were observed in toxicological evaluation experiments at a daily dose of 500 mg / kg.

[0004] Equation (Ⅰ).

[0005] Hydroxytyrosol possesses antioxidant capabilities, scavenging free radicals and protecting skin cells from oxidative stress damage, and is widely used in cosmetic formulation development. However, current hydroxytyrosol exhibits relatively poor antioxidant properties, and its instability under alkaline conditions has limited its wider application in cosmetics. Summary of the Invention

[0006] Therefore, it is necessary to provide a modified hydroxytyrosol, its preparation method, its application, and its cosmetics.

[0007] In a first aspect, a method for preparing modified hydroxytyrosol is provided, the method comprising the following steps:

[0008] In an oxygen-containing environment, hydroxytyrosol, collagen, alkaline protease, and arginine are reacted in an aqueous solvent with a pH of 9-10 under stirring conditions to prepare a reaction product containing the modified hydroxytyrosol.

[0009] The pH of the aqueous solvent is adjusted by arginine.

[0010] In an optional embodiment, the modified hydroxytyrosol comprises hydroxytyrosol grafted with hydrolyzed fragments of the collagen.

[0011] In an optional embodiment, the collagen includes collagen derived from fish skin.

[0012] In an optional embodiment, the hydrolyzed fragments of the collagen include glycine-proline-hydroxyproline fragments.

[0013] In an optional embodiment, the preparation method includes freeze-drying the reaction system after the reaction is completed, and the obtained freeze-dried product contains the modified hydroxytyrosol.

[0014] In an optional embodiment, the preparation method further conforms to at least one of (a) to (f):

[0015] (a) The mass ratio of the hydroxytyrosol to the collagen is 0.5:(0.1~1) based on the amount of feed.

[0016] (b) The mass ratio of the hydroxytyrosol to the alkaline protease is 0.5:(0.01~0.1) based on the amount of feed.

[0017] (c) The mass-to-volume ratio of the hydroxytyrosol to the aqueous solvent is 1 g: (8~15) mL, based on the amount of material fed.

[0018] (d) The reaction temperature is 40℃~50℃, and the reaction time is 5h~15h;

[0019] (e) The stirring speed of the reaction is 500~600 r / min;

[0020] (f) In the preparation method described above, 50% to 70% of hydroxytyrosol is converted into the modified hydroxytyrosol.

[0021] In an optional embodiment, the preparation method further includes: purifying the reaction product to obtain purified modified hydroxytyrosol.

[0022] In a second aspect, the reaction product prepared by the method for preparing the modified hydroxytyrosol described in the first aspect is provided; or the use of the modified hydroxytyrosol prepared by the method for preparing the modified hydroxytyrosol described in the first aspect in the preparation of a product for achieving at least one of the effects in (a) to (f):

[0023] (a) Scavenging free radicals;

[0024] (b) Reduce the level of inflammatory factors in the subject, said inflammatory factors including at least one of TNF-α and IL-6;

[0025] (c) Inhibits hyaluronidase activity;

[0026] (d) At least one of the following: antioxidant, anti-inflammatory, and anti-allergic;

[0027] (e) Soothe the subject's skin; and,

[0028] (f) Used for moisturizing the subject's skin.

[0029] In an optional implementation, the product includes cosmetics, which meet at least one of (i) and (ii):

[0030] (i) The cosmetic product has at least one of the following effects: antioxidant, soothing, and moisturizing; and,

[0031] (ii) The pH of the aqueous phase in the cosmetic is 7.5~10.

[0032] Thirdly, a cosmetic product is provided, comprising at least one of the reaction product prepared by the method for preparing modified hydroxytyrosol as described in the first aspect and the modified hydroxytyrosol prepared by the method for preparing modified hydroxytyrosol as described in the first aspect.

[0033] The preparation method of this application involves stirring under alkaline conditions at pH 9-10, allowing contact with oxygen in the environment. This oxidizes hydroxytyrosol in the reaction system into an active quinone intermediate, which then forms a covalent bond with an oligopeptide produced by alkaline protease hydrolysis of collagen via a Michael addition reaction, thereby forming a grafted product of hydroxytyrosol and peptides. The reaction product exhibits significantly enhanced free radical scavenging ability, inhibits the expression of inflammatory factors TNF-α and IL-6, and suppresses hyaluronidase activity, thus possessing soothing and antioxidant effects. It is stable in an alkaline environment and also has moisturizing properties, making it suitable as an active ingredient in cosmetics, imparting at least one of the following effects: antioxidant, soothing, and moisturizing. Attached Figure Description

[0034] To more clearly illustrate the technical solutions in the embodiments and examples of this application, and to more completely understand this application and its beneficial effects, the accompanying drawings used in the description of the embodiments or examples will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of this application. Those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0035] Figure 1 The infrared spectrum of unmodified hydroxytyrosol in Example 1;

[0036] Figure 2 The infrared spectrum of the modified hydroxytyrosol in Example 1;

[0037] Figure 3 The mass spectrum of the modified hydroxytyrosol in Example 1;

[0038] Figure 4 The hyaluronidase inhibition rate of each sample in Example 3;

[0039] Figure 5The curves showing the change in skin stratum corneum moisture content over time after application of each sample in Example 4;

[0040] Figure 6 The images show the samples of formulations 1 and 2 in Example 5 before and after pH adjustment. A is before pH adjustment, and B is after pH adjustment. Detailed Implementation

[0041] The present application will be further described in detail below with reference to the accompanying drawings, embodiments, and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. For example, features described or illustrated as part of one embodiment can be combined in a suitable manner in another embodiment to produce new embodiments. Furthermore, numerous details are set forth in the following description to provide a fuller understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the specification of this application is for descriptive purposes only and is not intended to be limiting of the application.

[0043] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0044] The terms “and / or,” “or / and,” and “and / or” as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. “Any and all combinations” includes any two related listed items, any more related listed items, or a combination of all related listed items. For example, “A and / or B” includes three parallel options: A, B, and “a combination of A and B.”

[0045] In this application, the terms "multiple", "various", "multiple times", "several", "several", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more or more.

[0046] In this application, "optionally", "optional", and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without".

[0047] In this application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass-volume percentage.

[0048] In this application, the technical features or solutions described in open-ended language include both closed-ended technical features or solutions consisting of the listed contents and open-ended technical features or solutions that include the listed contents.

[0049] In this application, where the method flow involves multiple steps, unless otherwise explicitly stated herein, there is no strict order restriction on the execution of these steps; they can be executed in any order other than those described. Moreover, any step may include multiple sub-steps or multiple stages, which are not necessarily completed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be performed alternately or simultaneously with other steps or parts of the sub-steps or stages of other steps.

[0050] In this application, the terms "first aspect," "second aspect," "third aspect," "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0051] In one aspect, some embodiments provide a method for preparing modified hydroxytyrosol, comprising the following steps:

[0052] In an oxygen-containing environment, hydroxytyrosol, collagen, alkaline protease, and arginine are reacted in an aqueous solvent with a pH of 9-10 under stirring conditions to prepare a reaction product containing modified hydroxytyrosol.

[0053] The pH of the aqueous solvent is regulated by arginine, and the pH of the aqueous solvent can be, for example, but not limited to, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or 10.

[0054] In this preparation method, collagen is hydrolyzed by alkaline protease to produce oligopeptides, and stirred under alkaline conditions (pH 9-10, arginine-regulated) and exposed to oxygen in the environment. Hydroxytyrosol in the reaction system is oxidized by oxygen in the environment to an active quinone intermediate (HT-2, 152 Da), which then reacts with the nucleophilic side chain in the peptide to form a covalent compound through Michael addition reaction, thereby forming a graft product of hydroxytyrosol and peptide.

[0055] The reaction product of this preparation method contains modified hydroxytyrosol, and in some embodiments, it also contains unmodified hydroxytyrosol that has not participated in the reaction. The reaction product of this preparation method contains arginine, which does not participate in the reaction, and collagen and its hydrolysis products are essentially absent, as well as arginine-grafted hydroxytyrosol with little or no residue. Since the remaining substances in the reaction product after removing the modified hydroxytyrosol have little impact on the use of the modified hydroxytyrosol, this preparation method may optionally purify or not purify the modified hydroxytyrosol in the reaction product.

[0056] In an optional embodiment, the modified hydroxytyrosol includes hydroxytyrosol grafted with hydrolyzed fragments of collagen.

[0057] In an optional embodiment, the collagen includes collagen derived from fish skin.

[0058] In an optional embodiment, the hydrolyzed fragments of collagen include gly-proline-hydroxyproline fragments (Gly-Pro-Hyp).

[0059] In an optional embodiment, a hydrolyzed fragment of collagen is grafted onto at least one site at C5 and C2 of hydroxytyrosol.

[0060] In an optional embodiment, the preparation method includes freeze-drying the reaction system after the reaction is completed, and the obtained freeze-dried product contains modified hydroxytyrosol.

[0061] In an optional embodiment, hydroxytyrosol and collagen are first added to the solvent, then the pH of the solvent is adjusted to 9-10 using a pH adjuster, then alkaline protease is added to the solvent, and the reaction is carried out while stirring under conditions where the solvent surface can be in contact with air, so that the hydroxytyrosol in the reaction system can react with oxygen in the air.

[0062] In an optional embodiment, the preparation method further conforms to at least one of (a) to (f):

[0063] (a) The mass ratio of hydroxytyrosol to collagen is 0.5:(0.1~1) based on the amount of feed, for example, but not limited to 0.5:0.1, 0.5:0.2, 0.5:0.3, 0.5:0.4, 0.5:0.5, 0.5:0.6, 0.5:0.7, 0.5:0.8, 0.5:0.9 or 0.5:1, preferably 0.5:0.4.

[0064] (b) The mass ratio of hydroxytyrosol to alkaline protease is 0.5:(0.01~0.1) based on the amount of feed; for example, it can be, but is not limited to, 0.5:0.01, 0.5:0.02, 0.5:0.03, 0.5:0.04, 0.5:0.05, 0.5:0.06, 0.5:0.07, 0.5:0.08, 0.5:0.09 or 0.5:0.1, preferably 0.5:(0.04~0.05).

[0065] (c) The mass-volume ratio of hydroxytyrosol to aqueous solvent is 1g:(8~15)mL, for example, but not limited to 1g:8mL, 1g:9mL, 1g:10mL, 1g:11mL, 1g:12mL, 1g:13mL, 1g:14mL or 1g:15mL, preferably 1g:10mL.

[0066] (d) The reaction temperature is 40℃~50℃ and the reaction time is 5h~15h; the reaction temperature can be, for example, but not limited to, 40℃, 42℃, 45℃, 47.5℃ or 50℃, preferably 40℃~45℃; the reaction time can be, for example, but not limited to, 5h, 6h, 8h, 10h, 12h or 15h.

[0067] (e) The stirring speed of the reaction is 500~600 r / min, and the stirring speed can be, for example, but not limited to, 500 r / min, 520 r / min, 550 r / min, 575 r / min or 600 r / min.

[0068] And, (f) in this preparation method, 50%~70% of hydroxytyrosol is converted into modified hydroxytyrosol.

[0069] In an optional embodiment, the preparation method further includes purifying the reaction product to obtain purified modified hydroxytyrosol.

[0070] Secondly, some embodiments provide the use of the reaction product prepared by the method for preparing the modified hydroxytyrosol of the first aspect; or the use of the modified hydroxytyrosol isolated from the reaction product prepared by the method for preparing the modified hydroxytyrosol of the first aspect in the preparation of a product for achieving at least one of the effects of (a) to (f):

[0071] (a) Free radical scavenging: Experimental verification shows that the reaction product obtained by the preparation method in the first aspect can scavenge free radicals, and the free radical scavenging ability is significantly improved compared with unmodified hydroxytyrosol. Exemplary products with free radical scavenging effects include, but are not limited to, standard reagents for in vitro antioxidant mechanism research, reagents for preparing antioxidant activity screening models, protective agents for oxidative stress cell models, and antioxidant additives for biological sample preservation.

[0072] (b) Reducing the levels of inflammatory factors in subjects, including at least one of TNF-α and IL-6, wherein the reaction product obtained by the method in the first aspect is experimentally verified to reduce the expression levels of inflammatory factors TNF-α and IL-6 in LPS-treated macrophages. Exemplary products that reduce the levels of inflammatory factors in subjects include, but are not limited to, molecular tools for studying inflammatory signaling pathways, reagents for preparing anti-inflammatory drug screening models, and regulators for in vitro studies of cytokine storms.

[0073] (c) Inhibition of hyaluronidase activity: Experimental verification shows that the reaction product obtained by the preparation method in the first aspect can inhibit hyaluronidase activity and reduce the degradation of hyaluronic acid. Exemplary products that inhibit hyaluronidase activity include, but are not limited to, standard inhibitors used for the detection of hyaluronidase activity.

[0074] (d) At least one of antioxidant, anti-inflammatory and anti-allergic properties, which can be used as an active ingredient in a drug or cosmetic having at least one of antioxidant, anti-inflammatory and anti-allergic properties.

[0075] (e) Soothes the skin of the test subject and can be used as an active ingredient in cosmetics with soothing effects.

[0076] And (f) can be used to moisturize the subject's skin and can be used as an active ingredient in cosmetics with moisturizing effects.

[0077] In an optional implementation, the product includes cosmetics, and the cosmetics meet at least one of (i) and (ii):

[0078] (i) The cosmetic has at least one of the functions of anti-oxidation, soothing and moisturizing. The reaction product obtained by the preparation method in the first aspect can scavenge free radicals, thereby giving the cosmetic an antioxidant capacity. The ability to inhibit the expression of inflammatory factors can give the cosmetic an anti-inflammatory capacity, thereby playing a soothing role. The modified hydroxytyrosol can reduce the degradation of hyaluronic acid by increasing the inhibition rate of hyaluronidase, thereby reducing the release level of histamine and inflammatory factors and exerting anti-allergic and soothing effects. At the same time, experimental verification shows that the formula containing modified hydroxytyrosol has a better moisturizing effect.

[0079] In addition, (ii) the pH of the aqueous phase in cosmetics is 7.5~10. Experiments have verified that modified hydroxytyrosol still has good stability in alkaline environment and can better exert its antioxidant, soothing and moisturizing effects in this system.

[0080] Fourthly, a cosmetic product is provided, comprising at least one of the reaction product prepared by the method for preparing modified hydroxytyrosol according to the first aspect and the modified hydroxytyrosol prepared by the method for preparing modified hydroxytyrosol according to the first aspect.

[0081] In an optional embodiment, the cosmetic also contains excipients acceptable for use in the cosmetic field, including but not limited to at least one of: carriers, emulsifiers, thickeners, stabilizers, preservatives, antioxidants, colorants, fragrances, pH adjusters, humectants, binders, and disintegrants.

[0082] In an optional implementation, the pH of the cosmetic product is 7.5 to 10.

[0083] In an optional embodiment, the cosmetic also contains butylene glycol, propylene glycol, polyglycerol-10, glycerin, beta-glucan, carbomer, allantoin, sodium hyaluronate, glucose, trehalose, triethanolamine, and water.

[0084] In an optional embodiment, the cosmetic contains, by weight percentage, 3% to 10% butylene glycol, 1% to 10% propylene glycol, 1% to 3% polyglycerol-10, 1% to 10% glycerin, 0.1% to 1% β-glucan, 0.1% to 2% carbomer, 0.01% to 5% allantoin, 0.01% to 5% sodium hyaluronate, 0.1% to 5% glucose, 0.1% to 5% trehalose, 0.1% to 5% triethanolamine, and 39% to 93.5% water.

[0085] In optional embodiments, cosmetics include, but are not limited to, toners, creams, lotions, serums, masks, and eye creams.

[0086] The following are some examples.

[0087] The embodiments of this application will be described in detail below with reference to some examples. It should be understood that these embodiments are only for illustrating this application and are not intended to limit the scope of this application. For experimental methods in the following embodiments where conditions are not specified, please refer to the guidelines given in this application first, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0088] In the following examples, the measurement parameters of the raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0089] The main reagents used in the following examples and comparative examples are as follows:

[0090] Hydroxytyrosol was purchased from Chongqing Boteng Pharmaceutical Technology Co., Ltd.

[0091] Alkaline protease, purchased from Xiasheng (Beijing) Biotechnology Development Co., Ltd., model FDY-2241, with an enzyme activity of 200,000 UI / g;

[0092] Acidic protease, purchased from Xiasheng (Beijing) Biotechnology Development Co., Ltd., FDY-2205, with an enzyme activity of 150,000 UI / g;

[0093] Neutral protease, purchased from Xiasheng (Beijing) Biotechnology Development Co., Ltd., FDG-2230, with an enzyme activity of 110,000 UI / g.

[0094] The collagen was purchased from Hainan Huayan Collagen Technology Co., Ltd., and it is derived from fish skin.

[0095] The content of modified hydroxytyrosol in the reaction products of Examples 1-4 and Comparative Examples 1-2 was detected by the following method: the content of unmodified hydroxytyrosol in the reaction products was detected by high performance liquid chromatography, and the yield of modified hydroxytyrosol from the reaction of hydroxytyrosol was (unmodified hydroxytyrosol before reaction - unmodified hydroxytyrosol after reaction) / unmodified hydroxytyrosol before reaction.

[0096] Example 1

[0097] This embodiment provides a method for preparing modified hydroxytyrosol using collagen combined with arginine, comprising the following steps:

[0098] (1) Add 0.5g of hydroxytyrosol (HT) and 0.4g of collagen to 10ml of water to form solution A;

[0099] (2) Add 0.1g of arginine to adjust the pH of the system to 9.0~10; add 0.05g of alkaline protease to the solution to form solution B;

[0100] (3) Stir thoroughly at 500 r / min under a 45℃ water bath to ensure full contact between the liquid surface and the air. The reaction time is 10 h to form solution C;

[0101] (4) After the reaction is complete, the sample is freeze-dried to obtain modified hydroxytyrosol, of which 66.3% of hydroxytyrosol reacts to generate modified hydroxytyrosol.

[0102] Infrared spectroscopy and mass spectrometry were used to detect unmodified and modified hydroxytyrosol, and the results are as follows: Figures 1-3 As shown, the stretching vibration frequency of the OH group of hydroxytyrosol is 3375 cm⁻¹. -1 The stretching vibration frequency of the modified hydroxytyrosol (OH) is 3378 cm⁻¹. -1 The modified sample showed a significant red shift in peak value compared to the unmodified raw material. This was particularly evident in the amide III band region (1330~1220 cm⁻¹). -1 The infrared spectrum mainly reflects the stretching vibration of carbon-nitrogen bonds (CN) and the bending vibration of nitrogen-hydrogen bonds (NH); compared with the infrared spectrum of the unmodified HT, the modified adduct at 1284 cm⁻¹... -1 A significantly stronger absorption peak was observed, confirming that the amino acid fragment was successfully grafted onto the hydroxytyrosol molecule.

[0103] Combined with infrared spectroscopy ( Figure 1 and Figure 2 It can be seen that the amino acid fragments after alkaline protease hydrolysis underwent covalent cross-linking with hydroxytyrosol. The process was carried out under alkaline conditions created by arginine and under long-term rapid stirring conditions to ensure full contact between the liquid surface and air. The hydroxytyrosol in the system was oxidized by oxygen in the environment to an active quinone intermediate (HT-2, 152Da), which then reacted with the nucleophilic side chain in the amino acid fragment to form a covalent complex through Michael addition reaction, thereby forming a graft product of hydroxytyrosol and amino acid.

[0104] From mass spectrum ( Figure 3 The m / z values ​​of 437.19 and 329.22 can be attributed to an addition of 152 to the peptide, where 152 corresponds to the quinone derivative after the oxidation of hydroxytyrosol, and 329.22 is likely the graft product of hydroxytyrosol and arginine. The m / z value of 437.19 is the most prominent peak. Subtracting 152 from the observed m / z value of 437.19 yields a calculated peptide mass of 285, which is consistent with the Gly-Pro-Hyp tripeptide fragment abundant in collagen peptides. This mass spectrometry data strongly demonstrates that hydroxytyrosol and collagen peptides form a stable covalent product.

[0105] Example 2

[0106] This embodiment provides a method for preparing modified hydroxytyrosol from collagen combined with arginine. The difference from Example 1 is that only the reaction temperature is changed, and the method includes the following steps:

[0107] (1) Add 0.5g of hydroxytyrosol (HT) and 0.4g of collagen to 10ml of water to form solution A;

[0108] (2) Add 0.1g of arginine to adjust the pH of the system to 9.0~10; add 0.05g of alkaline protease to the solution to form solution B;

[0109] (3) Stir thoroughly at 500 r / min under a 40℃ water bath to ensure full contact between the liquid surface and the air. The reaction time is 10 h to form solution C;

[0110] (4) After the reaction is complete, the sample is freeze-dried to obtain modified hydroxytyrosol, of which 60.3% of hydroxytyrosol reacts to generate modified hydroxytyrosol.

[0111] Example 3

[0112] This embodiment provides a method for preparing modified hydroxytyrosol from collagen combined with arginine. The difference from Example 1 is that only the stirring speed of the reaction is changed, and the method includes the following steps:

[0113] (1) Add 0.5g of hydroxytyrosol (HT) and 0.4g of collagen to 10ml of water to form solution A;

[0114] (2) Add 0.1g of arginine to adjust the pH of the system to 9.0~10; add 0.05g of alkaline protease to the solution to form solution B;

[0115] (3) Stir thoroughly at 600 r / min under a 45℃ water bath to ensure full contact between the liquid surface and the air. The reaction time is 10 h to form solution C;

[0116] (4) After the reaction is complete, the sample is freeze-dried to obtain modified hydroxytyrosol, of which 61.1% of hydroxytyrosol reacts to generate modified hydroxytyrosol.

[0117] Example 4

[0118] This embodiment provides a method for preparing modified hydroxytyrosol using collagen combined with arginine. The difference from Example 1 lies only in changing the amount of alkaline protease used, and includes the following steps:

[0119] (1) Add 0.5g of hydroxytyrosol (HT) and 0.4g of collagen to 10ml of water to form solution A;

[0120] (2) Add 0.1g of arginine to adjust the pH of the system to 9.0~10; add 0.04g of alkaline protease to the solution to form solution B;

[0121] (3) Stir thoroughly at 500 r / min under a 45℃ water bath to ensure full contact between the liquid surface and the air. The reaction time is 10 h to form solution C;

[0122] (4) After the reaction is complete, the sample is freeze-dried to obtain modified hydroxytyrosol, of which 59.7% of hydroxytyrosol reacts to generate modified hydroxytyrosol.

[0123] Comparative Example 1

[0124] This comparative example provides a method for preparing modified hydroxytyrosol using collagen combined with arginine. The difference from Example 1 is that an acidic protease is used instead of an alkaline protease, and the method includes the following steps:

[0125] (1) Add 0.5g of hydroxytyrosol (HT) and 0.4g of collagen to 10ml of water to form solution A;

[0126] (2) Add 0.1g of arginine to adjust the pH of the system to 9.0~10; add 0.05g of acidic protease to the solution to form solution B;

[0127] (3) Stir thoroughly at 500 r / min under a 45℃ water bath to ensure full contact between the liquid surface and the air. The reaction time is 10 h to form solution C;

[0128] (4) After the reaction is complete, the sample is freeze-dried to obtain modified hydroxytyrosol, of which 3.2% hydroxytyrosol reacts to generate modified hydroxytyrosol.

[0129] Comparative Example 2

[0130] This comparative example provides a method for preparing modified hydroxytyrosol using collagen combined with arginine. The difference from Example 1 is that a neutral protease is used instead of an alkaline protease, and the method includes the following steps:

[0131] (1) Add 0.5g of hydroxytyrosol (HT) and 0.4g of collagen to 10ml of water to form solution A;

[0132] (2) Add 0.1g of arginine to adjust the pH of the system to 9.0~10; add 0.05g of neutral protease to the solution to form solution B;

[0133] (3) Stir thoroughly at 500 r / min under a 45℃ water bath to ensure full contact between the liquid surface and the air. The reaction time is 10 h to form solution C;

[0134] (4) After the reaction is complete, the sample is freeze-dried to obtain modified hydroxytyrosol, of which 4.5% hydroxytyrosol reacts to generate modified hydroxytyrosol.

[0135] Comparative Example 3

[0136] This comparative example is a mixture containing hydroxytyrosol, arginine, and collagen. 0.5g of the modified product, 0.1g of arginine, and 0.4g of collagen were directly mixed.

[0137] Example of effect 1

[0138] DPPH scavenging experiment:

[0139] Equal volumes of distilled water were added to prepare 0.1 mg / mL samples of unmodified hydroxytyrosol and the samples prepared in Examples 1-4 and Comparative Examples 1-3. 2 mL of each hydroxytyrosol sample solution was added to 2 mL of DPPH (0.1 mM, methanol-soluble) solution, mixed thoroughly, and incubated at room temperature in the dark for 30 min. The absorbance was measured at 517 nm. Distilled water was used as the blank group, and vitamin C (Vc) was used as the positive control. All samples were measured in triplicate, and the average value was calculated. The scavenging ability is expressed as the scavenging rate, which was calculated using the following formula:

[0140] ;

[0141] A 空白 The absorbance of the blank group (2 mL of distilled water was used to replace the sample);

[0142] A 样品 The absorbance of the sample solution;

[0143] A 对照 The absorbance is calculated by replacing DPPH with 2 mL of methanol.

[0144] The results are shown in Table 1. The DPPH radical scavenging data in Table 1 show that, compared to unmodified hydroxytyrosol, modified hydroxytyrosol exhibited varying degrees of improvement in DPPH radical scavenging, with Example 1 showing the most significant improvement. This is attributed to the increased number of antioxidant active sites in modified hydroxytyrosol, which synergistically enhances its antioxidant activity. Specifically, the antioxidant activity of hydroxytyrosol mainly depends on the catechol hydroxyl group. After grafting amino acids, the amino and carboxyl functional groups of the amino acids form new active sites, which, together with the retained active hydroxyl groups of hydroxytyrosol, achieve a synergistic effect in enhancing antioxidant activity. In contrast, Comparative Examples 1-2, due to the addition of proteases that were not under optimal conditions, presumably did not undergo a grafting reaction with hydroxytyrosol, resulting in diluted purity and reduced antioxidant activity compared to the unmodified samples.

[0145] Table 1. Results of DPPH removal experiments

[0146]

[0147] Example 2

[0148] Test on the soothing efficacy of modified hydroxytyrosol:

[0149] 1. Test objective: This experiment is based on the LPS-stimulated RAW264.7 macrophage model to evaluate the soothing efficacy of the test sample by detecting changes in the levels of inflammatory factors (TNF-α, IL-6) secreted by the cells.

[0150] 2. Test Item: Soothing Efficacy Test - Detection of LPS-stimulated RAW264.7 macrophage inflammatory factor (TNF-α, IL-6) content.

[0151] 3. Test materials:

[0152] (1) Test system: RAW264.7 macrophages.

[0153] (2) Main reagents: high glucose DMEM culture medium, fetal bovine serum, PBS, MTT, DMSO, trypsin, LPS, dexamethasone, Mouse TNF-α ELISA Kit, Mouse IL-6 Valukine ELISA Kit.

[0154] (3) Main equipment: carbon dioxide incubator, biosafety cabinet, inverted fluorescence microscope, enzyme-linked immunosorbent assay (ELISA) reader.

[0155] 4. Testing Method:

[0156] (1) Cytotoxicity: Cell seeding: at 1×10 4 Cells were seeded at a density of cells per well into 96-well plates and incubated overnight in an incubator (37°C, 5% CO2).

[0157] (2) Experimental grouping: The experiment was set up with a zeroing group, a control group, a positive control group and a sample group. In the sample group, each sample was set with 8 concentration gradients, and 3 replicate wells were set under each concentration gradient.

[0158] (3) Solution preparation: Prepare working solutions of different concentrations according to the test concentration setting table.

[0159] (4) The optimal concentration of hydroxytyrosol was determined to be 0.0031% (m / V) based on cell viability screening.

[0160] 2. Inflammatory factor content test: The specific settings for experimental grouping are shown in Table 3.

[0161] Table 2 Experimental Design

[0162]

[0163] The experimental steps are as follows:

[0164] (1) Cell seeding: at 1×10 5Cells were seeded at a density of cells per well into 24-well plates and incubated overnight in an incubator (37°C, 5% CO2).

[0165] (2) Solution preparation: Prepare working solutions of different concentrations of test substance according to the experimental design table.

[0166] (3) Induction and drug administration: When the cell deposition rate in the 24-well plate reaches 40%~60%, 100 μL of 10×LPS working solution is added to each well according to the experimental design. The well is shaken left and right to mix the drug. The sample is added at the same time. The final LPS concentration is 1 μg / mL. Each group has 3 replicates. After drug administration, the wells are placed in an incubator (37℃, 5% CO2) for 24 h.

[0167] (4) Sample collection: After incubation, collect the cell culture supernatant into an EP tube (determine the amount of sample to be collected according to the detection index), and then freeze the sample in a -80℃ freezer.

[0168] (5) Detection of inflammatory factor content: The content of inflammatory factors in the cell supernatant was detected. The lower the average concentration value, the less inflammatory factors secreted by the cells in this sample, indicating that the soothing effect of the added ingredients in this sample is more obvious.

[0169] TNF-α level detection: The detection was performed according to the instructions of the Mouse TNF-α ELISA kit;

[0170] IL-6 level detection: Perform the detection according to the instructions of the MouseIL-6ELISA kit.

[0171] 5. Test Results:

[0172] (1) Results of TNF-α content test:

[0173] Based on the experimental method, cell supernatant was collected and TNF-α content was detected. The results are shown in Table 3.

[0174] Table 3 Results of TNF-α level detection (inflammatory factor)

[0175]

[0176] As shown in Table 3, compared with the BC group, the secretion of the inflammatory factor TNF-α in RAW264.7 macrophages in the NC group was significantly increased, indicating that the LPS stimulation model was successfully established in this experiment.

[0177] Compared with the NC group, the PC group showed a significant decrease in the secretion of the inflammatory factor TNF-α in RAW264.7 macrophages at a drug concentration of 100 μg / mL, indicating that the positive control test was effective.

[0178] Compared with the NC group, the modified hydroxytyrosol and the unmodified hydroxytyrosol in Example 1 significantly reduced the secretion of the inflammatory factor TNF-α in macrophages RAW264.7 at a concentration of 0.0031% (m / V), and the modified hydroxytyrosol reduced the amount of TNF-α by a greater margin than the unmodified hydroxytyrosol. The reason for this is that the amino acids grafted onto the modified HT also have anti-inflammatory and soothing effects. At the same time, the enhanced antioxidant properties of the modified product indirectly enhance the anti-inflammatory effect of the modified HT, resulting in a synergistic effect.

[0179] (2) IL-6 content test results:

[0180] Based on the experimental method, cell supernatant was collected and IL-6 content was detected. The results are shown in Table 4.

[0181] Table 4 Results of IL-6 inflammatory factor detection

[0182]

[0183] As shown in Table 4, compared with the BC group, the secretion of the inflammatory factor IL-6 by macrophages in the NC group was significantly increased, indicating that the LPS stimulation model was successfully established in this experiment.

[0184] Compared with the NC group, the PC group showed a significant decrease in the secretion of the inflammatory factor IL-6 by macrophages RAW264.7 at a drug concentration of 100 μg / mL, indicating that the positive control test was effective.

[0185] Compared with the NC group, the modified hydroxytyrosol and the unmodified hydroxytyrosol in Example 1 significantly reduced the secretion of the inflammatory factor IL-6 in macrophage RAW264.7 at a concentration of 0.0031% (m / V), and the modified hydroxytyrosol showed a greater reduction at the same concentration. The reason for this is that the amino acids grafted onto the modified HT also have anti-inflammatory and soothing effects. At the same time, the enhanced antioxidant properties of the modified product indirectly enhance the anti-inflammatory effect of the modified HT, resulting in a synergistic effect.

[0186] Example 3

[0187] Hyaluronidase inhibition assay to detect anti-allergic activity:

[0188] Designed according to Table 5: A is the sample group; B is the sample control group; C is the negative control group; D is the negative control group. Hyaluronidase inhibition rate = [1 - (AB) / (CD)] × 100%.

[0189] Table 5 Hyaluronidase Inhibition Experiment

[0190]

[0191] 1. After thoroughly mixing each reaction tube, incubate at 37°C in a water bath for 20 minutes; mix thoroughly again and continue incubating at 37°C for 40 minutes. Then remove the reaction tubes and let them stand at room temperature for 10 minutes.

[0192] 2. Mix each tube thoroughly again, transfer to a boiling water bath and heat for 30 minutes. After heating, immediately transfer to an ice bath, let stand and cool for 10 minutes, and then return to room temperature and let stand for 10 minutes.

[0193] 3. Shake each reaction tube thoroughly, add anhydrous ethanol to bring the volume to 8 mL, mix well, and develop the color at room temperature for 30 min. Finally, use a spectrophotometer to measure the absorbance of the sample at a wavelength of 530 nm.

[0194] Table 6 Details of Addition Amounts for Different Samples

[0195]

[0196] Hyaluronic acid (HA) is a core component of the extracellular matrix and intercellular matrix, playing a crucial role in maintaining skin elasticity, mediating cell adhesion, promoting wound healing, and regulating angiogenesis. Previous studies have shown a significant correlation between hyaluronidase inhibitory activity and histamine release inhibition activity in mast cells; active ingredients with histamine release inhibitory effects typically also exhibit strong hyaluronidase inhibitory activity.

[0197] This study evaluated the soothing efficacy of modified HT aqueous solution using a hyaluronidase inhibition assay. The results, as shown in the figure above, indicate that sample 1 exhibited the highest inhibition rate under the same concentration conditions, while samples 2 and 3 showed similar inhibition rates. The added components revealed that adding the same amount of modified HT significantly outperformed the same amount of unmodified HT + collagen + arginine, and also outperformed HT alone. These results suggest that the superior hyaluronic acid inhibition rate of modified HT is not due to a simple synergistic effect from the mixture of collagen, arginine, and HT, but rather to the enzymatically hydrolyzed modified HT mixture, including HT derivatives grafted with tripeptides and HT derivatives grafted with arginine. The increased number of covalently grafted active sites and the synergistic effect enhance the hyaluronidase inhibition rate of the HT derivatives, reduce hyaluronic acid degradation, and consequently decrease the release levels of histamine and inflammatory factors, ultimately exerting a certain anti-allergic and soothing effect.

[0198] Example of effect 4

[0199] Moisturizing efficacy assessment:

[0200] 1. Subject and Site Selection:

[0201] Ten qualified participants were recruited based on the pre-set screening criteria. The test area was selected on the inner side of each participant's left and right arms, 5 cm from the base of the palm, with a uniform test area of ​​3 cm × 3 cm on each side.

[0202] 2. Samples and Grouping:

[0203] Apply equal amounts of the following three samples to the test areas on both arms:

[0204] Sample group: Three formulation samples prepared according to Table 5 of Effect Example 3.

[0205] 3. Testing Process and Environment Control:

[0206] The test was conducted in a constant temperature and humidity environment: temperature (21±1)℃, relative humidity (50±5)%. After cleaning their arms, the subjects sat quietly in this environment for 30 minutes to achieve skin stabilization.

[0207] After the sitting period, the initial moisture content of the stratum corneum of the skin at each test site was measured immediately before application. After evenly applying the corresponding sample, the immediate moisture content of the skin at each site was measured at four time points: 0.5, 1, 1.5, and 2 hours. Each site and each time point was measured three times, and the arithmetic mean was taken as the final test value. The results were uniformly expressed as stratum corneum moisture content.

[0208] Depend on Figure 5 The data shows that the moisturizing effect of the HT-containing formula samples is generally poor, and the data begins to decline significantly after 2 hours. Sample 1 is significantly better than Sample 2 and Sample 3, indicating that simply physically mixing HT with collagen and arginine cannot achieve a significant synergistic effect. This is because polyphenols such as hydroxytyrosol have poor moisturizing effects. However, the hydroxytyrosol derivatives modified with collagen and arginine have a certain water-locking and moisturizing ability due to their amino acid groups. At the same time, the antioxidant properties of modified HT are enhanced, which can scavenge free radicals in the skin microenvironment and maintain the activity of moisturizing factors and other substances in the stratum corneum, thus synergistically enhancing the moisturizing effect.

[0209] Example 5

[0210] Stability in cosmetic formulations:

[0211] 1. Experimental Methods:

[0212] Table 7

[0213]

[0214] (1) According to the formula in Table 7, first disperse the carbomer until it is completely dissolved.

[0215] (2) Then dissolve butylene glycol, propylene glycol, polyglycerol-10, glycerol, β-glucan, allantoin, sodium hyaluronate, glucose and trehalose until all raw materials are completely dissolved.

[0216] (3) Formula 1 is further supplemented with unmodified hydroxytyrosol; Formula 2 is further supplemented with the modified hydroxytyrosol prepared in Example 1.

[0217] (4) Triethanolamine was added to adjust the pH of the system, and the pH value was measured to be 7.9.

[0218] (5) Add water to 100g to prepare an essence formula containing hydroxytyrosol.

[0219] (6) Before adjusting the pH value, take 2 mL of samples from both Formula 1 and Formula 2 as follows: Figure 6 (A); As a comparison, after adjusting the pH value, 2 mL of samples from both Formula 1 and Formula 2 were taken as follows: Figure 6 (B).

[0220] Hydroxytyrosol changes color under alkaline conditions primarily due to the oxidation of the catechol structure, producing colored quinones. When hydroxytyrosol is modified by amino acid grafting, the number of active hydroxyl groups oxidized to quinones in an alkaline environment is reduced, preventing further deactivation of the active hydroxyl groups. Unmodified hydroxytyrosol, however, shows significant and severe color change in an alkaline environment. While it may retain some antioxidant properties of active hydroxyl groups in the short term, its antioxidant properties decrease as the color deepens over time and more hydroxyl groups are deactivated. In cosmetic formulations involving hydroxytyrosol, its use in alkaline systems should be avoided as much as possible. However, sometimes, due to considerations such as overall formulation, skin feel, and cost, it is necessary to use alkaline formulations. Hydroxytyrosol derivatives modified with alkaline protease exhibit significantly improved stability and can better exert their antioxidant and soothing effects in such systems.

[0221] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0222] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A method for preparing modified hydroxytyrosol, characterized in that, Includes the following steps: In an oxygen-containing environment, hydroxytyrosol, collagen, alkaline protease, and arginine are reacted in an aqueous solvent with a pH of 9-10 under stirring conditions to prepare a reaction product containing the modified hydroxytyrosol. The pH of the aqueous solvent is adjusted by arginine.

2. The method for preparing modified hydroxytyrosol according to claim 1, characterized in that, The modified hydroxytyrosol includes hydroxytyrosol grafted with hydrolyzed fragments of the collagen.

3. The method for preparing modified hydroxytyrosol according to claim 1, characterized in that, The collagen includes collagen derived from fish skin.

4. The method for preparing modified hydroxytyrosol according to claim 1, characterized in that, The hydrolyzed fragments of the collagen include glycine-proline-hydroxyproline fragments.

5. The method for preparing modified hydroxytyrosol according to claim 1, characterized in that, The preparation method includes freeze-drying the reaction system after the reaction is completed, and the freeze-dried product obtained contains the modified hydroxytyrosol.

6. The method for preparing modified hydroxytyrosol according to claim 1, characterized in that, It also meets at least one of (a) to (f): (a) The mass ratio of the hydroxytyrosol to the collagen is 0.5:(0.1~1) based on the amount of feed. (b) The mass ratio of the hydroxytyrosol to the alkaline protease is 0.5:(0.01~0.1) based on the amount of feed. (c) The mass-to-volume ratio of the hydroxytyrosol to the aqueous solvent is 1 g: (8~15) mL, based on the amount of material fed. (d) The reaction temperature is 40℃~50℃, and the reaction time is 5h~15h; (e) The stirring speed of the reaction is 500~600 r / min; (f) In the preparation method described above, 50% to 70% of hydroxytyrosol is converted into the modified hydroxytyrosol.

7. The method for preparing modified hydroxytyrosol according to any one of claims 1 to 6, characterized in that, The preparation method further includes: purifying the reaction product to obtain purified modified hydroxytyrosol.

8. The reaction product prepared by the method of any one of claims 1 to 6; or the use of the modified hydroxytyrosol prepared by the method of claim 7 in the preparation of a product for achieving at least one of the effects in (a) to (f): (a) Scavenging free radicals; (b) Reduce the level of inflammatory factors in the subject, said inflammatory factors including at least one of TNF-α and IL-6; (c) Inhibits hyaluronidase activity; (d) At least one of the following: antioxidant, anti-inflammatory, and anti-allergic; (e) Soothe the subject's skin; and, (f) Used for moisturizing the subject's skin.

9. The application according to claim 8, characterized in that, The product includes cosmetics, which meet at least one of (i) and (ii): (i) The cosmetic product has at least one of the following effects: antioxidant, soothing, and moisturizing; and, (ii) The pH of the aqueous phase in the cosmetic is 7.5~10.

10. A cosmetic product, characterized in that, The product contains at least one of the reaction product prepared by the method of preparing modified hydroxytyrosol according to any one of claims 1 to 6 and the modified hydroxytyrosol prepared by the method of preparing modified hydroxytyrosol according to claim 7.

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