Preparation method of naringenin-7, 4 '-di-O-beta-D-glucopyranoside and application of naringenin-7, 4'-di-O-beta-D-glucopyranoside in cosmetics

Abstract

The invention relates to the technical field of biology, in particular to a preparation method of naringenin-7, 4 '-di-O-beta-D-glucopyranoside and application of the naringenin-7, 4'-di-O-beta-D-glucopyranoside in cosmetics, the structural formula of the naringenin-7, 4 '-di-O-beta-D-glucopyranoside is as follows: # imgabs0, and the naringenin derivative is prepared by taking naringenin as a raw material. The obtained product is high in purity and yield, meanwhile, the effect of the product is not transmitted to naringenin, and the product has excellent water solubility, bioavailability and cell affinity.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and more specifically, to a method for preparing naringenin-7,4'-di-O-β-D-pyranoglucoside and its application in cosmetics. Background Art Naringenin, as a natural organic compound, is mainly derived from natural resources such as the seed coat of the cashew nut of the Anacardiaceae family. In recent years, thanks to its wide range of pharmacological effects (antibacterial, anti-inflammatory, antioxidant, anti-tumor) and biological activities, it has been widely used in medicine, food, cosmetics and other fields, mainly as a drug precursor or auxiliary ingredient.

[0002] Related technologies also apply naringenin to cosmetics. Leveraging its antioxidant and anti-aging properties, it can be used in the preparation of anti-aging skin care products, sunscreens, and other products. By scavenging free radicals in the body, naringenin can protect cells from oxidative damage, thereby delaying the aging process. This is of great value in improving skin condition and reducing damage to the skin caused by the external environment. However, previous research has primarily focused on the extraction, purification, and pharmacological activity of naringenin. Problems inherent in naringenin, such as poor water solubility and low bioavailability, have not been effectively addressed, significantly limiting its potential application in cosmetics. Therefore, this paper provides a method for preparing naringenin-7,4'-di-O-β-D-pyranoglucopyranoside and its application in cosmetics. Summary of the Invention

[0003] To solve the above technical problems, the present invention provides a preparation method of naringenin-7,4'-di-O-β-D-pyranoglucoside and its application in cosmetics. By glycosidating naringenin, naringenin glycoside compounds can be formed. These compounds not only inherit the original biological activity and pharmacological effects of naringenin, but also have higher bioavailability and water solubility.

[0004] In a first aspect, the present application provides a method for preparing naringenin-7,4'-di-O-β-D-pyranoglucoside using naringenin as a raw material, comprising the following steps: S1. First, naringenin and 2,3,4,6-tetra-O-acetyl-β-D-pyranose acyl-2,2,2-trichloroimidoacetate were added to a solvent at room temperature to dissolve, and then trimethylsilyl trifluoromethanesulfonate was added dropwise at -20 to -15°C and the reaction was continued for 10 to 24 hours to obtain a reaction solution; S2. The reaction solution obtained in S1 was added dropwise to a sodium bicarbonate aqueous solution and stirred for 5-30 minutes. The mixture was then separated and the organic phase was washed with sodium chloride. After concentration to dryness, the organic phase was dissolved in dichloromethane and purified by normal phase preparation to obtain impurities, naringenin-4'-O-β-D-pyranoglucoside (OAc) and naringenin-7,4'-di-O-β-D-pyranoglucoside (OAc).

[0005] Preferably, the process further comprises a purification step of the product obtained in S2, specifically as follows: S3. Add naringenin-4'-O-β-D-pyranoglucopyranoside (OAc) and 2,3,4,6-tetra-O-acetyl-β-D-pyranoglucopyranosyl-2,2,2-trichloroimidoacetate obtained in S2 to a solvent at room temperature and dissolve them. Then, add trimethylsilyl trifluoromethanesulfonate dropwise at -20 to -15°C and continue the reaction for 10 to 24 hours to obtain a reaction solution. S4. The reaction solution obtained in S3 was added dropwise to a sodium bicarbonate aqueous solution and stirred for 5-30 minutes. The organic phase was then separated and washed with sodium chloride. After concentration to dryness, the organic phase was dissolved in dichloromethane and purified by normal phase preparative purification to collect naringenin-7,4'-di-O-β-D-pyranoglucoside (OAc); S5. The naringenin-7,4'-di-O-β-D-pyranoglucoside (OAc) obtained in S2 and S4 are combined, and the acetyl group is removed by sodium methoxide, and then the mixture is washed with ethanol and dried to obtain naringenin-7,4'-di-O-β-D-pyranoglucoside (OAc).

[0006] In a second aspect, the present application provides a method for preparing naringenin-7,4'-di-O-β-D-pyranoglucoside using naringenin-4'-O-β-D-pyranoglucoside (OAc) as a raw material, comprising the following steps: A1. Naringenin-4'-O-β-D-glucopyranoside (OAc) and 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside-2,2,2-trichloroimidoacetate were dissolved in a solvent at room temperature, and trimethylsilyl trifluoromethanesulfonate was added dropwise at -20 to -15°C and the reaction was continued for 10 to 24 hours to obtain a reaction solution. A2. The reaction solution obtained in A1 was added dropwise to a sodium bicarbonate aqueous solution and stirred for 5-30 minutes. The mixture was then separated and the organic phase was washed with sodium chloride. After concentration to dryness, the organic phase was dissolved in dichloromethane and purified by normal phase preparative purification to obtain naringenin-7,4'-di-O-β-D-pyranoside.

[0007] Preferably, the weight ratio of each material in S1 is as follows: The mass ratio of naringenin, 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl-2,2,2-trichloroimidoacetate, trimethylsilyl trifluoromethanesulfonate and solvent is (1-5):(3-18):(0.04-3.3):(5-50).

[0008] Preferably, the weight ratio of each material in S3 or A1 is as follows: The mass ratio of naringenin-4'-O-β-D-pyranoglucopyranoside (OAc), 2,3,4,6-tetra-O-acetyl-β-D-pyranoglucopyranosyl-2,2,2-trichloroimidoacetate, trimethylsilyl trifluoromethanesulfonate, and solvent is (0.7-5):(1.3-14.5):(0.03-3.3):(5-50).

[0009] Preferably, the solvent is selected from dichloromethane or acetonitrile.

[0010] In a third aspect, the present application provides a use of naringenin-7,4'-di-O-β-D-pyranoglucoside and / or the product obtained by any of the above preparation methods in cosmetics.

[0011] Preferably, the cosmetics are anti-inflammatory aging and anti-oxidative aging skin care products.

[0012] Preferably, the amount of naringenin-7,4'-di-O-β-D-pyranoglucoside used in the cosmetic is 0.05-5 wt%.

[0013] In summary, this application has the following beneficial effects: 1. This application provides two methods for synthesizing naringenin-7,4'-di-O-β-D-glucopyranoside, each prepared using naringenin or naringenin-4'-O-β-D-glucopyranoside (OAc) as a raw material. The resulting products have relatively stable and uniform properties. 2. The product obtained by the preparation method of this application, naringenin-7,4'-di-O-β-D-glucopyranoside, not only has basic efficacy comparable to naringenin, but also overcomes the shortcomings of naringenin, such as poor water solubility and low bioavailability, and is less cellular irritant. 3. This application is based on the discovery of the product's unique application in anti-aging and anti-stress, and on this basis, a number of anti-inflammatory aging and anti-oxidative aging skin care products have been developed. It only requires an addition amount of 0.05-5wt% to give the cosmetic system excellent effects, thereby meeting people's usage needs. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is the result of the H-NMR spectrum in Example 1; Figure 2 This is the result of the NMR carbon spectrum in Example 1; Figure 3 This is the mass spectrometry result of naringenin-7,4'-di-O-β-D-pyranoglucoside in Example 1; Figure 4 This is the H NMR spectrum result of naringenin-7,4'-di-O-β-D-glucopyranoside in Example 2; Figure 5 This is the mass spectrometry result of naringenin-7,4'-di-O-β-D-glucopyranoside in Example 2; Figure 6 This is the H-NMR spectrum result of naringenin-7,4'-di-O-β-D-glucopyranoside in Example 3; Figure 7 This is the mass spectrometry result of naringenin-7,4'-di-O-β-D-glucopyranoside in Example 3; Figure 8 This is a comparison test chart of the cellular anti-inflammatory effects of naringenin-7,4'-di-O-β-D-pyranoside and naringenin in Example 1; Figure 9 This is a comparison test chart of the antioxidant effects of naringenin-7,4'-di-O-β-D-pyranoside and naringenin in zebrafish in Example 1; Figure 10 This is a diagram of the cell anti-aging test of naringenin-7,4'-di-O-β-D-pyranoglucoside in Example 1 (red arrows indicate typical β-galactosidase-positive cells). DETAILED DESCRIPTION

[0015] Below in conjunction with embodiment 1-3 and accompanying Figure 1-10 To further explain this application in detail, the raw materials used in this application are all common commercially available materials unless otherwise specified in the table below: Example 1 A naringenin-7,4'-di-O-β-D-glucopyranoside, whose structural formula is shown in formula (I): The synthetic route of the formula (I) is as follows: Specifically, it is prepared by the following preparation steps: S1. At room temperature, weigh the corresponding weights of naringenin and 2,3,4,6-tetra-O-acetyl-β-D-pyranose glucopyranosyl-2,2,2-trichloroimidoacetate; Then, the mixture was added to 1 kg of a stirred solvent (dichloromethane), stirred for 1 hour, and then trimethylsilyl trifluoromethanesulfonate was added dropwise at -20--15°C and the reaction was continued for 10 hours to obtain a reaction solution; The weights of naringenin, 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl-2,2,2-trichloroimidoacetate, and trimethylsilyl trifluoromethanesulfonate in S1 are 40.0 g, 328.79 g, and 26.97 g, respectively; S2. The reaction solution obtained in S1 was added dropwise to 400 ml of a 3% sodium bicarbonate aqueous solution, stirred for 10 min after addition, allowed to stand for 10 min, and the lower organic phase was collected. After separation, the organic phase was washed with sodium chloride, and then vacuum concentrated at an external temperature of 40° C. and a vacuum degree of -0.07 MPa (a conventional chemical operation step, using a rotary evaporator for concentration) until dry, and then dissolved with dichloromethane; Finally, normal phase preparative purification was performed using a DAC-200 high pressure preparative chromatograph with a petroleum ether / ethyl acetate volume ratio of 3 / 1 to collect impurities; a petroleum ether / ethyl acetate volume ratio of 2 / 1 to collect naringenin-4'-O-β-D-pyranoside (OAc); and a petroleum ether / ethyl acetate volume ratio of 1 / 1 to collect naringenin-7,4'-di-O-β-D-pyranoside (OAc), which were then concentrated separately for later use. S3. At room temperature, weigh the corresponding weights of naringenin-4'-O-β-D-pyranoglucopyranoside (OAc) and 2,3,4,6-tetra-O-acetyl-β-D-pyranoglucopyranoside-2,2,2-trichloroimidoacetate obtained in S2 and add them to 800 g of a stirring solvent (dichloromethane), and stir for 1 hour. Then, add trimethylsilyl trifluoromethanesulfonate dropwise at -20°C and continue the reaction for 10 hours to obtain reaction solution B. The weights of naringenin-4'-O-β-D-pyranoglucopyranoside (OAc), 2,3,4,6-tetra-O-acetyl-β-D-pyranoglucopyranoside-2,2,2-trichloroimidoacetate, and trimethylsilyl trifluoromethanesulfonate in the above S3 are 80.0 g, 60.0 g, and 2.68 g, respectively. S4. Add the reaction solution B obtained in S3 dropwise into 200 mL of 3% sodium bicarbonate aqueous solution and stir for 5 min. After separation, wash the organic phase with sodium chloride, concentrate to dryness, and dissolve in dichloromethane. Finally, perform normal phase preparative purification: collect naringenin-7,4'-di-O-β-D-pyranoside (OAc) at 1 / 1. S5. Combine the naringenin-7,4'-di-O-β-D-pyranoglucoside (OAc) obtained in S2 and S4 and dissolve them in methanol at a weight ratio of 1:4. Then, add sodium methoxide solution dropwise at 5°C and keep warm for 2 hours. Add glacial acetic acid dropwise under stirring to adjust the pH to 6-7. Filter and rinse once with 1 Vol of ethanol. Dry to obtain the final product naringenin-7,4'-di-O-β-D-pyranoglucoside. The nuclear magnetic resonance detection results are as follows: Figure 1-2 Shown: Nuclear magnetic hydrogen spectrum analysis 1 HNMR(400MHz,D2O)δ7.28(s,2H,H-2',H-6'),7.04(s,2H,H-3',H-5'),6.05(d,2H,H-8-H-6),5.16(s,1H ,H-1),4.95(d,J=8.0Hz,1H,H-1”),4.84(s,1H,H-1”),3.81(d,J=12.0Hz,1H,H-2”),3.74(d,J=12.0Hz, 1H,H-2”), 3.63(d,J=12.0Hz,1H,H-3”),3.59-3.54(m,2H,H-3”-H-4”),3.51-3.46(m,2H,H-4”-H-5”),3.44-3.38(m,4H,H-6”),3.35-3.29(t,1H,H-5”),2.93(br,1H,H-2),2.60(d,J=16.0Hz,1H,H-2), C NMR analysis 13 C NMR(400MHz,D2O)δ197.9(C-3),164.7(C-7),162.7(C-5),162.4(C-9),157.2(C-4' ),132.0(C-1'),128.4(C-2',C-6'),116.8(C-3',C-5'),103.7(C-4),100.2(C-6), 99.2(C-8),97.1(C-1”),96.2(C-1”),78.6(C-1),76.1(C-3”),75.4(C-2”),72.72(C-4”),65.2(C-5”),60.4(C-6”),29.6(C-2) The structure is correct, and the molecular weight of the product is 595.16682(MH - ), which deviates from the theoretical value (595.16684) by 0.03 ppm. Therefore, it can be further determined that the product is naringenin-7,4'-di-O-β-D-pyranoglucoside. Figure 3 shown.

[0016] Example 2 A naringenin-7,4'-di-O-β-D-glucopyranoside, whose structural formula is shown in formula (I): The synthetic route of the formula (I) is as follows: Specifically, it is prepared by the following preparation steps: The weights of S1, naringenin-4'-O-β-D-glucopyranoside (OAc), 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl-2,2,2-trichloroimidoacetate, and trimethylsilyl trifluoromethanesulfonate were 80.0 g, 60.0 g, and 2.68 g, respectively; S2. Add the reaction solution B obtained in S1 dropwise into 200 mL of sodium bicarbonate aqueous solution (3%) and stir for 5 min. After separation, wash the organic phase with sodium chloride, concentrate to dryness, and dissolve in dichloromethane. Finally, perform normal phase preparative purification: collect naringenin-7,4'-di-O-β-D-pyranoside (OAc) at 1 / 1. S3. Naringenin-7,4'-di-O-β-D-pyranoglucoside (OAc) was dissolved in methanol at a weight ratio of 1:4, and then sodium methoxide solution was added dropwise at 5°C. The mixture was kept warm for 2 hours. Glacial acetic acid was added dropwise under stirring to adjust the pH to 6-7. The mixture was filtered and rinsed once with 1 Vol of ethanol. The final product, naringenin-7,4'-di-O-β-D-pyranoglucoside, was obtained by drying. The results of nuclear magnetic resonance (NMR) analysis were as follows: Figure 4 Shown: NMR hydrogen spectrum analysis 1 HNMR(400MHz,D2O)δ7.33(d,J=8.0Hz,2H,H-2',H-6'),7.06(d,J=8.0Hz,2H,H-3',H-5'),6.10(d,2H ,H-8-H-6),5.26(d,J=16.0Hz,1H,H-1),4.99(d,J=8.0Hz,1H,H-1”),4.92(d,J=8.0Hz,1H,H-1”),3.8 The structure is correct, the NMR carbon spectrum is consistent with that of Example 1, which will not be described in detail. The molecular weight of the product is 595.16669 (MH) after mass spectrometry. - ), which deviates from the theoretical value (595.16684) by 0.25 ppm. Therefore, it can be further determined that the product is naringenin-7,4'-di-O-β-D-pyranoglucoside. Figure 5 shown.

[0017] Example 3 A naringenin-7,4'-di-O-β-D-glucopyranoside, whose structural formula is shown in formula (I): The synthetic route of the formula (I) is as follows: Specifically, it is prepared by the following preparation steps: S1. Naringenin-7,4'-di-O-β-D-pyranoglucoside (OAc) was dissolved in methanol at a weight ratio of 1:4, and then sodium methoxide solution was added dropwise at 5°C. The mixture was kept warm for 2 hours. Glacial acetic acid was added dropwise under stirring to adjust the pH to 6-7. The mixture was filtered and rinsed once with 1 Vol of ethanol. The final product, naringenin-7,4'-di-O-β-D-pyranoglucoside, was obtained by drying. The results of nuclear magnetic resonance (NMR) analysis were as follows: Figure 6 Shown: Nuclear magnetic hydrogen spectrum analysis 1 HNMR(400MHz,D2O)δ7.28(d,J=8.0Hz,2H,H-2',H-6'),7.04(d,J=8.0Hz,2H,H-3',H-5'),6.06(d,2H ,H-8-H-6),5.18(d,J=12.0Hz,1H,H-1),4.96(d,J=8.0Hz,1H,H-1”),4.85(d,J=8.0Hz,1H,H-1”),3.8 The structure is correct, the NMR carbon spectrum is consistent with that of Example 1, which will not be described in detail. The molecular weight of the product is 595.16681 (MH) after mass spectrometry. - ), which deviates from the theoretical value (595.16684) by 0.05 ppm. Therefore, it can be further determined that the product is naringenin-7,4'-di-O-β-D-pyranoglucoside. Figure 7 shown.

[0018] Combined with the NMR and mass spectrometry results of Examples 1-3 above, it can be seen that naringenin-7,4'-di-O-β-D-glucopyranoside (OAc) can be prepared regardless of whether naringenin, naringenin-4'-O-β-D-glucopyranoside (OAc) or naringenin-7,4'-di-O-β-D-glucopyranoside (OAc) is used as the raw material.

[0019] In addition, since the dosage ratio of each raw material component can be obtained by trial and error within a limited number of times, and the effects after adjustment within the range are predictable, they are not listed one by one, and only the group with the optimal ratio is selected. As for the conditions such as temperature, washing, concentration, and purification, they basically have no effect on the final product, so the embodiments are not listed one by one. Therefore, the undisclosed groups should not be regarded as limiting the scope of protection of this application.

[0020] Performance testing Naringenin-7,4'-di-O-β-D-glucopyranoside and naringenin prepared in Example 1 (the group with the highest purity) were selected for comparative testing. The specific testing items, reference standards, and steps are as follows: 1) Water solubility comparison test Weigh several portions of naringenin, naringin, and naringenin-7,4'-di-O-β-D-pyranoglucoside, each containing 1 mg. Place the solution at 25℃±2℃. Add one portion of naringenin, naringin, or naringenin-7,4'-di-O-β-D-pyranoglucoside to 1 mL of aqueous solution every 5 minutes. Record the amount of solution added when the solution is not completely dissolved. If solute particles or droplets are visible to the naked eye, the solution is considered to be incompletely dissolved.

[0021] 2) Cytotoxicity comparison test Keratinocytes in logarithmic growth phase (purchased from Jiangsu KeyGen Biotech Co., Ltd.) were added to a 96-well plate (2*10 4 / well), and when the cells adhered and the density reached 80-90%, the cells in the wells were washed with PBS (purchased from Beijing Solebow Technology Co., Ltd.), and different concentrations of naringenin-7,4'-di-O-β-D-pyranoglucopyranoside and naringenin were added. The drug concentration gradient was 0 μM, 7.8 μM, 15.625 μM, 31.25 μM, 62.5 μM, 125 μM, 250 μM, 500 μM, and 1000 μM.

[0022] After 24 hours of culture, add 10 μL of CCK8 (Cell Counting Kit-8 reagent, purchased from Dalian Meilun Biotechnology Co., Ltd.) to each well. After incubation for 1-4 hours, measure absorbance using a microplate reader to calculate changes in cell viability. Relative cell viability (%) = (sample absorbance / blank absorbance) * 100%.

[0023] 3) Comparative test of cell anti-inflammatory effect Mouse macrophage RAW264.7 cells (purchased from Wuhan Punosai Life Science Co., Ltd.) were added to each well (1*10 5 The cells were plated in 24-well plates and allowed to adhere overnight before being divided into blank control group, model group and experimental group. The blank control group was added with normal complete culture medium (89% DMEM, 10% fetal bovine serum, 1% double antibody); Model group: 0.05 mg / mL SLS (sodium lauryl sulfate); naringenin group: 0.05 mg / mL SLS + 100 μM naringenin; naringenin-7,4'-di-O-β-D-pyranoglucopyranoside group: 0.05 mg / mL SLS + 100 μM naringenin-7,4'-di-O-β-D-pyranoglucopyranoside. The medium was changed for all groups by discarding all the original culture medium and adding 500 μL of the corresponding culture medium according to the group.

[0024] After incubation for 24 hours, the culture medium of each group was collected and centrifuged to obtain the supernatant, and the IL-6 content was tested by ELISA. The anti-inflammatory effect was characterized by inhibiting the production of IL-6.

[0025] 4) Comparative test of antioxidant effects in zebrafish 72 hpf AB zebrafish embryos were cultured and divided into a normal group, a model group, a naringenin group, and a naringenin-7,4'-di-O-β-D-glucopyranoside group. The components of each group are as follows: Normal group: E3 culture medium; Model group: E3 culture medium containing 25 μg / mL LPS (lipopolysaccharide); Naringenin group: E3 culture medium containing 25 μg / mL LPS + 100 μM naringenin; Naringenin-7,4'-di-O-β-D-glucopyranoside group: E3 culture medium containing 25 μg / mL LPS and 100 μM naringenin-7,4'-di-O-β-D-glucopyranoside.

[0026] Then the cells were placed in a 27.5°C incubator and cultured for 48 hours. The zebrafish status was observed regularly during the period. After the incubation, DCFH-DA (2',7'-dichlorofluorescein diacetate) was used for staining for 40 minutes. The ROS (reactive oxygen species) of the zebrafish in each group was observed using a fluorescence microscope. Finally, ImageJ was used to count the green fluorescence intensity of the zebrafish in each group.

[0027] 5) Comparative test of cell anti-aging effects The anti-aging efficacy of naringenin-7,4'-di-O-β-D-pyranoglucoside was tested by β-galactosidase staining experiment. 3 Each well) was plated in a six-well plate and allowed to adhere to the wall overnight; Add the corresponding reagents according to the following groups: Blank group: Normal medium replacement (84% DMEM, 15% fetal bovine serum, 1% double antibody) was performed, all the original medium was discarded, and 2 mL of fresh medium was added; Model group: 10 μg / mL AAPH (azobisisobutylamidine hydrochloride), discard all the original culture medium, and add 2 mL of fresh culture medium containing 10 μg / mL AAPH; Naringenin-7,4'-di-O-β-D-glucopyranoside group: 10 μg / mL AAPH + 100 μM naringenin-7,4'-di-O-β-D-glucopyranoside. All the original culture medium was discarded, and 2 mL of fresh culture medium containing 10 μg / mL AAPH + 100 μM naringenin-7,4'-di-O-β-D-glucopyranoside was added.

[0028] After incubation for 24 h, the cells were stained according to the instructions of the β-galactosidase staining kit (Biyuntian, C0602), and the β-galactosidase-positive cell rate was counted.

[0029] 6) Moisturizing effect comparison test Take HaCaT cells (1*10 5 Cells (100 μM) were seeded into 6-well plates and allowed to adhere overnight. Complete medium was replaced as usual in the blank group, while complete medium containing 100 μM naringenin or 100 μM naringenin-7,4'-di-O-β-D-pyranoside was added to the experimental groups. The cells were cultured at 37°C and 5% CO2 for 24 h. Total RNA was extracted from the cells of each group after the incubation. The expression levels of FLG and AQP3 genes in the cells were then detected according to the real-time fluorescence quantitative PCR protocol. Among them, the protein expressed by AQP3 is mainly responsible for the transport of water and glycerol, directly affecting the hydration of the stratum corneum; FLG is an important donor of natural moisturizing factor (NMF), and Caspase14 is responsible for degrading it into NMF, thereby exerting a moisturizing effect.

[0030] The naringenin-7,4'-di-O-β-D-glucopyranoside obtained in Example 1 was extracted and tested for its water solubility, cytotoxicity, cellular anti-inflammatory effect, in vivo antioxidant effect, cellular anti-aging effect, and moisturizing effect according to the above measurement procedures. The test results are recorded in Tables 1-6 below.

[0031] Table 1: Water solubility test results As can be seen from Table 1 above, the water solubility of naringenin-7,4'-di-O-β-D-pyranoglucoside obtained in Example 1 is much stronger than that of naringenin and naringin, and its solubility in water is greater than 10 mg / mL. This property is of great significance for the application extension of naringenin and its derivatives, and effectively overcomes the original application barriers.

[0032] Table 2: Cytotoxicity test results As shown in Table 2 above, within the tested concentration range, cell viability decreased significantly when the naringenin concentration was above 500 μM, while naringenin-7,4'-di-O-β-D-pyranoglucoside had no inhibitory effect on cell viability at concentrations of 1 mM and below. Therefore, naringenin-7,4'-di-O-β-D-pyranoglucoside has lower cytotoxicity and higher safety than naringenin.

[0033] Table 3: Cellular anti-inflammatory test results From the above table 3 and Figure 4 It can be seen that after the above-mentioned groups were treated with SLS, the IL-6 content in the model group increased significantly, proving that macrophages respond to external stimuli and can be used as an anti-inflammatory test tool. Naringenin and naringenin-7,4'-di-O-β-D-pyranoglucopyranoside can both significantly inhibit the production of IL-6, but there is no significant difference in the anti-inflammatory effect among the groups. Therefore, this part can be regarded as the original efficacy of naringenin being retained.

[0034] Table 4: In vivo antioxidant effect test results From the above table 4 and Figure 5 It can be seen that the green fluorescence intensity in the model group was significantly higher than that in the blank group, indicating that: 1. the fluorescent probe is sensitive; 2. LPS can be used as a modeling agent to induce an increase in ROS in zebrafish; Both naringenin and naringenin-7,4'-di-O-β-D-pyranoglucoside have significant inhibitory effects on reactive oxygen species, and the effect of naringenin-7,4'-di-O-β-D-pyranoglucoside is significantly stronger than that of naringenin, proving that the bioavailability of naringenin is significantly improved after glycosylation.

[0035] Table 5: Cell anti-aging test results From the above table 5 and Figure 6It can be seen that after AAPH treatment in the model group, a large number of cells underwent aging due to the oxidative stress effect it produced. Naringenin-7,4'-di-O-β-D-pyranoglucoside can significantly inhibit this process, indicating that naringenin-7,4'-di-O-β-D-pyranoglucoside has anti-aging effects.

[0036] Table 6: Cell moisturizing efficacy test results As shown in Table 6 above, naringenin-7,4'-di-O-β-D-pyranoglucoside can significantly promote the expression of FLG and AQP3, and the effect is significantly better than that of naringenin. This proves that naringenin-7,4'-di-O-β-D-pyranoglucoside has a more significant moisturizing effect than naringenin.

[0037] In summary, combined with Table 1-6 and Figure 1-10 The new derivative of naringenin provided in this application - naringenin-7,4'-di-O-β-D-pyranoglucoside, not only has basic anti-inflammatory effects that are comparable to naringenin, but also overcomes the defects of naringenin such as poor water solubility, low bioavailability, and low moisturizing effect, and is less cellular irritating.

[0038] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed, but as long as they are within the scope of the claims of the present application, they are protected by the patent law.

Claims

1. A method for preparing naringenin-7,4'-di-O-β-D-glucopyranoside, characterized in that: The method comprises the following preparation steps: S1. First, naringenin and 2,3,4,6-tetra-O-acetyl-β-D-pyranose acyl-2,2,2-trichloroimidoacetate were added to a solvent at room temperature to dissolve, and then trimethylsilyl trifluoromethanesulfonate was added dropwise at -20 to -15°C and the reaction was continued for 10 to 24 hours to obtain a reaction solution; S2. The reaction solution obtained in S1 was added dropwise to a sodium bicarbonate aqueous solution and stirred for 5-30 min. The mixture was then separated and the organic phase was washed with sodium chloride. After concentration to dryness, the organic phase was dissolved in dichloromethane and purified by normal phase preparation to obtain impurities, naringenin-4'-O-β-D-pyranoglucoside (OAc) and naringenin-7,4'-di-O-β-D-pyranoglucoside (OAc).

2. The method for preparing naringenin-7,4'-di-O-β-D-glucopyranoside according to claim 1, characterized in that: The method further includes a purification step for the product obtained in S2, as follows: S3. Add naringenin-4'-O-β-D-glucopyranoside (OAc) and 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside-2,2,2-trichloroimidoacetate obtained in S2 to a solvent at room temperature and dissolve them. Then, add trimethylsilyl trifluoromethanesulfonate dropwise at -20 to -15°C and continue the reaction for 10 to 24 hours to obtain a reaction solution. S4. The reaction solution obtained in S3 is added dropwise to a sodium bicarbonate aqueous solution and stirred for 5-30 minutes. The organic phase is then separated and washed with sodium chloride. After concentration to dryness, the organic phase is dissolved in dichloromethane and purified by normal phase preparative purification to collect naringenin-7,4'-di-O-β-D-pyranoside (OAc); S5. Combine the naringenin-7,4'-di-O-β-D-pyranoglucoside (OAc) obtained in S2 and S4, remove the acetyl group by sodium methoxide, and then rinse with ethanol and dry to obtain naringenin-7,4'-di-O-β-D-pyranoglucoside.

3. A method for preparing naringenin-7,4'-di-O-β-D-glucopyranoside, characterized in that: The method comprises the following preparation steps: A1. Add naringenin-4'-O-β-D-glucopyranoside (OAc) and 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside-2,2,2-trichloroimidoacetate to a solvent at room temperature and dissolve them. Then, add trimethylsilyl trifluoromethanesulfonate dropwise at -20 to -15°C and continue the reaction for 10 to 24 hours to obtain a reaction solution. A2. The reaction solution obtained in A1 was added dropwise to a sodium bicarbonate aqueous solution and stirred for 5-30 minutes. The organic phase was then separated and washed with sodium chloride. After concentration to dryness, the organic phase was dissolved in dichloromethane and purified by normal phase preparative purification to obtain naringenin-7,4'-di-O-β-D-pyranoside (OAc).

4. The method for preparing naringenin-7,4'-di-O-β-D-glucopyranoside (OAc) according to claim 1, wherein: The weight ratio of each material in S1 is as follows: The mass ratio of naringenin, 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl-2,2,2-trichloroimidoacetate, trimethylsilyl trifluoromethanesulfonate and solvent is (1-5):(3-18):(0.03-3.3):(5-50).

5. The method for preparing naringenin-7,4'-di-O-β-D-pyranoglucoside according to any one of claims 1 or 3, characterized in that: The weight ratios of the materials in S3 or A1 are as follows: The mass ratio of naringenin-4'-O-β-D-glucopyranoside (OAc), 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoyl-2,2,2-trichloroimidoacetate, trimethylsilyl trifluoromethanesulfonate, and solvent is (0.7-5):(3-14.5):(0.03-3.3):(5-50).

6. The method for preparing naringenin-7,4'-di-O-β-D-glucopyranoside according to any one of claims 2 or 3, characterized in that: The solvent is selected from dichloromethane or acetonitrile.

7. Use of the product obtained by any one of the preparation methods according to claims 1 to 6 in cosmetics.

8. The use of naringenin-7,4'-di-O-β-D-glucopyranoside in cosmetics according to claim 7, characterized in that: The cosmetics are anti-inflammatory and anti-oxidative aging skin care products.

9. The use of naringenin-7,4'-di-O-β-D-glucopyranoside in cosmetics according to claim 8, characterized in that: The amount of naringenin-7,4'-di-O-β-D-pyranoglucoside used in cosmetics is 0.05-5wt%.

Images