Preparation method and application of resveratrol and derivatives thereof

Abstract

The invention provides an anti-inflammatory and / or acne-removing and / or oil-controlling and / or antioxidant and / or slow-release whitening composition, which comprises resveratrol, a hydroxyl derivative thereof, an acetylated derivative thereof or a salt thereof. The invention provides application of resveratrol, a hydroxyl derivative thereof, an acetylated derivative thereof or a salt thereof in preparation of drugs, cosmetics or skin care products for resisting inflammation and / or removing acnes and / or controlling oil and / or resisting oxidation and / or slowly-releasing and whitening. The invention provides a preparation method of a resveratrol acetylated derivative. The inventor finds that the resveratrol, the hydroxyl derivative, the acetylated derivative or the salt of the resveratrol show potential application prospects in the field of skin care products with the effects of resisting inflammation, removing acnes, controlling oil, resisting oxidation and / or slowly-releasing and whitening and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of cosmetics, and relates to resveratrol derivatives and their biotransformation preparation methods. The present invention also relates to the use of resveratrol and its derivatives in preparing medicines, cosmetics or skin care products with anti-inflammatory, acne / acne mark removal, oil control, antioxidant and / or sustained-release whitening effects. The present invention also relates to medicines, cosmetics or skin care products containing acetylated resveratrol derivatives. Background Art

[0002] With the continuous development of the skin care and cosmetics industry, various functional raw materials are constantly being explored, the raw material reserves are constantly expanding, and people's requirements for facial skin care are getting higher and higher. However, the skin sensitivity of modern people has increased dramatically. Due to improper use of cosmetics, excessive cleaning, light, skin sensitivity, etc., many skin problems occur, such as redness, itching, peeling or inflammatory reactions, etc., and acne or acne may further lead to skin inflammation and other problems. At the same time, temperature changes, external stimulation, seasonal changes, etc. can also cause different degrees of skin allergies, redness and other inflammatory reactions in people with sensitive skin. Therefore, it is very important to develop new, efficient, low-cost, anti-inflammatory, oil-control and other multi-functional raw materials.

[0003] At present, the effective raw materials for anti-inflammatory and oil control in the market are single, mainly based on formula compositions. For example, patent CN112315882 A discloses a soothing, repairing and moisturizing facial cream and its preparation method, in which phenoxyethanol, which is more irritating to sensitive skin, is used as a preservative and stabilizer. However, these raw materials themselves have no effect. Similarly, some irritating raw materials in many formulas or compositions in the prior art can induce a series of inflammations, and this series of inflammations are usually recurrent and persistent, and are difficult to cure, requiring the use of external application drugs and oral conditioning methods. However, such methods are costly, have strong irritation, and are prone to side effects.

[0004] Therefore, there is an urgent need to develop a high-efficiency, low-cost, and multi-functional raw material that has strong anti-inflammatory, acne / acne scar removal, oil control, antioxidant and / or sustained-release whitening effects. Summary of the invention

[0005] In view of the deficiencies in the prior art, the object of the present invention is to provide an acetylated resveratrol derivative and a bioconversion preparation method thereof. The present invention also provides the use of resveratrol and acetylated resveratrol derivatives for preparing medicines, cosmetics or skin care products with anti-inflammatory, acne / acne scar removal, oil control, anti-oxidation and / or sustained-release whitening effects. The inventors unexpectedly discovered that in addition to the whitening effect, resveratrol and its acetylated derivatives also have multiple effects such as anti-inflammatory, acne / acne scar removal, oil control, anti-oxidation and / or sustained-release whitening. The inventors also unexpectedly discovered that a series of acetylated resveratrol derivatives can be obtained by using a more gentle and friendly bioconversion method.

[0006] The objective of the present invention is achieved through the following technical solutions:

[0007] The first aspect of the present invention relates to an anti-inflammatory and / or acne-removing and / or oil-controlling and / or anti-oxidative and / or sustained-release whitening composition, wherein the composition comprises one or more of resveratrol, resveratrol hydroxy derivatives, resveratrol acetylated derivatives or salts thereof.

[0008] In some embodiments, the resveratrol, resveratrol hydroxy derivative, resveratrol acetylated derivative or salt thereof is used as an anti-inflammatory and / or anti-acne and / or oil control and / or anti-oxidation and / or sustained-release whitening ingredient.

[0009] In some embodiments, the resveratrol, resveratrol hydroxy derivative, resveratrol acetylated derivative or salt thereof is used as the sole anti-inflammatory and / or anti-acne and / or oil control and / or anti-oxidation and / or sustained-release whitening ingredient.

[0010] In some embodiments, the hydroxy derivative of resveratrol is oxidized resveratrol.

[0011] In some embodiments, the acetylated resveratrol derivative is triacetylated resveratrol and / or tetraacetylated oxidized resveratrol.

[0012] In some embodiments, the composition comprises triacetylated resveratrol and / or tetraacetylated oxidized resveratrol.

[0013] In some embodiments, the composition is a pharmaceutical composition.

[0014] In some embodiments, the composition is a cosmetic composition.

[0015] In some embodiments, the composition is a skin care composition.

[0016] In some embodiments, the weight of the resveratrol, the hydroxy derivative of resveratrol, the acetylated derivative of resveratrol or the salt thereof accounts for 0.01%-5% of the total weight of the composition.

[0017] In some embodiments, the composition further comprises a pharmaceutically or cosmetically acceptable excipient.

[0018] In some embodiments, the pharmaceutical composition is selected from one or more of a cream, a gel, a patch, a spray, an ointment, a plaster, a lotion, a liniment, a paste, or a poultice.

[0019] In some embodiments, the cosmetic composition or skin care composition is selected from one or more of lotion, skin softener, toner, astringent, emulsion, massage cream, nourishing cream, moisturizing cream, hand cream, foundation, essence, facial mask, soap, facial cleanser, facial cleanser, facial cream, body lotion or shower gel.

[0020] The second aspect of the present invention relates to the use of resveratrol, resveratrol hydroxy derivatives, resveratrol acetylated derivatives or salts thereof in the preparation of medicines, cosmetics or skin care products for anti-inflammatory and / or acne removal and / or oil control and / or anti-oxidation and / or sustained-release whitening.

[0021] In some embodiments, the resveratrol, resveratrol hydroxy derivatives, resveratrol acetylated derivatives or salts thereof are used as anti-inflammatory and / or anti-acne and / or oil control and / or anti-oxidation and / or sustained-release whitening ingredients in medicines, cosmetics or skin care products.

[0022] In some embodiments, the resveratrol, resveratrol hydroxy derivative, resveratrol acetylated derivative or salt thereof is used as the only anti-inflammatory and / or anti-acne and / or oil-control and / or anti-oxidation and / or sustained-release whitening ingredient in medicines, cosmetics or skin care products.

[0023] In some embodiments, the hydroxy derivative of resveratrol is oxidized resveratrol.

[0024] In some embodiments, the acetylated resveratrol derivative is triacetylated resveratrol and / or tetraacetylated oxidized resveratrol.

[0025] In some embodiments, the medicine, cosmetic or skin care product comprises triacetylated resveratrol and / or tetraacetylated oxidized resveratrol.

[0026] In some embodiments, the amount of resveratrol, resveratrol hydroxy derivative, resveratrol acetylated derivative or salt thereof added to the medicine, cosmetic or skin care product is 0.01%-5% by weight.

[0027] In some embodiments, the medicament is selected from one or more of a cream, a gel, a patch, a spray, an ointment, a plaster, a lotion, a liniment, a paste, or a poultice.

[0028] In some embodiments, the cosmetics or skin care products are selected from one or more of lotion, skin softener, toner, astringent, emulsion, massage cream, nourishing cream, moisturizing cream, hand cream, foundation, essence, facial mask, soap, facial cleanser, facial cleanser, facial cream, body lotion or shower gel.

[0029] In some embodiments, the anti-inflammatory includes preventing and / or treating skin inflammation.

[0030] In some embodiments, the skin inflammation is skin inflammation associated with acne.

[0031] The third aspect of the present invention relates to a biotransformation preparation method of acetylated resveratrol derivatives, wherein the method uses a polar organic solvent as a solvent, lipase as a catalyst, and vinyl acetate as an acetyl donor to acetylate substrate resveratrol and / or oxidized resveratrol.

[0032] In some embodiments, the ratio of the substrate to the acetyl donor is 1:5 to 1:30. In some embodiments, the ratio of the substrate to the acetyl donor is 1:10 to 1:20. In some embodiments, the ratio of the substrate to the acetyl donor is 1:12 to 1:18. In some embodiments, the ratio of the substrate to the acetyl donor is 1:15.

[0033] In some embodiments, the duration of the reaction is 12-96 hours, preferably 24-72 hours, and more preferably 24-48 hours.

[0034] In some embodiments, the concentration of the lipase is 50-300 mg / ml, preferably 100-200 mg / ml, more preferably 150-200 mg / ml.

[0035] In some embodiments, the method further comprises the step of post-processing the product. In some embodiments, the post-processing includes, but is not limited to, filtration, concentration, chromatography separation and drying.

[0036] In some embodiments, the lipase is Novozym 435 lipase.

[0037] The conditions such as enzyme concentration, temperature, and substrate ratio will affect the conversion rate. The applicant has found through extensive research that the best conversion rate can be obtained when the conversion is carried out under the above-mentioned specific conditions.

[0038] In some embodiments, the solvent is selected from ethyl acetate, methanol, ethanol, 2-methyl-2-butanol, or a mixture of one or more thereof.

[0039] In some embodiments, the acetylated resveratrol derivative is triacetylated resveratrol or tetraacetylated oxidized resveratrol.

[0040] The beneficial effects of the present invention are as follows:

[0041] Different from the traditional chemical synthesis method, the present invention adopts Novozym 435 lipase to prepare the acetylated product of resveratrol by biotransformation in an organic solvent. The preparation method is mild and non-irritating, low in cost, and the prepared product has better biocompatibility. Since it does not contain harmful or irritating solvents or by-products in chemical synthesis, it can be directly added to the product. The present invention uses acetyl-modified resveratrol acetylated derivatives as active substances, which are mild to the skin, less irritating, low in cytotoxicity, good in structural stability, good in skin permeability and retention, and has excellent sustained release. The acetyl-modified resveratrol acetylated derivatives of the present invention have excellent anti-inflammatory, oil control, sustained-release whitening, anti-oxidation and other effects, can effectively relieve anti-inflammatory effects, and can also relieve acne or various inflammatory reactions caused by pore blockage.

[0042] The inventors have proved through experimental evidence that the acetylated resveratrol derivative or its salt has at least the following effects:

[0043] 1) The acetylated resveratrol derivatives of the present invention, triacetylated resveratrol and tetraacetylated oxidized resveratrol, use acetyl groups to protect the phenolic hydroxyl groups, which can effectively improve their structural stability, transdermal effect (i.e., bioavailability), and reduce cytotoxicity. At the same time, after entering the cells, these compounds can be hydrolyzed by intracellular esterases to release resveratrol or oxidized resveratrol, thereby maintaining their original efficacy while having a sustained release effect;

[0044] 2) The acetylated resveratrol derivatives of the present invention, triacetylated resveratrol and tetraacetylated oxidized resveratrol, have a good inhibitory effect on IL-6 and NO inflammatory factors in RAW cells. The IL-6 inflammatory factor inhibits IC 50 The values ​​were 2.03μg / ml and 8.96μg / ml, respectively, and the NO inhibition IC 50 The values ​​were 3.4μg / ml and 4.69μg / ml respectively. Compared with resveratrol and oxidized resveratrol, the small molecules modified with acetyl groups have similar anti-inflammatory activity and more application advantages.

[0045] 3) The acetylated resveratrol derivatives of the present invention, triacetylated resveratrol and tetraacetylated oxidized resveratrol, can effectively alleviate inflammation induced by acne or pimples. Through the test of TNF-α inflammatory factor induced by Propionibacterium acnes (PA) in THP-1 cell line, the TNF-α inhibition rate of 10μg / ml triacetylated resveratrol was as high as 47.81%, while the TNF-α inhibition rate of tetraacetylated oxidized resveratrol was 30.59%, which also had similar, sustained-release anti-inflammatory effects compared with unmodified resveratrol and oxidized resveratrol;

[0046] 4) The acetylated resveratrol derivatives of the present invention, triacetylated resveratrol and tetraacetylated oxidized resveratrol, have a good oil control effect on human sebaceous gland cells (SZ95 system), with oil control efficiencies of 33.52% at 5 μg / ml and 36.42% at 0.5 μg / ml, respectively, which are better than the positive control epigallocatechin gallate EGCG (10 μg / ml inhibition rate 31%);

[0047] 5) Compared with resveratrol and oxidized resveratrol, the acetylated resveratrol derivatives of the present invention, triacetylated resveratrol and tetraacetylated oxidized resveratrol, in addition to having similar whitening effects, also have a sustained-release whitening effect. 16 F 10 In the cell melanin inhibition model test, the general whitening functional raw materials (such as 3,3',4',7-tetrahydroxyflavone) have an obvious upward trend in melanin within 48h-72h, while the small molecules after acetyl modification and protection need to be slowly hydrolyzed by intracellular esterase, so they can continuously release the functional raw materials, so that the melanin level can be maintained at a low level within 48-72h, and have a sustained-release whitening effect.

[0048] 6) The acetylated resveratrol derivatives of the present invention, triacetylated resveratrol and tetraacetylated oxidized resveratrol, also have good antioxidant effects. According to the human Hacat epidermal cell ROS test, the ROS active oxygen inhibition rate of 30 μg / ml triacetylated resveratrol is 36.06%, and the ROS active oxygen inhibition rate of 25 μg / ml tetraacetylated oxidized resveratrol is 47.61%. Compared with unmodified resveratrol and oxidized resveratrol, they have similar antioxidant activity, and compared with the positive control EGCG (40 μg / ml inhibition rate 47.5%), the effect is better.

[0049] 7) The acetylated resveratrol derivatives of the present invention, triacetylated resveratrol and tetraacetylated oxidized resveratrol, have better transdermal and skin retention effects, can stay in the skin for a long time to exert their effects, and can effectively improve their bioavailability in epidermal cells. The transdermal rate of resveratrol / oxidized resveratrol small molecule monomers is about 64% (32°C, 24h), while the transdermal efficiency of acetylated small molecules can reach 90%+ (32°C, 24h). At the same time, the transdermal results can also assist in proving the conversion of acetylated products in epidermal cells, and have a sustained release effect.

[0050] In summary, the resveratrol and its derivatives of the present invention, especially the acetylated derivatives triacetylated resveratrol and tetraacetylated oxidized resveratrol, have anti-inflammatory, acne / acne mark removal, oil control, antioxidant and / or sustained-release whitening effects, and have shown important scientific research value and potential application prospects in the field of skin care products. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 The results of the IL-6 and NO inflammatory factor tests on RAW cells according to Example 2 of the present invention are shown.

[0052] Figure 2 The test results of TNF-α inflammatory factor in THP-1 cell line induced by Propionibacterium acnes (PA) according to Example 3 of the present invention are shown.

[0053] Figure 3 The results of the oil control test on human sebaceous gland cells (SZ95 series) according to Example 4 of the present invention are shown.

[0054] Figure 4 B according to Example 5 of the present invention is shown 16 F 10 Melanin inhibition test results.

[0055] Figure 5 The results of the Hacat cell ROS reactive oxygen species inhibition test according to Example 6 of the present invention are shown.

[0056] Figure 6 The observation results of the 14-day stability test at 60° C. according to Example 7 of the present invention are shown, wherein the active ingredient addition amount is 0.01%. DETAILED DESCRIPTION

[0057] definition

[0058] The terms "weight %" and "weight percentage" are used interchangeably herein. If not otherwise specified, percentage refers to weight percentage.

[0059] Unless otherwise indicated, all component or composition levels are in reference to the active portion of that component or composition and are exclusive of impurities, for example, residual solvents or by-products, which may be present in commercially available sources of such component or composition.

[0060] Unless otherwise indicated, all temperatures herein are in degrees Celsius (° C.) Unless otherwise indicated, all measurements herein are made at 20° C., atmospheric pressure, and 50% relative humidity.

[0061] It should be understood that each maximum numerical limit given throughout this specification includes each lower numerical limit, as if such lower numerical limits were expressly written herein. Each minimum numerical limit given throughout this specification will include each higher numerical limit, as if such higher numerical limits were expressly written herein. Each numerical range given throughout this specification will include each narrower numerical range falling within such wider numerical range, as if such narrower numerical ranges were all expressly written herein.

[0062] As used herein, the term "acne" and "pimples" are used interchangeably, and is referred to as "pimples" in the cosmetic field and as "acne" in medicine.

[0063] As used herein, the term "skin care product" means any substance and any component thereof that is intended to be rubbed, poured, sprinkled, sprayed, introduced or otherwise applied to a mammalian body or any part thereof to provide a cosmetic benefit. Skin care products may include materials generally recognized as safe (GRAS) by the National Food and Drug Administration, food additives, and materials used in non-cosmetic consumer products (including over-the-counter drugs). Skin care products may include, but are not limited to: (i) chemicals, compounds, small or large molecules, extracts, preparations, or combinations thereof that are known to induce or cause at least one effect (positive or negative) on skin tissue; (ii) chemicals, compounds, small molecules, extracts, preparations, or combinations thereof that are unclear whether they have an effect on skin tissue and are found to induce or cause an effect on skin tissue using the provided methods and systems; and (iii) compounds or combinations of compounds that provide acute and / or chronic beneficial effects to the skin or cell types that are normally present therein when applied to the skin. Skin care products can regulate and / or improve skin or its associated cells (e.g., improve skin elasticity; improve skin hydration; improve skin condition; and improve cell metabolism).

[0064] As used herein, the term "active ingredient" refers to a compound that provides a benefit or improvement to the skin when applied to the skin. It should be understood that active ingredients are not only for application to the skin, but also for application to hair, nails and other mammalian keratinous tissues.

[0065] As used herein, the term "cosmetically acceptable excipients" refers to excipients that are not necessary active ingredients in cosmetics and are suitable for use in humans and / or animals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), and have a reasonable benefit / risk ratio. Typically, cosmetically acceptable excipients may include moisturizers, emulsifiers, thickeners, chelating agents, emollients, and the like.

[0066] As used herein, the term "pharmaceutically acceptable excipient" refers to an excipient that is not a necessary active ingredient in a drug itself and is suitable for administration to humans and / or animals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), and has a reasonable benefit / risk ratio. Typically, pharmaceutically acceptable excipients may include fillers, stabilizers, diluents, adhesives, surfactants, solvents, etc.

[0067] Active ingredients and other ingredients useful herein may be classified or described herein according to their cosmetic and / or therapeutic benefits or their postulated modes of action. However, it should be understood that in some cases, active ingredients and other ingredients useful herein may provide more than one cosmetic and / or therapeutic benefit, or operate via more than one mode of action. Therefore, the classification herein is for convenience only and is not intended to limit the ingredients to the specifically indicated application or applications listed.

[0068] Resveratrol is a compound with the IUPAC name 5-[2-(4-hydroxyphenyl)vinyl]benzene-1,3-diol. The compound can also be represented by the following aliases: 3,5,4'-trihydroxy-trans-stilbene; trans-3,5,4'-trihydroxystilbene; 3,4',5-stilbenetriol; trans-resveratrol; (E)-5-(p-hydroxystyryl)benzene-diol; (E)-5-(4-hydroxystyryl)benzene-1,3-diol.

[0069] Oxyresveratrol is a compound with the IUPAC name 4-[(E)-2-(3,5-dihydroxyphenyl)vinyl]benzene-1,3-diol. This compound may also be referred to by the following aliases: 2,3',4,5'-tetrahydroxy-trans-stilbene; 2,3',4,5'-tetrahydroxystilbene; 4-[(E)-2-(3,5-dihydroxyphenyl)vinyl]benzene diol; tetrahydroxystilbene.

[0070] Triacetylated resveratrol, which can also be represented as trans-triacetyl-resveratrol; acetyl-resveratrol; acetyl-trans-resveratrol; trans-resveratrol triacetate; 5-[(1E)-2-[4-(acetyloxy)phenyl]vinyl]-1,3-benzenediol-1,3-diacetate; 4-[(E)-2-(3,5-diacetoxyphenyl)vinyl]phenyl acetate; resveratrol 3,5,4'-triacetate; 3,5,4'-tri-O-acetylated resveratrol; 3,5,4'-triacetoxy-trans-stilbene; and trans-3,5,4'-triacetoxystilbene, can also be represented by the structural formula of the following chemical formula 1.

[0071] [Chemical formula 1]

[0072]

[0073] Tetraacetyl oxidized resveratrol, which can also be called acetyl-oxidized resveratrol; trans-tetraacetyl oxidized resveratrol; acetyl-trans-oxidized resveratrol; trans-oxidized resveratrol tetraacetate; 4-[(E)-2-(3,5-diacetoxyphenyl)vinyl]benzene-1,3-diacetate; 2,3',4,5'-tetraacetoxy-trans-stilbene; 2,3',4,5'-tetraacetoxystilbene; and tetraacetoxystilbene, etc., can also be represented by the structural formula of the following chemical formula 2.

[0074] [Chemical formula 2]

[0075]

[0076] Resveratrol or its hydroxy derivatives, acetylated derivatives can be used in the form of its salts permitted for use in cosmetics or pharmacy. As salts, acid-added salts formed by free acids permitted for use in cosmetics or pharmacy. As free acids, organic acids and inorganic acids can be used, as inorganic acids, hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, tartaric acid, etc. can be used, as organic acids, methanesulfonic acid, p-toluenesulfonic acid, acetic acid, trifluoroacetic acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, mandelic acid, propionic acid, citric acid, lactic acid, glycolic acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, vitamin C, carbonic acid, vanillic acid, hydroiodic acid, etc. can be used, but are not limited thereto.

[0077] Furthermore, a metal salt acceptable for use in cosmetics or pharmacy can be prepared by using a base.

[0078] The above-mentioned salts of resveratrol or its hydroxyl derivatives or acetylated derivatives that are permitted for use in cosmetics or pharmacy include, unless otherwise specified, most acidic or basic salts that can exist in resveratrol or its acetylated derivatives of hydroxyl derivatives. For example, salts permitted for use in cosmetics or pharmacy include sodium, calcium and potassium salts of hydroxyl groups, and other pharmaceutically permitted salts of amino groups include hydrobromide, sulfate, hydrogen sulfate, phosphate, hydrogen phosphate, dihydrogen phosphate, acetate, succinate, citrate, tartrate, lactate, mandelate, methanesulfonate, methanolate and p-toluenesulfonate, which can be prepared by a salt preparation method known to those skilled in the art.

[0079] Example

[0080] The present invention can be implemented through the following embodiments, but the present invention is not limited thereto.

[0081] The percentages involved in the embodiments of the present invention are all weight percentages unless otherwise indicated.

[0082] The instruments and equipment used in the embodiments of the present invention are all conventional instruments and equipment in the field and can be replaced by instruments and equipment that meet the corresponding standards.

[0083] Unless otherwise specified, the Chinese herbal medicine raw materials and reagents used in the embodiments of the present invention are all commercially available analytically pure reagents.

[0084] The human sebaceous gland cell SZ95 cell line was obtained from Qingqi (Shanghai) Biotechnology Development Co., Ltd.;

[0085] Mouse B 16 F 10 The melanin inhibition model was obtained from the Cell Bank of the Chinese Academy of Sciences;

[0086] RAW cells were obtained from Fuheng Biotechnology Co., Ltd.;

[0087] THP-1 cell line was obtained from the Cell Bank of the Chinese Academy of Sciences;

[0088] Human epidermal Hacat cells were obtained from Zhejiang Meisen Cell Technology Co., Ltd.

[0089] The raw materials and instruments and equipment mentioned in the present invention are all commonly used raw materials and instruments and equipment in the field, which are only examples and are not intended to limit the scope of protection of the present invention. Those skilled in the art can select equivalent raw materials and related instruments and equipment based on the disclosure of the present invention.

[0090] Example 1 Preparation of triacetylated resveratrol and tetraacetylated oxidized resveratrol

[0091] A series of acetylated derivatives were transformed using 1000 PLU / g of Novozym 435 lipase (Visionox lipase) (from Candida Antarctica). Substrate: resveratrol / oxidized resveratrol, vinyl acetate (1:15 eq.), enzyme concentration 150 mg / ml, solvent: 2-methyl-2-butanol, temperature: 40°C, reaction time 24-48 h. After the reaction, the liquid was collected by suction filtration, and the converted product was obtained by rotary evaporation. The impurities were separated by column chromatography and dried for standby use. The conversion rate of acetylated resveratrol was as high as over 90%, while the conversion rate of acetylated oxidized resveratrol was 50%.

[0092] Example 2 RAW cell IL-6 and NO inflammatory factor test

[0093] Mouse macrophage RAW cells were used for testing, and the cells were plated in 96-well plates using DMEM++ (Thermo Fisher Scientific (Gibco)) at a density of 4x10 4 Each well was plated with 100 μL of the solution and cultured overnight in a 37°C constant temperature incubator (5% CO2 concentration).

[0094] After 24 hours, the original culture medium was aspirated, and the cells were washed 1-2 times with PBS buffer solution. 100 μL of DMEM++ culture medium was added to each well, and the LPS (lipopolysaccharide) stimulator prepared with the culture medium was added, 50 μL per well, with a final concentration of 1 μg / ml.

[0095] The sample was prepared with culture medium, the sample included 0.05 μg / ml and 0.1 μg / ml dexamethasone solution as positive control, culture medium as blank control, resveratrol with concentrations of 0.5 μg / ml, 1 μg / ml, 2.5 μg / ml, 5 μg / ml, and 1 μg / ml, oxidized resveratrol, triacetylated resveratrol, tetraacetylated oxidized resveratrol solution, added to 96-well plate, 50 μL per well, 200 μL / well in total. Added to 96-well plate, 50 μL per well, 200 μL / well in total. Cultured in 37°C constant temperature incubator for 24 hours (5% CO2 concentration).

[0096] After 24 hours, 50 μL of supernatant was taken from each well and added to a new 96-well plate, and NO detection was performed using a NO detection kit (Biyuntian Biotechnology Co., Ltd.). The remaining 150 μL of supernatant was collected and stored, and the lower layer of cells was used to measure the activity of cck8 cells. The collected supernatant was used to detect IL-6 inflammatory factors using the mouse IL-6 uncoated ELISA kit from ThermoFisher Scientific.

[0097] The results are shown in Table 1 and Figure 1 As shown in Figure 2, acetyl-modified triacetyl-resveratrol and tetraacetylated oxidized-resveratrol have excellent anti-inflammatory effects, and the IL-6 inflammatory factor inhibits IC 50 The values ​​were 2.03μg / ml and 8.96μg / ml, respectively, and the NO inhibition IC 50 The values ​​were 3.4μg / ml and 4.69μg / ml respectively. Compared with resveratrol and oxidized resveratrol, the acetyl-modified small molecules have similar anti-inflammatory activity.

[0098] Table 1 IL-6 inflammatory factor inhibition IC 50

[0099]

[0100] Example 3 Propionibacterium acnes (PA)-induced TNF-α inflammatory factor test in THP-1 cell line

[0101] The human monocytic leukemia THP-1 cell line was used for testing, and the cells were plated in 96-well plates using medium 1640++ (Thermo Fisher Scientific (Gibco)) at a density of 5x10 4 / well, add phorbol ester (PMA) to make the cells adhere to the wall, the final concentration is 50ng / ml, 100μL per well, and culture in a 37℃ constant temperature incubator overnight (5% CO2 concentration). After 24h, aspirate the original culture medium, wash 1-2 times with PBS buffer solution, add 90μL 1640++ culture medium to each well, and add the inactivated PA (0.05μg / ml) solution prepared with the culture medium, 5μL per well.

[0102] The sample was prepared with culture medium, the sample included 0.1 μg / ml dexamethasone solution as a positive control, culture medium as a blank control, and resveratrol at concentrations of 0.5 μg / ml, 1 μg / ml, 2.5 μg / ml, 5 μg / ml, and 1 μg / ml, 2.5 μg / ml, 5 μg / ml, 10 μg / ml, respectively. Oxidized resveratrol, triacetylated resveratrol, and tetraacetylated oxidized resveratrol solutions were added to a 96-well plate, 5 μL per well, for a total of 100 μL / well. Cultured in a 37°C constant temperature incubator for 24 hours (5% CO2 concentration). After 24 hours, the supernatant was collected and stored, and the lower layer of cells was used to measure the activity of cck8 cells. The collected supernatant was used to detect TNF-α inflammatory factors using the human TNF-α ELISA kit of Aibixin (Shanghai) Biotechnology Co., Ltd.

[0103] The results are shown in Table 2 and Figure 2As shown, the TNF-α inhibition rate of 10μg / ml triacetylated resveratrol is as high as 47.81%, while the TNF-α inhibition rate of tetraacetylated oxidized resveratrol is 30.59%. Compared with unmodified resveratrol and oxidized resveratrol, they have similar, sustained-release anti-inflammatory effects and can effectively relieve the inflammatory response caused by acne or acne.

[0104] Table 2 THP-1 cell TNF-α inhibition rate

[0105]

[0106] Example 4 Human sebaceous gland cells (SZ95 system) oil control test

[0107] The human sebaceous gland cell line SZ95 was used for testing. The cells were plated in 96-well plates using DMEM++ (ThermoFisher Scientific (Gibco)) at a density of 1x10 4 Each well was plated with 100 μL of the solution and cultured overnight in a 37°C constant temperature incubator (5% CO2 concentration).

[0108] After 24 hours, the original culture medium was aspirated, and the cells were washed with PBS buffer solution for 1-2 times. The stimulant linoleic acid (LA) solution prepared with the culture medium was added to a final concentration of 0.006% (g / ml), 100 μL per well.

[0109] The samples were prepared with culture medium, including 10 μg / ml or 20 μg / ml epigallocatechin gallate (EGCG) solution as a positive control, culture medium as a blank control, and resveratrol solutions at concentrations of 0.5 μg / ml, 1 μg / ml, and 2 μg / ml, oxidized resveratrol solutions at concentrations of 5 μg / ml, 10 μg / ml, and 20 μg / ml, triacetylated resveratrol solutions at concentrations of 0.5 μg / ml, 1 μg / ml, and 5 μg / ml, and tetraacetylated oxidized resveratrol solutions at concentrations of 0.5 μg / ml, 1 μg / ml, and 5 μg / ml, respectively. The samples were added to a 96-well plate, 100 μL per well, for a total of 200 μL / well.

[0110] After incubation at 37°C for 24 h (5% CO2 concentration), all the solution in the well plate was aspirated, and the well plate was washed 1-2 times with PBS buffer. Nile red staining solution prepared in PBS (final concentration 1 μg / ml) was added, 100 μL per well, and incubated at 37°C for 20 min (5% CO2 concentration). The fluorescence was detected by a microplate reader at an excitation wavelength of 485 nm and an emission wavelength of 565 nm.

[0111] The results are shown in Table 3 and Figure 3As shown, the oxidized resveratrol derivatives have a good oil control effect, with oil control efficiencies of 33.52% at 5 μg / ml and 36.42% at 0.5 μg / ml, respectively. The effect is better than the positive control epigallocatechin gallate EGCG (10 μg / ml inhibition rate 31%).

[0112] Table 3 SZ95 cell oil control inhibition rate

[0113]

[0114] Example 5B 16 F 10 Melanin inhibition test

[0115] Mouse melanocyte B 16 F 10 The cell lines were tested for sustained-release whitening efficacy, and the cells were plated in 24-well plates with DMEM++ (Thermo Fisher Scientific (Gibco)) at a density of 2-3x10 4 Each well was cultured with 500 μL per well and incubated overnight in a 37°C constant temperature incubator (5% CO2 concentration).

[0116] After 24 hours, the original culture medium was aspirated, and the cells were washed 1-2 times with PBS buffer solution. The melanin stimulator α-MSH (Shanghai Myrel Biochemical Technology Co., Ltd.) solution prepared with phenol red-free DMEM++ culture medium was added to a final concentration of 1 μM, 450 μL per well.

[0117] Then, a sample was prepared with DMEM culture medium, the sample including kojic acid (KA) at a concentration of 100 μg / ml as a positive control, culture medium as a blank control, 3,3',4',7-tetrahydroxyflavone solution at concentrations of 5 μg / ml, 10 μg / ml, 25 μg / ml, 50 μg / ml, and 100 μg / ml, triacetylated resveratrol at concentrations of 5 μg / ml, 10 μg / ml, 25 μg / ml, and 100 μg / ml, and tetraacetylated oxidized resveratrol at concentrations of 5 μg / ml, 10 μg / ml, 25 μg / ml, and 100 μg / ml, respectively. The samples were added to a 96-well plate, 50 μL per well, and a total of 500 μL / well.

[0118] The plates were cultured in a 37°C constant temperature incubator for 48-72 h (5% CO2 concentration), 100 μL / well of the supernatant was taken out and placed in a new 96-well plate, and the absorbance was detected at a wavelength of 405 nm using an enzyme-labeled instrument.

[0119] The results are as follows Figure 4As shown, it shows the comparison of the whitening efficacy test at 48h and 72h. (a) For 3,3',4',7-tetrahydroxyflavone: Compared with 48h, at 72h, the melanin of the blank group increased by 43.05%, the melanin of the positive control 100μg / ml kojic acid increased by 19.84%, and the melanin of 25μg / ml 3,3',4',7-tetrahydroxyflavone increased by 22.01%; (b) For triacetylated resveratrol: Compared with 48h, at 72h, the blank group increased by 38.4%, while 25μg / ml triacetylated resveratrol only increased by 13%, which means that triacetylated resveratrol is continuously hydrolyzed by esterase in the cell to slowly release resveratrol, and it can still have a good whitening effect after 72h; (c) Tetraacetylated oxidized resveratrol obtained similar results to triacetylated resveratrol. The results show that compared with resveratrol and oxidized resveratrol, the oxidized resveratrol derivatives not only have similar whitening effects, but also have a sustained-release whitening effect. The general whitening raw materials (such as 3,3',4',7-tetrahydroxyflavone) have an obvious upward trend in melanin within 48h-72h, while the small molecules after acetyl modification and protection require slow hydrolysis by intracellular esterase, so they can continuously release the effective raw materials, so that the melanin level can be maintained at a low level within 48-72h, and have a sustained-release whitening effect.

[0120] Example 6 Human epidermal cell Hacat cell ROS antioxidant test

[0121] Human epidermal Hacat cells were used for testing, 96-well plates were plated with DMEM++, and the density was 2x10 4 Each well was plated with 100 μL of the solution and cultured overnight in a 37°C constant temperature incubator (5% CO2 concentration).

[0122] After 24 hours, the original culture medium was aspirated, washed 1-2 times with PBS buffer solution, and samples were prepared with the culture medium, including 20 μg / ml or 40 μg / ml epigallocatechin gallate (EGCG) solution as a positive control, culture medium as a blank control, and resveratrol solutions with concentrations of 3.75 μg / ml, 7.5 μg / ml, 15 μg / ml, and 30 μg / ml, respectively, oxidized resveratrol solutions with concentrations of 1 μg / ml, 2.5 μg / ml, 5 μg / ml, 10 μg / ml, and 20 μg / ml, respectively, triacetylated resveratrol solutions with concentrations of 3.75 μg / ml, 7.5 μg / ml, 15 μg / ml, 30 μg / ml, and 60 μg / ml, respectively, and tetraacetylated oxidized resveratrol solutions with concentrations of 5 μg / ml, 10 μg / ml, 25 μg / ml, and 50 μg / ml, respectively.

[0123] Add 100 μL to each well of a 96-well plate. After incubation at 37°C for 24 h (5% CO2 concentration), all the solution in the well plate was aspirated, the wells were washed 1-2 times with PBS buffer, 100 μL of H2O2 solution prepared in PBS (final concentration 200 μM) was added to each well, and incubated at 37°C for 1 h. After that, the wells were washed 1-2 times with PBS buffer, and 100 μL of fluorescent probe DCFH-DA (Sigma-Aldrich (Shanghai) Trading Co., Ltd.) + Hoechst solution (Thermo Fisher Scientific (invitrogen)) prepared in DMEM (final concentrations of 10 μg / ml and 5 μg / ml, respectively) was added to each well, and incubated at 37°C for 20 min. The fluorescence was detected by dual-channel microplate reader, with the detection wavelengths of 485 nm (excitation) and 525 nm (emission) for DCGH-DA and 350 nm (excitation) and 461 nm (emission) for Hoechst.

[0124] The results are shown in Table 4 and Figure 5 As shown, the ROS reactive oxygen species inhibition rate of 30 μg / ml triacetylated resveratrol was 36.06%, and the ROS reactive oxygen species inhibition rate of 25 μg / ml tetraacetylated oxidized resveratrol was 47.61%. Compared with unmodified resveratrol and oxidized resveratrol, they have similar antioxidant activity, and compared with the positive control EGCG (40 μg / ml inhibition rate 47.5%), the effect is better.

[0125] Table 4 Hacat cell ROS antioxidant inhibition rate

[0126]

[0127]

[0128] Example 7 Stability Test

[0129] 8 bottles of 0.01 wt% resveratrol and its derivatives (resveratrol, oxidized resveratrol, triacetylated resveratrol and tetraacetylated oxidized resveratrol) solutions were prepared with 50 wt% butanediol solution, one bottle was kept in a 4°C refrigerator, and the rest were placed in a 60°C oven. Starting from the second day, one bottle was taken out every day for the first 4 days to measure the pH value, and one bottle was taken out every 2-3 days thereafter to measure the pH value. All samples were collected after 14 days to test their efficacy.

[0130] The results are shown in Tables 5-8 and Figure 6After being placed at 60℃ for 14 days, which is equivalent to being placed at room temperature (25℃) for 158.4 days (according to the calculation formula T25℃=T60℃*2^3.5), that is, 5.3 months later, the test efficacy of the acetylated samples triacetylated resveratrol and tetraacetylated oxidized resveratrol remained almost unchanged, indicating their good stability, while the unmodified resveratrol and oxidized resveratrol began to change color slightly on the second day, indicating that the samples modified with acetyl groups have a more stable structure.

[0131] Table 5 Stability analysis of resveratrol

[0132]

[0133]

[0134] Table 6 Stability analysis of oxidized resveratrol

[0135]

[0136] Table 7 Stability analysis of triacetylated resveratrol

[0137]

[0138]

[0139] Table 8 Stability analysis of tetraacetylated resveratrol

[0140]

[0141] Example 8 Cytotoxicity Test

[0142] The experiment was plated according to the cell density and culture medium required for different cell experiments, as follows:

[0143] Mouse macrophage RAW cells were tested, plated in 96-well plates, culture medium DMEM++, density 4x10 4 pcs / well, 100 μL per well.

[0144] Human monocytic leukemia THP-1 cell line was tested, 96-well plate, medium 1640++, density 5x10 4 Phorbol myristate amine (PMA) was added to allow the cells to adhere to the wall, with a final concentration of 50 ng / ml, 100 μL per well.

[0145] The human sebaceous gland cell line SZ95 was tested, 96-well plates were plated, the culture medium was DMEM++, the density was 1x10 4 Each well was plated with 100 μL of the solution and cultured overnight in a 37°C constant temperature incubator (5% CO2 concentration).

[0146] Mouse melanocyte B 16 F 10 The cell line was tested for sustained-release whitening efficacy, 24-well plates were plated, DMEM++ medium, density 2-3x10 4 pcs / well, 500 μL per well.

[0147] Human epidermal Hacat cells were tested in 96-well plates in DMEM++ medium at a density of 2x10 4 Each well was plated with 100 μL of the solution and cultured overnight in a 37°C constant temperature incubator (5% CO2 concentration).

[0148] After 24 hours, the original culture medium was aspirated and washed 1-2 times with PBS buffer solution. The samples were prepared with culture medium, resveratrol solutions with concentrations of 2μg / ml, 5μg / ml, 10μg / ml and 25μg / ml, oxidized resveratrol solutions with concentrations of 10μg / ml and 20μg / ml, triacetylated resveratrol solutions with concentrations of 5μg / ml, 10μg / ml, 50μg / ml and 100μg / ml, and tetraacetylated oxidized resveratrol solutions with concentrations of 20μg / ml, 25μg / ml, 50μg / ml and 100μg / ml. Add 100μL to each well of a 96-well plate. After incubation at 37°C for 24 h (5% CO2 concentration), all the solution in the well plate was aspirated, and the well plate was washed 1-2 times with PBS buffer. 100 μL of cck8 solution prepared in DMEM (AbMole BioScience) was added to each well. After incubation at 37°C for 1 h (5% CO2 concentration), the absorbance was read at 450 nm.

[0149] The results are shown in Table 9, indicating that the cytotoxicity of the acetylated active substance is about 2-13 times lower than that of the unmodified active substance.

[0150] Table 9 Cytotoxicity test

[0151]

[0152] Example 9 Transdermal Absorption Test in Franz Diffusion Cell

[0153] Porcine skin (Bama Xiang pig skin, Shanghai Zimeng Technology Co., Ltd.) was immersed in PBS (Cytiva, SH30028.02) for thawing;

[0154] Fixation: Add a rotor to the diffusion cell, use filter paper to absorb the moisture on the surface of the pig skin, cut off the excess part of the pig skin edge according to the shape of the diffusion cell, and fix the pig skin on the Franz diffusion cell (caliber 20mm, capacity 7mL);

[0155] Sample addition: Use a syringe to fill the diffusion cell with PBS (receiving solution, depending on the solubility of the transdermal compound, water-soluble compounds use PBS as the receiving solution, and compounds with poor water solubility can be added with 5% Tween 80 in PBS as the receiving solution), drive out the bubbles by inverting the diffusion cell, add PBS to the red line position, and evenly spread 1 mL of sample on the pig skin (the sample solvent is used as a blank control);

[0156] Transdermal: Place the diffusion cell in a transdermal diffusion tester (SV-12D transdermal diffusion tester, Shanghai Yuyan Scientific Instrument Co., Ltd.), set the instrument to 32±0.5℃, 400rpm / min, and transdermal for 24h (the transdermal time can be determined according to the test requirements);

[0157] Treatment: 1) Collect receiving solution; 2) Rinse the surface sample of pig skin with PBS, absorb the surface moisture with filter paper, cut off the edge of the pig skin, keep the pig skin involved in the transdermal part, cut it into pieces, and add 2mL of solvent (the solvent depends on the solubility of the transdermal compound, PBS is selected for water-soluble compounds, and suitable organic solvents are selected for compounds with poor water solubility);

[0158] Extraction: Ultrasonication was used for 30 min to extract the target compounds from pig skin;

[0159] Detection: The target compound was detected by high performance liquid phase reaction with the receiving solution and the pig skin extract, and the transdermal amount and the retention amount were calculated. The results are shown in Tables 10 and 11, indicating that the transdermal amount of the acetylated product was higher. The transdermal rate of resveratrol / oxidized resveratrol small molecule monomer was about 64% (32°C, 24h), while the transdermal efficiency of acetylated small molecules could reach more than 90% (32°C, 24h), which means better bioavailability. In addition, it can be seen from the transdermal experiment that triacetylated resveratrol can be slowly converted into resveratrol in epidermal cells, which helps to prove the sustained release effect of the acetylated product. Acetylated oxidized resveratrol also has the same effect.

[0160] The inventors also unexpectedly discovered that, compared with pterostilbene, acetylated resveratrol has a stronger retention ability and can stay in the skin for a long time to exert its effects.

[0161] Table 10

[0162]

[0163]

[0164] Table 11

[0165]

[0166] 100μg / mL resveratrol will precipitate

[0167] It should be understood that this specification is not limited to the technical solutions shown above, and various modifications and changes can be made to various technical solutions without departing from its scope. This specification is intended to cover any modification, use or adaptive change of the present invention, which follows the general principles of this specification and includes common knowledge or common technical means in the technical field. This specification and embodiments are only regarded as exemplary, and the scope of protection of the present invention is defined by the appended claims.

Claims

1. An anti-inflammatory and / or acne-removing and / or oil-controlling and / or anti-oxidative and / or sustained-release whitening composition, wherein: The composition comprises resveratrol, one or more of its hydroxyl derivatives, acetylated derivatives or salts thereof.

2. The composition according to claim 1, wherein The resveratrol hydroxy derivative is oxidized resveratrol; Optionally, the acetylated resveratrol derivative is triacetylated resveratrol and / or tetraacetylated oxidized resveratrol; Optionally, the composition comprises triacetylated resveratrol and / or tetraacetylated oxidized resveratrol.

3. The composition according to claim 2, wherein The weight of the resveratrol, its hydroxyl derivative, acetylated derivative or its salt accounts for 0.01%-5% of the total weight of the composition.

4. The composition according to any one of claims 1 to 3, wherein The composition is a pharmaceutical composition, a cosmetic composition or a skin care composition; the composition optionally comprises pharmaceutically or cosmetically acceptable excipients.

5. The composition according to claim 4, wherein The pharmaceutical composition is selected from one or more of creams, gels, patches, sprays, ointments, plasters, lotions, liniments, pastes or mud compresses; the cosmetic composition or skin care composition is selected from one or more of lotions, skin softeners, toners, astringents, lotions, massage creams, nourishing creams, moisturizing creams, hand creams, foundations, essences, facial masks, soaps, facial cleansers, cleansing milks, cleansing creams, body lotions or shower gels.

6. Use of resveratrol, its hydroxyl derivatives, acetylated derivatives or salts thereof in the preparation of medicines, cosmetics or skin care products for anti-inflammatory and / or acne removal and / or oil control and / or anti-oxidation and / or sustained-release whitening.

7. The use according to claim 6, wherein The resveratrol hydroxy derivative is oxidized resveratrol; Optionally, the acetylated resveratrol derivative is triacetylated resveratrol and / or tetraacetylated oxidized resveratrol; Optionally, the medicine, cosmetic or skin care product contains triacetylated resveratrol and / or tetraacetylated oxidized resveratrol; Optionally, the resveratrol, its hydroxyl derivatives, acetylated derivatives or salts thereof are added in an amount of 0.01%-5% by weight in medicines, cosmetics or skin care products.

8. The use according to claim 7, wherein The medicine is selected from one or more of creams, gels, patches, sprays, ointments, plasters, lotions, liniments, pastes or mud compresses; the cosmetics or skin care products are selected from one or more of lotions, softeners, toners, astringents, lotions, massage creams, nourishing creams, moisturizers, hand creams, foundations, essences, facial masks, soaps, facial cleansers, cleansing milks, cleansing creams, body lotions or shower gels.

9. The composition according to any one of claims 1 to 5 or the use according to any one of claims 6 to 8, wherein the acetylated resveratrol derivative is prepared by a biotransformation method, wherein the method uses a polar organic solvent as a solvent, a lipase as a catalyst, and vinyl acetate as an acetyl donor to acetylate the substrate resveratrol and / or oxidized resveratrol; Optionally, the ratio of the substrate to the acetyl donor is 1:5 to 1:30, preferably 1:10 to 1:20, more preferably 1:12 to 1:18, most preferably 1:15; Optionally, the reaction duration is 12-96 hours, preferably 24-72 hours, more preferably 24-48 hours; Optionally, the concentration of the lipase is 50-300 mg / ml, preferably 100-200 mg / ml, more preferably 150-200 mg / ml; Optionally, the method further comprises the step of post-processing the product.

10. The composition or use according to claim 9, wherein The lipase is Novozym 435 lipase; Optionally, the solvent is selected from ethyl acetate, methanol, ethanol, 2-methyl-2-butanol or a mixture of one or more thereof; Optionally, the acetylated resveratrol derivative is triacetylated resveratrol or tetraacetylated oxidized resveratrol.

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