1, 3-dibenzyl phenol compound, preparation method and application of 1, 3-dibenzyl phenol compound in whitening and anti-aging cosmetics

Abstract

The invention belongs to the technical field of cosmetics and medicines, and particularly discloses a 1, 3-dibenzyl phenol compound, a preparation method and application of the 1, 3-dibenzyl phenol compound in preparation of whitening and anti-aging cosmetics. The invention designs and synthesizes 4, 4 '-(1, 3-phenylene bis (methylene)) bis 2-methylphenol (compound 2) and 3, 3'-(1, 3-phenylene bis (methylene)) bis 1, 2-benzenediol (compound 3) for the first time on the basis that a diaryl heptene polyphenol compound with alpha-glucosidase inhibitory activity in ottelia acuminata is used as a parent. The invention discloses a compound 1-3 which has remarkable activity in the aspects of inhibiting alpha-glucosidase and scavenging free radicals. The 4, 4 '-(1, 3-phenylene bis (methylene)) diphenol and the compound 2 have remarkable activity in the aspect of inhibiting tyrosinase, and the compound has the cell melanin synthesis inhibition rate higher than that of a classic whitening component phenethyl resorcinol in the market, so that the compound has good development and application prospects in the field of whitening, brightening and anti-aging cosmetics.

Description

Technical Field

[0001] The invention belongs to the technical field of cosmetics and medicines, and particularly relates to 1,3-bisbenzylphenol compounds, a preparation method and application thereof in the preparation of whitening and anti-aging cosmetics. Background Art

[0002] Oxygen free radicals produced during cell metabolism can cause cumulative damage to intracellular biomacromolecules, causing cell aging and loss of proliferation capacity. This imbalance of oxidation and antioxidant systems is considered to be an important cause of skin aging. Tyrosinase, also known as polyphenol oxidase, is an oxidoreductase widely found in animals, plants, microorganisms and the human body. It is the key rate-limiting enzyme in melanin synthesis and is closely related to the occurrence of excessive melanin deposition such as freckles and brown spots on human skin. In recent years, its application in the fields of medicine and beauty has attracted widespread attention at home and abroad. At present, most of the skin whitening and brightening agents on the market are based on the inhibition of tyrosinase, such as α-arbutin, kojic acid, 4-butylresorcinol, etc., or based on antioxidants, such as vitamin C, vitamin E and its derivatives, to achieve whitening effects. α-arbutin is a hydroquinone derivative, which has a cytotoxic effect on melanocytes and a strong irritant to the skin. Long-term use may cause permanent white spots on the skin and has been included in the list of banned ingredients for cosmetics in many countries. Kojic acid inhibits the catalytic activity of tyrosinase by chelating the copper ions of tyrosinase. Its use in commercialized skin and hair lighteners can easily lead to contact allergies, and may also cause skin lesions or liver cancer. Vitamin C and vitamin E are important antioxidants for the human body. They do not directly inhibit tyrosinase, but reduce the colored intermediates in the biosynthesis of melanin, and have only an insufficient whitening and brightening effect on the skin. These unfavorable factors limit their widespread application in the whitening and anti-freckle cosmetics market.

[0003] Earlier, Xu Gang's research group isolated a series of diarylheptanoid polyphenol compounds from the aquatic plant Ottelia acuminata var. acuminata, an edible vegetable of the Bai nationality in Dali, Yunnan. These compounds have significant α-glucosidase inhibitory activity and have potential therapeutic value for diabetes (Liu HX, Ma JZ, Ye YS, Zhao JJ, Wan SJ, Hu XY, Xu G. α-Glucosidase inhibitive diarylheptanoids from Ottelia acuminata var. acuminata, a traditional vegetable of Bai Nationality in Yunnan, Natural Products and Bioprospecting 2022, 12: 22). Studies have shown that tyrosinase is a protein with a sugar chain. During its maturation, its original sugar chain must undergo a series of modification and shearing before it can be converted into mature tyrosinase and exert its normal biological function. α-glucosidase I and II are the key enzymes in the sugar chain modification and shearing process. (Mehta A, Zitzmann N, Rudd PM, Block TM, Dwek RA.α-Glucosidase inhibitors as potential broad based anti-viral agents, FEBS Letters, 1998, 430(1): 17-22). When α-glucosidase is inhibited, the sugar chain modification on the glycoprotein is blocked, and active tyrosinase cannot be produced, and the formation of melanin is correspondingly reduced. (Takahashi H, Parsons PG, Rapid and reversible inhibition of tyrosinase activity by glucosidase inhibitors in human melanoma cells, the Journal of Investigative Dermatology, 1992, 98(4): 481-487.). Therefore, inhibiting α-glucosidase to reduce the maturation of tyrosinase, so as to achieve the purpose of reducing the formation of melanin and skin pigmentation, is a new possible way to whiten and remove spots. Considering that the content of diarylheptene polyphenol compounds from natural sources is low, their structural characteristics are cis- and trans-olefin structures, their molecular stability is poor, they are easily isomerized by double bond shift through ene reactions, they are difficult to synthesize, and their molecules are relatively flexible.To solve the drug source problem, we used this type of natural product as a lead compound and as a template, and used conformational restriction and bio-isosteric replacement strategies to simplify and replace its open-chain flexible molecular structure with a rigid benzene ring polyphenol structure analogue. At the same time, in order to improve the antioxidant activity, we added the phenolic hydroxyl group on the benzene ring to synthesize a series of 1,3-bis-benzylphenol compounds (compounds 1-3), and studied their tyrosinase inhibitory activity, α-glucosidase inhibitory activity, antioxidant activity and inhibitory activity of cell melanogenesis. Among them, although compound 1 is a known compound (Larry Q. Reyes, Salumeh Issazadeh, Jane Zhang, Buu Dao, and Russell J. Varley, Synthesis of Tri-Aryl Methane Epoxy Resin Isomers and Their Cure with Aromatic Amines, Macromolecular Materials and Engineering, Macromol. Mater. Eng. 2020, 305 (2), 1900546.), there is no report on its biological activity. The antioxidant activity, tyrosinase inhibitory activity, and α-glucosidase inhibitory activity of compounds 1, 2, and 3 were discovered for the first time, and compounds 2 and 3 are new compounds. This type of ingredient has a higher cell melanin synthesis inhibition rate than the classic whitening ingredient phenylethyl resorcinol in the market, and has a good application prospect in whitening, anti-aging and diabetes treatment. Summary of the invention

[0004] In view of the deficiencies in the prior art, the technical problem to be solved by the present invention is to provide a 1,3-bisbenzylphenol compound, a preparation method and an application thereof in the preparation of whitening and anti-aging cosmetics.

[0005] In order to solve the technical problem of the present invention, the present invention provides the following technical solutions:

[0006] The first aspect of the technical solution of the present invention is to provide a 1,3-bis-benzylphenol compound, the structural formula of which is shown in the structural formula of the following compound 2 and compound 3.

[0007]

[0008] The compound 2 is named 4,4'-(1,3-phenylenebis(methylene))bis-2-methylphenol.

[0009] The compound 3 is named 3,3'-(1,3-phenylenebis(methylene))bis-1,2-benzenediol.

[0010] The second aspect of the technical solution of the present invention is to provide a preparation method for the above-mentioned 1,3-bisbenzylphenol compounds, which comprises the following steps: 1,3-di(bromomethyl)benzene or 1,3-di(chloromethyl)benzene is mixed with o-cresol or 1,2-diphenol in a molar ratio of 1:(5-10), aluminum chloride is used as a catalyst, and under nitrogen protection, the mixture is heated at 100-120°C for a period of time (preferably, the mixture is heated at 110°C for 2h), and then purified by column chromatography to obtain the obtained mixture.

[0011] The third aspect of the technical solution of the present invention is to provide the application of the above-mentioned 1,3-bis-benzylphenol compounds. Specifically:

[0012] Application of the above 1,3-bisbenzylphenol compounds in the preparation of α-glucosidase inhibitors.

[0013] Application of the above 1,3-bisbenzylphenol compounds in the preparation of drugs for treating diabetes.

[0014] The above-mentioned 1,3-bisbenzylphenol compounds are used in the preparation of anti-skin oxidation / aging products, and the anti-oxidation mainly refers to the removal of intracellular active oxygen free radicals.

[0015] The use of the above 1,3-bisbenzylphenol compounds in the preparation of skin care products or skin disease treatment drugs for reducing melanin production, preventing or treating pigmentation diseases. The pigmentation diseases are preferably freckles, chloasma, stretch marks, age spots or melanoma.

[0016] In particular, the use of the above compound 2 in the preparation of tyrosinase inhibitors.

[0017] The fourth aspect of the technical solution of the present invention is to provide the application of compound 1. Specifically:

[0018] Application of compound 1 in the preparation of tyrosinase inhibitors.

[0019] Application of compound 1 in the preparation of α-glucosidase inhibitors.

[0020] Application of compound 1 in the preparation of drugs for treating diabetes.

[0021] The application of compound 1 in the preparation of anti-skin oxidation / aging products, wherein the anti-oxidation mainly refers to the removal of intracellular active oxygen free radicals.

[0022] The use of compound 1 in the preparation of skin care products or skin disease treatment drugs for reducing melanin production, preventing or treating pigmentation diseases. The pigmentation diseases are preferably freckles, chloasma, stretch marks, age spots and / or melanoma.

[0023] The compound 1 is named 4,4'-(1,3-phenylenebis(methylene))diphenol;

[0024] The compound 1 has the following structural formula:

[0025]

[0026] The fifth aspect of the technical solution of the present invention is to provide a whitening and / or anti-skin oxidation / aging skin care composition or pharmaceutical composition, comprising an effective amount of at least one of the compounds 1, 2, and 3 shown in the structural formula as an active ingredient, and one or more acceptable carriers in the cosmetic field or the pharmaceutical field.

[0027] The pharmaceutical composition mentioned above refers to a pharmaceutical composition for treating skin diseases.

[0028] The skin care composition or skin disease treatment pharmaceutical composition is related to whitening and / or anti-skin oxidation / aging effects. Whitening refers to inhibiting tyrosinase activity, and / or inhibiting α-glucosidase activity, and / or inhibiting cellular melanin production and pigmentation; anti-skin oxidation / aging refers to removing active oxygen free radicals in cells, especially those generated by ultraviolet light.

[0029] The sixth aspect of the technical solution of the present invention is to provide a skin care composition or a pharmaceutical composition comprising an effective amount of at least one of Compound 1, Compound 2, and Compound 3 as an active ingredient for use in the preparation of whitening and / or anti-aging skin care products or skin disease treatment drugs.

[0030] Compound 1, Compound 2 and Compound 3 have good skin lightening and whitening effects (i.e., for example, they have strong α-glucosidase and / or tyrosinase inhibitory effects in a cell in vitro test system, reducing the production of cellular melanin); they also have good antioxidant effects (i.e., for example, they have strong free radical scavenging effects in a cell in vitro test system); specifically, Compounds 1 and 2 have high tyrosinase inhibitory activity, α-glucosidase inhibitory activity and antioxidant activity, and also have a significantly higher cell melanin synthesis inhibition rate than the classic whitening ingredient phenethylresorcinol on the market; Compound 3 has extremely excellent α-glucosidase inhibitory activity and antioxidant activity, and also has a slightly better cell melanin synthesis inhibition rate than the classic whitening ingredient phenethylresorcinol on the market. Compounds 1, Compound 2 and Compound 3 can be prepared in a highly pure form; they are acceptable to dermatology and toxicology; they also show good stability to light effects, and therefore can be preferably used as an effective ingredient for whitening and anti-aging cosmetics.

[0031] The skin care products or skin disease treatment drugs are related to whitening and / or anti-skin oxidation / aging effects. Whitening refers to inhibiting tyrosinase activity, and / or inhibiting α-glucosidase activity, and / or inhibiting cell melanin production and pigmentation; anti-skin oxidation / aging refers to removing active oxygen free radicals in cells, especially those generated by ultraviolet light.

[0032] The skin care products or dermatological treatments described herein can improve the aesthetic and / or cosmetic appearance of the skin. These improvements can be manifested in any of the following ways: reduction of dermatological aging symptoms, which are attributed to, for example, chronological aging, hormonal aging and / or photoaging; reduction of skin fragility; reduction of pore size; prevention and / or reversal of collagen and / or elastin loss; improvement of the effects of estrogen imbalance; prevention of skin atrophy; prevention and / or reduction of the appearance and / or depth of lines and / or wrinkles, including fine lines and / or wrinkles; prevention, reduction and / or treatment of hyperpigmentation; improvement of liver color, skin clarity and / or firmness; prevention, reduction and improvement of skin sagging; promotion of antioxidant activity; improvement of skin toughness, plumpness, elasticity, suppleness and / or softness; increase of procollagen and / or collagen production; improvement of skin texture and / or promotion of restoration of original texture state; promotion of skin barrier repair and / or function; improvement of skin contour appearance; restoration of skin radiance and / or brightness; minimization of fatigue and stress response The invention can be used to improve the skin's dermatological symptoms, such as skin eruptions and / or resistance to stress reactions caused by environmental influences (pollution, temperature changes); replenish the essential nutrients and / or components in the skin that are reduced due to aging and / or menopause; enhance the connection between skin cells; increase cell proliferation and / or multiplication; enhance the skin cell metabolism that is reduced due to aging and / or menopause; delay cell aging; inhibit enzymes in the skin that accelerate skin cell aging; minimize skin dryness and / or increase skin moisture; minimize skin discoloration, including dark circles; promote and / or accelerate cell renewal; increase skin thickness; increase skin elasticity and / or elasticity; increase epidermal exfoliation with or without the help of alpha-hydroxy acids or other peeling agents; prevent and reverse the loss of glycosaminoglycans (GAGs), collagen and / or elastin; improve microcirculation; reduce and / or prevent the formation of fat masses; reduce acne formation; can be used as skin and hair lighteners or anti-age spot agents. In particular, the pharmaceutical composition of the present invention can improve the aesthetic appearance, health and vitality of the skin. Such an improvement can be manifested in at least one of: reduced melanin production, treatment of pigmentation disorders, preferably freckles, melasma, stretch marks, age spots or melanoma; prevention and / or reversal of collagen and / or elastin loss; improved skin texture; improved skin tone, clarity, and / or firmness; promotion / acceleration of cell renewal; and increased skin thickness.

[0033] The seventh aspect of the technical solution of the present invention is to provide a pharmaceutical composition for treating diabetes, which includes an effective amount of at least one of Compound 1, Compound 2, and Compound 3 as an active ingredient, and one or more acceptable carriers in the pharmaceutical field.

[0034] The diabetes therapeutic drug exerts its effect by inhibiting the activity of α-glucosidase.

[0035] The eighth aspect of the technical solution of the present invention is to provide a pharmaceutical composition comprising an effective amount of at least one of the compounds 1, 2 and 3 as an active ingredient, for use in the preparation of a drug for treating diabetes.

[0036] The acceptable carriers in the cosmetic field mentioned above refer to conventional cosmetic carriers in the cosmetic field, and these carriers include emulsifiers (such as oil-in-water, water-in-oil, water-in-silicone oil, silicone oil-in-water, water-in-oil-in-water, oil-in-water-in-oil, silicone oil-in-water-in-oil, etc.), creams, lotions, liquids (such as aqueous solutions or water-alcohol solutions), anhydrous bases (such as lipsticks or powders, etc.), gels, ointments, milks, creams, sprays, solids, eye creams, etc. The composition of the present invention can be made into various forms of cosmetics, including sunscreen, sunless tanning products, hair care products (such as shampoo, conditioner, hair dye, bleach, hair straightener and perm), nail polish, moisturizing cream, skin lotion and skin cream, lipstick and lip balm, cleanser, toner, facial mask, deodorant, antiperspirant, keratin exfoliant, shaving products (cream, aftershave), wet wipes, tanning lotion, bath gel, body oil, foot care products (powder, spray), foundation cream, rouge, eye shadow and eyeliner, lip gloss, mascara, baby products (baby skin cream, body oil, shampoo, talcum powder, wet wipes). In addition, these cosmetics can also be used as stay-on cosmetics or makeup remover creams.

[0037] The acceptable carriers in the pharmaceutical field mentioned above include one or more anesthetics, antiallergens, antifungals, antimicrobials, anti-inflammatory agents, antioxidants, preservatives, chelating agents, colorants, decolorizing agents, softeners, emulsifiers, exfoliating agents, film-forming agents, fragrances, wetting agents, insect repellents, lubricants, moisturizing agents, pharmaceutical agents, light stabilizers, preservatives, skin protectants, skin penetration enhancers, sunscreens, stabilizers, surfactants, thickeners, viscosity regulators, vitamins, or any combination thereof.

[0038] The present invention has the following advantages and beneficial effects:

[0039] 1. Based on the diarylheptene polyphenol compounds with α-glucosidase inhibitory activity in sea cauliflower, a series of 1,3-bisbenzylphenol compounds were designed and synthesized for the first time: 4,4'-(1,3-phenylenebis(methylene))bis-2-methylphenol (compound 2), 3,3'-(1,3-phenylenebis(methylene))bis-1,2-benzenediol (compound 3).

[0040] 2. It was disclosed for the first time that 1,3-bisbenzylphenol compounds 1-3 have significant activity in inhibiting α-glucosidase and scavenging free radicals, compounds 1 and 2 have significant activity in inhibiting tyrosinase, and compounds 1-3 have a higher cell melanin synthesis inhibition rate than the classic whitening ingredient phenethyl resorcinol on the market.

[0041] 3. Compounds 1-3 reported in the present invention have simple synthesis steps, high yield, low manufacturing cost, good safety and stability, and have good development and application prospects in the field of whitening, brightening and anti-aging cosmetics. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 :Effects of three compounds and positive drug kojic acid on tyrosinase activity.

[0043] Figure 2 : Inhibitory effects of three compounds and positive acarbose on α-glucosidase activity.

[0044] Figure 3 : The scavenging effects of three compounds and positive drug vitamin E on ABTS free radicals.

[0045] Figure 4 :Effects of three compounds and positive drug phenethylresorcinol on B16 cell activity.

[0046] Figure 5 : Effects of compounds 1-3 and phenethylresorcinol on melanin synthesis in B16 cells.

[0047] Figure 6 :Compound 1 1 H NMR spectrum (600 MHz, CD3OD).

[0048] Figure 7 :Compound 1 13 C NMR spectrum (150 MHz, DMSO-d6).

[0049] Figure 8 :Compound 2 1 H NMR spectrum (600 MHz, DMSO-d6).

[0050] Fig. 9 :Compound 213 C NMR spectrum (150 MHz, DMSO-d6).

[0051] Fig.10 :Compound 3 1 H NMR spectrum (500 MHz, DMSO-d6).

[0052] Fig.11 :Compound 3 13 C NMR spectrum (125 MHz, DMSO-d6). DETAILED DESCRIPTION

[0053] The following specific examples can further illustrate the present invention, but do not limit the scope of protection requested by the claims of the present invention in any way.

[0054] Example 1: Synthesis of Compound 1-3

[0055] 1. Synthesis of compound 1: 1,3-di(bromomethyl)benzene (1.5 g, 5.73 mmol), phenol (6.2 mL, 57.3 mmol), and aluminum chloride (195 mg, 1.5 mmol) were weighed in sequence, heated at 110° C. under nitrogen protection until the raw materials disappeared, reacted for 2 hours, cooled to room temperature, extracted with saturated sodium carbonate aqueous solution and ethyl acetate three times each, then dried over sodium sulfate, filtered and dried by suction, passed through a silica gel column, and purified with ethyl acetate / petroleum ether (EA:PE=1:200, v / v) to obtain the product compound 1 (473.3 mg, yield 42%, white solid).

[0056]

[0057] 4,4'-(1,3-phenylenebis(methylene))diphenol (Compound 1): white solid, 1 H NMR (600MHz, CD3OD, δ, ppm, J / Hz): 9.17 (s, 2H), 7.15 (t, J = 10, 1H), 7.04 (s, 1H), 6.98-6.95 (m, 6H), 6.66-6.64 (m, 4H), 3.76 (s, 4H). 13 C NMR (150MHz, DMSO-d6, δ, ppm): 155.9, 142.4, 131.8, 130.0, 129.3, 128.8, 126.5, 115.6, 40.7. HRMS(ESI-)calcd for C 20 H 17 O2[MH]-=289.1234, found 289.1246. 1 H NMR spectrum, 13C NMR spectrum Figure 6-7 shown.

[0058] 2. Synthesis of compound 2: Weigh 1,3-di(bromomethyl)benzene (0.3 g, 1.15 mmol), o-cresol (0.6 g, 5.75 mmol), and aluminum chloride (39 mg, 0.29 mmol) in sequence, and heat to 110°C under nitrogen protection until the raw materials disappear. React for 2 hours, cool to room temperature, and directly pass through a silica gel column chromatography with ethyl acetate / petroleum ether (EA:PE=1:7.5, v / v) as eluent to obtain the product compound 2 (40.7 mg, yield 26%, brown solid).

[0059]

[0060] 4,4'-(1,3-phenylenebis(methylene))bis(2-methylphenol) (Compound 2): brown solid, 1 H NMR (600MHz, DMSO-d6, δ, ppm, J / Hz): 9.05 (s, 2H), 7.15 (t, J = 7.6Hz, 1H), 7.05 (s, 1H), 6.97 (d, J = 7. 6Hz, 2H), 6.87 (s, 2H), 6.80 (dd, J = 8.2, 2.3Hz, 2H), 6.67 (d, J = 8.1Hz, 2H), 3.73 (s, 4H), 2.06 (s, 6H). 13 C NMR(150MHz,DMSO-d6,δ,ppm):153.5,142.0,131.3,130.9,128.8,128.3,126.7,126.0,123.6,114.5,40.4,16.0.HRMS(ESI-)calcd for C 22 H 21 O2[MH]-=317.1547, found 317.1561. 1 H NMR spectrum, 13 C NMR spectrum Figure 8-9 shown.

[0061] 3. Synthesis of compound 3: Weigh 1,3-di(bromomethyl)benzene (1.5 g, 5.73 mmol), 1,2-benzenediol (6.3 g, 57.2 mmol), and aluminum chloride (195 mg, 1.5 mmol) in sequence, and heat to 110°C under nitrogen protection until the raw materials disappear. React for 2 hours, cool to room temperature, and directly pass through a silica gel column chromatography with ethyl acetate / petroleum ether (EA:PE=1:2, v / v) as eluent to obtain the product compound 3 (124.8 mg, yield 32%, light yellow solid).

[0062]

[0063] 3,3'-(1,3-phenylenebis(methylene))bis-1,2-benzenediol (Compound 3): pale yellow solid, 1 H NMR (500MHz, DMSO-d6, δ, ppm, J / Hz): 8.74 (s, 2H), 8.64 (s, 2H), 7.15 (t, J = 7.6Hz, 1H), 7.01 (s, 1H), 6.95 (dd ,J=7.6,1.7Hz,2H),6.61(d,J=8.0Hz,2H),6.53(d,J=2.1Hz,2H),6.44(dd,J=8.0,2.1Hz,2H),3.69(s,4H). 13 C NMR(125MHz,DMSO-d6,δ,ppm):145.5,143.9,142.4,132.5,129.4,128.7,126.6,119.8,116.5,115.9,41.0.HRMS(ESI)calcd for C 20 H 18 NaO4[M+Na] + =345.1097, found 345.1097. 1 HNMR spectrum, 13 C NMR spectrum Figure 10-11 shown.

[0064] Example 2: Study on the inhibition of tyrosinase activity by compounds 1-3

[0065] 1. Solution preparation

[0066] Phosphate buffer (0.2M, pH 6.8): Accurately weigh 2.84 g of Na2HPO4 and 2.4 g of NaH2PO4, dissolve them in purified water to 100 mL respectively, mix them in equal volumes and adjust the pH to 6.8.

[0067] L-DOPA solution: Accurately weigh 3.95 mg of L-DOPA and dissolve it in 10 mL of phosphate buffer to prepare a 2 mM L-DOPA solution.

[0068] Tyrosinase solution: Accurately weigh 1 mg of tyrosinase (enzyme activity of 500 U / mg) and dissolve it in 5 mL of phosphate buffer (0.2 M, pH 6.8) to prepare a tyrosinase solution with an enzyme activity of 100 U / mL.

[0069] Sample working solution: accurately weigh 10 mg of compound 1-3 and kojic acid respectively and place them in 500 μL ultrapure water, then slowly add 1M NaOH until the solution is clear and transparent, and finally make up to 1 mL with purified water to prepare a 10 mg / mL stock solution; then use the above-mentioned phosphate buffer to dilute the test compounds and the positive control drug kojic acid to the concentrations of 31.2, 62.5, 125, 250.00 and 500.00 μg / mL of sample working solution.

[0070] 2. Tyrosinase activity assay

[0071] Four test groups were set up: sample group A1, sample negative control group A2, enzyme standard group B1 and enzyme negative control group B2; 3 parallels were set for each sample, and a 200 μL reaction system was prepared according to Table 1. The corresponding volume of phosphate buffer, sample solutions of various concentrations to be tested (final concentrations of 3.12, 6.25, 12.5, 25 and 50 μg / mL), tyrosinase solution (final concentration of 20 U / mL) and reaction substrate L-DOPA (final concentration of 1.2 mM) were added to the 96-well plate in sequence, incubated in a microplate constant temperature oscillator at 37°C for 30 minutes, and then the absorbance of each test group at 475 nm was measured in a multifunctional microplate reader (SPARK 10M, TECAN). The inhibition rate of the test compound on tyrosinase was calculated as follows:

[0072] Tyrosinase inhibition rate (%) = [1-(A1-A2) / (B1-B2)] × 100

[0073] Table 1 Tyrosinase activity inhibition test reaction system preparation (volume / μL)

[0074]

[0075] like Figure 1 As shown in the figure, compound 1, compound 2 and the positive control drug kojic acid all showed a dose-dependent inhibitory effect on tyrosinase activity, and compound 2 had the best inhibitory effect. The half-inhibitory concentration IC of compound 1, compound 2 and kojic acid on tyrosinase was calculated by nonlinear fitting using Graphpad Prism8.0 software. 50 The concentrations of compound 3 and compound 3 were 10.43 μg / mL (35.96 μM), 4.36 μg / mL (13.68 μM) and 18.61 μg / mL (131.6 μM), respectively. 50 >50μg / mL). The results showed that compounds 1 and 2 had good inhibitory activity against tyrosinase, and their tyrosinase inhibitory effects were 3.7 times and 9.6 times that of kojic acid, respectively, and they are expected to be used as browning inhibitors for skin whitening and brightening.

[0076] Example 3: Study on the inhibition of α-glucosidase activity by compounds 1-3

[0077] 1. Solution preparation

[0078] Phosphate buffer (0.2M, pH 6.8): Accurately weigh 2.84 g of Na2HPO4 and 2.4 g of NaH2PO4, dissolve them in purified water to 100 mL respectively, mix equal volumes of the two and adjust the pH to 6.8.

[0079] α-Glucosidase solution: α-glucosidase powder from Saccharomyces cerevisiae was prepared into 1U / mL α-glucosidase solution with phosphate buffer (0.2M, pH 6.8) and stored at -20℃.

[0080] Substrate PNPG solution: Use an analytical balance to accurately weigh 211 mg of 4-nitrobenzene-α-D-pyranoglucoside (PNPG), add 70 mL of the above phosphate buffer to dissolve evenly, prepare a 10 mM substrate storage solution, and store at -20°C away from light.

[0081] Positive control working solution: Acarbose was selected as the positive control in this experiment. 103.3 mg of acarbose powder was accurately weighed and fully dissolved and mixed with 1 mL of the above phosphate buffer to prepare a 160 mM acarbose stock solution. The stock solution was then diluted with the above phosphate buffer to a positive control working solution with concentrations of 2.5, 5, 10, 20, 40, 80 and 160 mM.

[0082] Test compound working solution: accurately weigh 2.90 mg of compound 1, 3.19 mg of compound 2 and 3.23 mg of compound 3, respectively, and place them in 500 μL of ultrapure water, slowly add 1M NaOH until the solution is clear and transparent, and finally make up to 1 mL with purified water to prepare a 10 mM compound stock solution. Then dilute it with the above phosphate buffer to a concentration of 0.062, 0.125, 0.25, 0.5, 1 and 2 mM for compound 1 working solution, 0.025, 0.05, 0.1, 0.2, 0.4 and 0.8 mM for compound 2 working solution, and 3.12, 6.25, 12.5, 25, 50 and 100 μM for compound 3 working solution.

[0083] 2. α-glucosidase activity assay

[0084] Four test groups were set up: blank group A, enzyme standard group B, sample negative control group C, sample group D; 3 parallels were set for each sample. According to Table 2, a 600 μL reaction system was prepared, and the corresponding volume of phosphate buffer, working solution of each concentration of the sample to be tested (the final concentration was 20 times diluted by the working solution of the test compound), α-glucosidase solution (final concentration 0.005U / mL) and reaction substrate PNPG (final concentration 0.5mM) were added to the 96-well plate in turn, and mixed thoroughly. After the reaction system was incubated at 37°C for 50 minutes, 60 μL of 0.1mol / LNa2CO3 solution was added to terminate the reaction. The 96-well plate to be tested was placed in a multifunctional microplate reader (SPARK 10M, TECAN) to measure the absorbance of each test group at 405nm. The inhibition rate of α-glucosidase was calculated as follows:

[0085] α-glucosidase inhibition rate (%) = [1-(DC) / (BA)] × 100

[0086] Table 2 α-glucosidase activity inhibition test reaction system preparation (volume / μL)

[0087]

[0088] like Figure 2 As shown, the three compounds and the positive control drug acarbose all had a dose-dependent inhibitory effect on α-glucosidase activity. The half-inhibitory concentration IC of compound 1, compound 2, compound 3 and acarbose on α-glucosidase was calculated by nonlinear fitting using Graphpad Prism 8.0 software. 50 The above experimental results show that all three compounds have good inhibitory activity against α-glucosidase, and the inhibitory effect is significantly better than that of the positive control drug acarbose. It is expected that the three compounds can reduce the production of mature active tyrosinase by effectively inhibiting α-glucosidase, thereby achieving the purpose of reducing skin pigmentation.

[0089] Example 4: Study on the Antioxidant Activity of Compounds 1-3

[0090] Mix equal volumes of 7.4mmol / L ABTS solution and 2.6mmol / L K2S2O8 solution and keep them at room temperature in the dark for 16h to prepare ABTS free radical stock solution. Dilute the ABTS free radical stock solution with phosphate buffer solution (pH 7.4, 10mmol / L) so that its absorbance at a wavelength of 734nm reaches 0.70±0.05 to prepare ABTS working solution. Take 200μL ABTS working solution and mix it with 10μL phosphate buffer solution (pH7.4, 10mmol / L), and measure the absorbance at 734nm as A0. Accurately weigh 10mg of compound 1 and dissolve it in 500μL purified water, slowly add 1mol / L NaOH until the solution is clear and transparent, and finally make up to 1mL with purified water to prepare a 10mg / mL mother solution. Then dilute the mother solution to be tested to the corresponding concentration with phosphate buffer solution (pH7.4, 10mmol / L). The positive control drug Trolox (water-soluble vitamin E) was directly prepared into a 10 mg / mL stock solution with pure water and then diluted with water to the corresponding concentration. 10 μL of sample solutions of different concentrations were mixed with 200 μL of ABTS working solution to make the final concentration of the positive drug vitamin E 0.9, 1.79, 3.58, 7.15, 10.73, 14.30, 25 and 50 μg / mL, the final concentration of compound 1 was 0.62, 1.25, 2.50, 5.00, 10, 20, 40 and 50 μg / mL, and the final concentration of compound 2 and compound 3 was 0.78, 1.56, 3.12, 6.25, 12.5, 25 and 50 μg / mL. The mixed sample solution was allowed to stand at room temperature for 10 minutes, and the absorbance (Ai) was measured at a wavelength of 734 nm. At the same time, 10 μL of the sample solution of the corresponding concentration was mixed with 200 μL of 10 mmol / L pH 7.4 phosphate buffer solution, and the background absorbance Aj at a wavelength of 734 nm was measured. Three parallels were set for each sample concentration. The formula for calculating the scavenging rate of the sample for ABTS free radicals is as follows:

[0091] ABTS free radical scavenging rate (%) = [1-(Ai-Aj) / A0] × 100

[0092] like Figure 3 As shown in the figure, within the test concentration range, the tested compounds all showed good scavenging effects on ABTS free radicals. Graphpad Prism 8.0 software was used for nonlinear fitting to calculate the IC values ​​of compounds 1-3 for scavenging ABTS free radicals. 50 The half maximal inhibitory concentrations were 6.78 μg / mL (i.e. 23.22 μM), 2.56 μg / mL (i.e. 8.02 μM) and 2.10 μg / mL (i.e. 6.49 μM), respectively. The IC of the positive control drug vitamin E50 The value is 4.75μg / mL (i.e. 11.05μM). The above experimental results show that compounds 1-3 have good free radical scavenging ability, among which compounds 2 and 3 have better antioxidant activity than the positive control drug vitamin E, which is expected to help cells resist oxidative stress damage and delay skin aging.

[0093] Table 3 IC50 of the related activities of the three compounds 50 value

[0094]

[0095] Example 5: Cellular activity test of compounds 1-3

[0096] Treatment of test samples:

[0097] Sample group: Compounds 1-3 and phenethyl resorcinol were dissolved in DMSO and diluted with 1640 culture medium to a mother solution with a concentration of 38.6 μmol / L. The mother solution was then diluted with 1640 culture medium to a series of concentration samples of 19.3 μmol / L, 9.65 μmol / L, 4.825 μmol / L, and 2.413 μmol / L for use.

[0098] Negative control group: 1640 basal culture medium.

[0099] Cell viability test:

[0100] B16 cells were plated in 96-well plates. After 24 hours, the culture medium was discarded and basal culture medium containing different concentrations of test samples was added. After 24 hours, OD490 nm was detected by MTT method, and the effect of test samples on B16 cell activity was analyzed by t-test.

[0101] The statistical analysis software was SPSS, and the comparison between the test sample and the negative control was performed using the independent sample t test. The above statistical analyses were all two-tailed tests, with a significance level of α = 0.05. *P < 0.05, * indicates that the test sample has a significant difference compared with the negative control group at this concentration. The experimental results are as follows Figure 4 .

[0102] Depend on Figure 4 It can be seen that when the concentration of the three compounds was 19.3 μmol / L, the cell activity was >90% and there was no cytotoxicity.

[0103] Example 6: Compound 1-3 cell melanin synthesis inhibition experiment

[0104] 19.3μmol / L compound 1-3 and phenethyl resorcinol were used as experimental samples, and 1640 basal medium was used as negative control. B16 cells were plated on a 6-well plate. After 24 hours of culture, the 1640 basal medium containing different concentrations of test samples was replaced. After two changes of medium, the cells were washed twice with PBS, and 200μL of 0.25% trypsin was added to each well to digest the cells. The cells were collected in a centrifuge tube and centrifuged for 5 minutes. 200μL of 1M NaOH containing 10% DMSO was added to each tube to lyse the cells, shaken evenly, and transferred to a 96-well plate. The absorbance at 405nm of each well was detected by an ELISA instrument. The formula for calculating the inhibition rate of cell melanin synthesis is:

[0105]

[0106] Where: T—absorbance of test sample well;

[0107] C—average of three absorbance values ​​of the negative control group;

[0108] The statistical analysis software was SPSS, and the comparison between the test sample and the negative control was performed using the independent sample t test. The above statistical analyses were all two-tailed tests, and the significance level was α = 0.05. *P < 0.05; **P < 0.01, * indicates that the test sample has a significant difference compared with the negative control group at this concentration. The results are shown in Figure 5 .

[0109] Depend on Figure 5 It can be seen that the inhibition rates of 19.3 μmol / L of compound 1-3 and phenethyl resorcinol on B16 cell melanin synthesis are 52.230%, 40.881%, 34.798% and 32.791%, respectively, which have a significant effect on inhibiting cell melanin synthesis (P<0.05), and the inhibition rate of compound 1-3 on B16 cell melanin synthesis is significantly higher than that of phenethyl resorcinol, a classic whitening ingredient on the market, indicating that compound 1-3 has excellent whitening effect.

Claims

1. A 1,3-bis-benzylphenol compound, the structural formula of which is shown in the structural formula of the following compound 2 or compound 3:

2. The method for preparing the 1,3-bisbenzylphenol compound according to claim 1, It is characterized in that The preparation method comprises the following steps: 1,3-di(bromomethyl)benzene or 1,3-di(chloromethyl)benzene is mixed with o-cresol or 1,2-diphenol in a substance ratio of 1:(5-10), aluminum chloride is used as a catalyst, and the mixture is heated at 100-120°C for a period of time under nitrogen protection, and then purified by column chromatography to obtain the product.

3. Use of the 1,3-bisbenzylphenol compound according to claim 1 in the preparation of α-glucosidase inhibitors or drugs for treating diabetes, Application in the preparation of anti-skin oxidation and / or aging products, Application of the invention in the preparation of skin care products or skin disease treatment drugs for reducing melanin production and Application in the preparation of skin care products or skin disease treatment drugs for preventing or treating pigmentation diseases.

4. Use of the compound 2 named 4,4'-(1,3-phenylenebis(methylene))bis-2-methylphenol in claim 1 in the preparation of tyrosinase inhibitors.

5. Use of compound 1 in the preparation of tyrosinase inhibitors or α-glucosidase inhibitors or drugs for treating diabetes, Application in the preparation of anti-skin oxidation and / or aging products, The use of compound 1 in the preparation of a skin care product or a skin disease treatment drug for reducing melanin production and in the preparation of a skin care product or a skin disease treatment drug for preventing or treating pigmentation diseases, the structural formula of the compound 1 is as follows:

6. A skin care product composition or pharmaceutical composition for whitening and / or anti-skin oxidation / aging, It is characterized in that Comprising an effective amount of compound 1 as an active ingredient Compound 2 Compound 3 At least one of, and one or more acceptable carriers in the field of cosmetics.

7. Comprising an effective amount of compound 1 as an active ingredient Compound 2 Compound 3 Use of at least one of the skin care composition or pharmaceutical composition in the preparation of whitening and / or anti-aging skin care products or skin disease treatment drugs.

8. A drug composition for treating diabetes, It is characterized in that Comprising an effective amount of compound 1 as an active ingredient Compound 2 Compound 3 At least one of the following, and one or more acceptable carriers in the pharmaceutical field.

9. Comprising an effective amount of compound 1 as an active ingredient Compound 2 Compound 3 Use of at least one of the pharmaceutical compositions in the preparation of drugs for treating diabetes.

10. The whitening and / or anti-skin oxidation / aging skin care composition or pharmaceutical composition according to claim 6, It is characterized in that Whitening refers to inhibiting tyrosinase activity, and / or inhibiting α-glucosidase activity, and / or inhibiting cellular melanin production and pigmentation; anti-skin oxidation / aging refers to removing active oxygen free radicals in cells.

11. The use according to claim 7, It is characterized in that Whitening refers to inhibiting tyrosinase activity, and / or inhibiting α-glucosidase activity, and / or inhibiting cellular melanin production and pigmentation; anti-skin oxidation / aging refers to removing active oxygen free radicals in cells.

12. The use according to claim 3 or 5 or 11 or the composition according to claim 10, It is characterized in that The pigmentation disease is freckles, chloasma, stretch marks, age spots and / or melanoma.

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