Senecio acyl paeoniflorin as well as preparation method and application thereof

Abstract

The invention relates to the field of cosmetics, in particular to senecio acyl paeoniflorin as well as a preparation method and application thereof. The climbing groundsel acyl paeoniflorin provided by the invention comprises a compound as shown in a formula (I), and the compound as shown in the formula (I) comprises 4 '-O-climbing groundsel acyl paeoniflorin (1), 3'-O-climbing groundsel acyl paeoniflorin (2) and 6 '-O-climbing groundsel acyl paeoniflorin (3). Research finds that the prepared compound has a protection effect on HSF cells and HaCaT cells damaged by H2O2, promotes the HSF cells to secrete hyaluronic acid (HA) and I-type collagen (Col I), has DPPH free radical scavenging activity and has tyrosinase inhibition activity, so that the compound has anti-aging and whitening effects and can be applied to the field of cosmetics.

Description

Technical Field

[0001] The present invention relates to the field of cosmetics, and in particular to senecioyl-peonidin, a preparation method and application thereof. Background Art

[0002] Youth and whitening are the eternal themes that people pursue, specifically the pursuit of whitening and radiance of human skin and delaying skin aging. Skin is the largest organ on the human body surface. It is in direct contact with the external environment for a long time and is easily damaged by irritants and light (mainly ultraviolet rays) in the external environment, making the skin prone to aging, damage, pigmentation, etc., thus affecting the beauty of the human body.

[0003] Melanin is the most important type of pigment that affects skin whiteness. After melanin is produced in melanocytes, mature melanosomes will be transferred along the extension direction of melanocyte dendrites and transmitted to the surrounding keratinocytes, thereby regulating skin color and protecting against ultraviolet radiation. Melanin transport blockers can reduce the transfer rate of melanosomes to keratinocytes, reduce the melanin content of each epidermal cell layer, and achieve a whitening effect. Such as niacinamide, azelaic acid, green tea extract, etc. After melanin is distributed in keratinocytes, it will fall off with the keratinocytes. Melanin deposition on the skin surface will cause visible skin color changes or spots. Therefore, by promoting the metabolism of the skin's stratum corneum and accelerating the shedding of aging keratinocytes, the metabolism of melanin can be accelerated, thereby improving the skin's skin color and appearance.

[0004] Skin damage is mainly manifested as intracellular water loss, oxidative free radical damage, reduced cell matrix secretion, etc. Skin aging reduces the skin's protective ability and regulatory ability to the body, so that the skin cannot adapt to changes in the internal and external environment, and changes in the overall appearance of color, color, shape, texture, etc. occur. Skin aging is divided into endogenous aging and exogenous aging. Endogenous aging refers to the natural aging of the skin with age. It manifests as whitening of the skin, the appearance of fine wrinkles, decreased elasticity, sagging skin, etc. The main cause of exogenous aging is photoaging caused by sun exposure. It manifests as wrinkles, sagging skin, roughness, yellowish or grayish yellow skin discoloration, capillary dilation, pigment spot formation, etc. Therefore, ingredients with both anti-aging and whitening effects have broad application prospects in the field of cosmetics. However, there is currently no relevant report on the senecioyl paeoniflorin of the present invention and its function. Summary of the invention

[0005] Therefore, the technical problem to be solved by the present invention is a senecioyl-peonidin and a preparation method and use thereof. The senecioyl-peonidin has anti-aging and whitening effects, and the materials of the preparation method are easily available, easy to operate, and suitable for industrial production.

[0006] To this end, the present invention provides the following technical solutions:

[0007] The embodiment of the present invention discloses a senecioyl-peonidin, comprising: a compound as shown in formula (I):

[0008]

[0009] Where: R 1 =R 3 =H, R 2 for

[0010] R 1 =R 2 =H, R 3 for

[0011] R 2 =R 3 =H, R 1 for

[0012] The present invention discloses a method for preparing the senecioylpaeoniflorin, comprising:

[0013] Dissolving compound a in an organic solvent, adding organic tin, a basic catalyst and compound b, and subjecting the mixture to an esterification condensation reaction to obtain a compound represented by formula (I);

[0014] The synthetic route is as follows:

[0015]

[0016] Where: R 1 =R 3 =H, R 2 for

[0017] R 1 =R 2 =H, R 3 for

[0018] R 2 =R 3 =H, R 1 for

[0019] In some embodiments, the molar ratio of compound 1, organotin, alkaline catalyst and compound 2 is 1:1-2:1-2:1-2, preferably 1:1:1.2:1;

[0020] And / or, the organotin includes but is not limited to any one of dimethyltin dichloride, dibutyltin dichloride, butyltin trichloride, and dibutyltin oxide.

[0021] And / or, the basic catalyst includes, but is not limited to, any one of triethylamine, trimethylamine, triethylenediamine, and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU).

[0022] In some embodiments, the reaction conditions of the esterification condensation reaction are room temperature for 0.5 to 5 hours, preferably 1 hour. In some embodiments, the room temperature is 10-30°C, which can be any one of 10, 15, 20, 25, 30°C or a range between any two values. The reaction time can be any one of 0.5, 1, 2, 3, 4, 5 hours or a range between any two values.

[0023] In some embodiments, the organic solvent is selected from any one of acetone, dioxane, tetrahydrofuran, ethylene glycol dimethyl ether, N,N-dimethylformamide, and dimethyl sulfoxide.

[0024] In some embodiments, after the esterification condensation reaction is completed, the reaction is quenched, extracted, dried, and purified.

[0025] In some embodiments, in the purification step, the column chromatography conditions are as follows: the weight of the silica gel used for silica gel column chromatography is 1:1 of the sample, and methanol-chloroform is eluted at a volume ratio of 2.5-5:97.5-95.

[0026] The embodiments of the present invention disclose that the senecioyl-peonidin has any of the following uses:

[0027] (1) Use in soothing, repairing or protecting the skin or in preparing products for soothing, repairing or protecting the skin;

[0028] (2) Use as an antioxidant or in the preparation of products with antioxidant effects;

[0029] (3) Use in anti-skin aging and / or skin moisturizing or in the preparation of anti-skin aging and / or skin moisturizing products;

[0030] (4) Use in promoting the secretion of collagen and / or hyaluronic acid by the skin or in preparing products that secrete collagen and / or hyaluronic acid by the skin;

[0031] (5) Use in skin whitening, melanin inhibition or in the preparation of skin whitening or melanin inhibition products.

[0032] In some embodiments, including: in soothing, repairing or protecting H 2 O 2 Damaged skin or preparations to soothe, repair or protect 2 O 2 Use in products for damaged skin;

[0033] and / or, use as an antioxidant for scavenging DPPH free radicals or in the preparation of a product with an antioxidant effect for scavenging DPPH free radicals;

[0034] and / or, use as a tyrosinase inhibitor or in the preparation of a product having the effect of inhibiting tyrosinase;

[0035] And / or, the product includes cosmetics, medicines or health foods.

[0036] An embodiment of the present invention discloses a cosmetic comprising the senecio acyl paeoniflorin. The content of the 6'-O-acyl paeoniflorin is 0.001wt% or more, such as 0.001, 0.01, 0.1, 1, 5, 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99, 99.9wt% or any range between any two values.

[0037] In some embodiments, the cosmetic further comprises auxiliary materials, carriers and / or excipients acceptable in the field of cosmetics.

[0038] Furthermore, the cosmetic auxiliary material matrix includes but is not limited to: 1. Moisturizers, such as propylene glycol, etc.; 2. Thickeners, such as stearic acid, lauryl alcohol, xanthan gum, carbomer, carboxymethyl cellulose, hydroxyethyl cellulose, etc.; 3. Emulsifiers, such as cetearyl glucoside, PEG-100 stearate, Tweens, etc.; 4. Emollients, such as caprylic / capric triglyceride, dicaprylyl carbonate, squalane, tocopheryl acetate, cyclopentasiloxane, caprylyl polymethicone, isododecane, polydimethylsiloxane, etc.; 5. Fragrance; 6. Pigment.

[0039] The cosmetics include those having relative fluidity and the appearance of which can be any one of a water solution, a cream, a lotion, a washing liquid and a facial mask.

[0040] Furthermore, in some embodiments, the cosmetics include but are not limited to one of a facial mask, essence, spray, moisturizing cream, cleanser, toner, moisturizing lotion, sunscreen, BB cream, shampoo, shower gel, and skin care gel.

[0041] The technical solution of the present invention has the following advantages:

[0042] 1. The present invention provides a senecioyl paeoniflorin, comprising: a compound as shown in formula (I), wherein the compound as shown in formula (I) comprises 4'-O-senecioyl paeoniflorin (1), 3'-O-senecioyl paeoniflorin (2), and 6'-O-senecioyl paeoniflorin (3). The present invention has found that: the prepared compound has a good effect on H 2 O 2 The compound has a protective effect on damaged HSF cells and HaCaT cells, promotes HSF cells to secrete hyaluronic acid (HA) and type I collagen (ColⅠ), has DPPH free radical scavenging activity, and has tyrosinase inhibitory activity. Therefore, the compound has anti-aging and whitening effects and can be used in the field of cosmetics.

[0043] 2. The present invention provides a method for preparing senecioylpaeoniflorin, wherein the raw materials of the compound preparation method are readily available, easy to operate, and suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0045] Figure 1 is the 4'-O-senecioyl-peonidin in Example 1 of the present invention 1 H NMR spectra;

[0046] Figure 2 is the 4'-O-senecioyl-peonidin in Example 1 of the present invention 13 C NMR spectra;

[0047] Figure 3 is the 3'-O-senecioyl-peonidin in Example 1 of the present invention 1 H NMR spectra;

[0048] Figure 4 is the 3'-O-senecioyl-peonidin in Example 1 of the present invention 13 C NMR spectra;

[0049] Figure 5 is the 6'-O-senecioyl-peonidin in Example 1 of the present invention 1 H NMR spectra;

[0050] Figure 6 is the 6'-O-senecioyl-peonidin in Example 1 of the present invention 13 C NMR spectra;

[0051] Figure 7 The different compounds in Example 1 of the present invention have different effects on H 2 O 2 The results of the protective effect on injured HaCaT cells;

[0052] Figure 8 The different compounds in Example 1 of the present invention have different effects on H 2 O 2 The result of the protective effect of damaged HSF cells;

[0053] Fig. 9 This is the result of different compounds in Example 2 of the present invention promoting the secretion of type I collagen (Col Ⅰ) by HaCaT cells;

[0054] Fig.10 This is the result of different compounds in Example 2 of the present invention promoting the secretion of hyaluronic acid (HA) by HSF cells;

[0055] Fig.11 is the result of DPPH radical scavenging activity of different compounds in Example 3 of the present invention;

[0056] Fig.12 is the result of tyrosinase inhibitory activity of different compounds in Example 4 of the present invention;

[0057] Above Figure 7-Figure 12 The data statistical method used in this study is the t-test method for statistical analysis. DETAILED DESCRIPTION

[0058] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0059] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

[0060] Example 1

[0061] This embodiment provides a method for preparing senecioyl-peonidin, comprising: the compound structure of senecioyl-peonidin is as shown in formula (I):

[0062]

[0063] Where: R1 =R 3 =H, R 2 for

[0064] R 1 =R 2 =H, R 3 for

[0065] R 2 =R 3 =H, R 1 for

[0066] The synthetic route is as follows:

[0067]

[0068] Where: R 1 =R 3 =H, R 2 for

[0069] R 1 =R 2 =H, R 3 for

[0070] R 2 =R 3 =H, R 1 for

[0071] Preparation method:

[0072] Paeoniflorin (0.2 mmol, compound a, CAS: 23180-57-6) was dissolved in 5 mL of dry acetone, and dimethyltin dichloride (0.2 mmol), triethylamine (33 μL, 0.24 mol) and 3,3-dimethylacryloyl chloride (22 μL, 0.2 mmol, compound b, CAS: 3350-78-5) were added in sequence under stirring. After reacting at room temperature for 1 h, 10 mL of water was added to quench the reaction, and n-butanol was used for extraction (10 mL × 3). The organic phase was dried over anhydrous sodium sulfate, adsorbed on silica gel 1.5 times the weight of the sample, evaporated to dryness and dissolved at room temperature, and chromatographed on a silica gel column 15 times the weight of the sample, with a methanol-chloroform gradient elution (volume ratio of 5:95). The same fractions were combined by TLC detection to separate 4'-O-senicoyl-peonidin (31 mg, yield 28%), 3'-O-senicoyl-peonidin (22 mg, yield 20%) and 6'-O-senicoyl-peonidin (39 mg, yield 35%).

[0073] When R 1 =R3 =H, R 2 for It is 4'-O-senecioyl-peonidin (denoted as compound 1), and its structural formula is as follows:

[0074]

[0075] Molecular formula: C 28 H 34 O 12

[0076] Molecular weight: 562

[0077] Appearance: White powder

[0078] HRESIMS m / z 607.2033 (calculated 607.2032, [M+HCOO] - ).

[0079] like Figure 1 As shown, 1 H NMR (600MHz,pyridine-D 5 ):δ H 8.15(2H,dd,J=7.2,1.2Hz,H-2” / H-6”),7.50-7.47(m,1H,H-4”),7.34-7.31(m,2H,H-3” / H-5”),7.03(q,1H,H-3”’),5.96(s,1H,H-9), 5.77(s,1H,H-2"'),5.63(m,1H,H-4'),5.27(d,J=12.0Hz,1H,H-8a),5.20(d,J=7.8Hz,H-1'),5.12(d,J=12.0Hz,1H,H-8a),4.34-4.31( m,1H,H-5'),4.21-4.19(m,1H,H-6'a),4.14-4.08(m,2H,H-6'b),4.02-3.99(m,1H,H-2'),3.10-3.09(m,1H,H-5),2.95-2.92(m,1H,H- 7a), 2.48 (d, J = 12.0Hz, H-3a), 2.36 (d, J = 12.0Hz, H-3b), 2.31 (d, J = 12.6Hz, H-7b), 2.12 (d, J = 1.2Hz, H-5"'), 1.67 (s, 6H, H-10, H-4"');

[0080] like Figure 2 As shown, 13 C NMR (150MHz,pyridine-D 5): δ89.5(C-1),86.5(C-2),45.2(C-3),106.5(C-4),44.4(C-5),72.2(C-6),24.0(C-7),62 .0(C-8),102.2(C-9),20.3(C-10),100.9(C-1'),75.6(C-2'),77.0(C-3'),72.2(C-4'),76 .3(C-5'),62.7(C-6'),131.1(C-1”),130.4(C-2” / C-6”),129.3(C-3” / C-5”),133.8(C-4” ),167.1(C-7”’),166.6(C-1”’),117.0(C-2”’),157.9(C-3”’),27.5(C-4”’),20.6(C-5”’).

[0081] When R 1 =R 2 =H, R 3 for It is 3'-O-senecioyl-peonidin (denoted as compound 2), and its structural formula is as follows:

[0082]

[0083] Molecular formula: C 28 H 34 O 12

[0084] Molecular weight: 562

[0085] Appearance: White powder

[0086] HRESIMS m / z 607.2040 (calculated 607.2032, [M+HCOO] - )

[0087] like Figure 3 As shown, 1 H NMR (600MHz,pyridine-D 5 ):δ H8.13(2H,dd,J=7.2,1.2Hz,H-2” / H-6”),7.47-7.45(m,1H,H-4”),7.33-7.30(m,2H,H-3” / H-5”),5.95-5.92(m, 2H,H-9,H-2"),5.20-5.15(m,4H,H-8a,H-8b,H-1',H-3'),4.54-4.51(m,1H,H-6'a),4.39-4.31(m,2H,H-6'b,H -4'),4.14-4.12(m,1H,H-2'),3.98-3.95(m,1H,H-5'),3.09-3.08(m,1H,H-5),2.86-2.83(m,1H,H-7a),2.50( d,J=12.0Hz,H-3a),2.33-2.30(m,2H,H-7b,H-3b),2.12(s,3H,H-5”'),1.70(s,3H,H-10),1.59(s,3H,H-4”’);

[0088] like Figure 4 As shown, 13 C NMR (150MHz,pyridine-D 5 ): δ89.6(C-1),86.4(C-2),45.2(C-3),106.4(C-4),44.3(C-5),72.1(C-6),23.8(C-7),61 .8(C-8),102.1(C-9),20.4(C-10),100.9(C-1'),73.5(C-2'),78.9(C-3'),70.2(C-4'),78 .8(C-5'),62.8(C-6'),131.0(C-1”),130.4(C-2” / C-6”),129.3(C-3” / C-5”),133.8(C-4” ),167.1(C-7”’),167.0(C-1”’),117.7(C-2”’),156.6(C-3”’),27.4(C-4”’),20.5(C-5”’).

[0089] R 2 =R 3 =H, R 1 for It is 6'-O-senecioyl-peonidin (denoted as compound 3), and its structural formula is as follows:

[0090]

[0091] Molecular formula: C 28 H 34 O 12

[0092] Molecular weight: 562

[0093] Appearance: White powder

[0094] HRESIMS m / z 607.2034 (calcd. 607.2032, [M+HCOO] - )

[0095] like Figure 5 As shown, 1 H NMR (600MHz,pyridine-D 5 ):δ H 8.14(2H,d,J=7.8Hz,H-2” / H-6”),7.50-7.47(m,1H,H-4”),7.33-7.31(m,2H,H-3” / H-5”),7.03(q,1H,H-3”’),5.96(s ,1H,H-9),5.88(s,1H,H-2"'),5.21-4.98(m,4H,H-8a,H-8b,H-6'a,H-1'),4.87-4.84(m,1H,H-6'b),4.22-4.19(m,1H, H-5'),4.05-4.00(m,3H,H-2',H-3',H-4'),3.12-3.11(m,1H,H-5),2.88-2.86(m,1H,H-7a),2.58(d,J=12.6Hz,H-3a) ,2.39(d,J=12.0Hz,H-7b),2.30(d,J=12.0Hz,H-3b),2.22(s,3H,H-5"'),1.73(d,J=7.2Hz,H-4"'),1.72(s,3H,H-10);

[0096] like Figure 6 As shown, 13 C NMR (150MHz,pyridine-D 5): δ89.4(C-1),86.5(C-2),45.2(C-3),106.5(C-4),44.3(C-5),71.9(C-6),23.4(C-7) ,61.9(C-8),102.1(C-9),20.3(C-10),100.8(C-1'),75.3(C-2'),78.7(C-3'),72.2(C- 4'),75.7(C-5'),64.1(C-6'),131.1(C-1”),130.4(C-2” / C-6”),129.3(C-3” / C-5”),1 33.8(C-4”’),166.9(C-1”’),117.0(C-2”’),157.7(C-3”’),27.5(C-4”’),20.6(C-5”’).

[0097] Experimental Example 1 Effect of three kinds of Senecio acyl paeoniflorin on H 2 O 2 The damaged HSF cells and HaCaT cells had a protective effect.

[0098] 1. Materials and Methods

[0099] 1.1 Materials and samples

[0100] Human skin fibroblasts (HSF) and human immortalized epidermal cells (HaCaT) were purchased from Beijing Beina Chuanglian Biotechnology Co., Ltd.; culture medium (Dulbecco's Modified Eagle Medium, DMEM) was purchased from Thermo Fisher Scientific (Suzhou, China); serum (fetal bovine serum, FBS) was purchased from Life Technologies (NY, USA); hydrogen peroxide (H 2 O 2 ) was purchased from Xilong Science (Sichuan, China); MTT was purchased from Biotian Biotechnology (Shanghai, China).

[0101] Samples: 4'-O-Senecioyl-peonidin (Compound 1), 3'-O-Senecioyl-peonidin (Compound 2), 6'-O-Senecioyl-peonidin (Compound 3) prepared in Example 1; paeonidin.

[0102] 1.2 Instruments

[0103] Full wavelength scanning multifunctional microplate reader (Thermofisher Varioskan LUX, USA); carbon dioxide incubator (Thermo 3111, USA); biological safety cabinet (Thermo 1300 series A2, USA).

[0104] 1.3 Experimental process

[0105] 1). Take cells in logarithmic phase, discard old culture medium, wash twice with PBS, and discard PBS;

[0106] 2) Digest the cells with 0.25% trypsin. When the cell outline becomes darker and tends to become round under the microscope, quickly remove the trypsin.

[0107] 3). Use DMEM complete medium containing 10% FBS to terminate digestion and resuspend the cells. Take 10 μL of cell suspension, count with a cell counter, and adjust the cell concentration to 3×10 4 / mL, inoculated in 96-well plates, added 100 μL of cell suspension to each well, and incubated at 37°C, 5% CO 2 Incubate in an incubator for 24 h to allow the cells to adhere;

[0108] 4). Aspirate the culture medium and add 1mM H 2 O 2 Add to the model injury wells (model group), add 100 μL to each well, add the same amount of complete medium to the control group, and continue incubating in the incubator for 1 hour;

[0109] 5). Absorb and discard H 2 O 2 , washed twice with PBS, discarded PBS, and added the diluted sample (concentration of 10 μg / mL, diluent is culture medium) to the plate model injury wells (experimental group), added 100 μL to each well, set 3 replicates for each concentration, and continued incubation in the incubator for 24 h;

[0110] 6). Aspirate the culture medium, add the prepared MTT solution (1 mg / mL), add 100 μL to each well, and incubate in the incubator for 4 hours;

[0111] 7). Aspirate the MTT solution, add DMSO, add 100 μL to each well, and incubate in the incubator for 10 minutes;

[0112] 8). The absorbance was measured at 490 nm using an ELISA reader and calculated using the formula: repair rate = (experimental group - model group) / (control group - model group) × 100%. The statistical software GraphPad prism 5 was used to plot the graph. The experiment was repeated 3 times.

[0113] 2. Results:

[0114] like Figure 7-Figure 8 As shown, compounds 1-3 have 2 O 2At a concentration of 10 μg / mL, compounds 1-3 had a certain protective effect on H 2 O 2 The repair rates of damaged HaCaT cells were 18.3%, 29.6% and 16.0% respectively; 2 O 2 The repair rates of damaged HSF cells were 14.0, 14.2 and 12.5%, respectively, which were higher than the repair rate of paeoniflorin (11.7%).

[0115] Experimental Example 2 Effects of three senecioyl-peonidins on promoting the secretion of hyaluronic acid (HA) and type I collagen (ColⅠ) by HSF cells.

[0116] 1. Materials and Methods

[0117] 1.1 Materials and samples

[0118] Human skin fibroblasts (HSF) were purchased from Beijing Beina Chuanglian Biotechnology Co., Ltd.; culture medium (Dulbecco's Modified Eagle Medium, DMEM) was purchased from Thermo Fisher Scientific (Suzhou, China); serum (fetal bovine serum, FBS) was purchased from Life Technologies (NY, USA); (Sichuan, China); hyaluronic acid quantitative detection kit (HA ELISA Kit) was purchased from Wuhan Huamei Bioengineering Co., Ltd. (Wuhan, China); type Ⅰ collagen quantitative detection kit (Col Ⅰ ELISA Kit) was purchased from Wuhan Huamei Bioengineering Co., Ltd. (Wuhan, China).

[0119] Samples: 4'-O-Senecioyl-peonidin (Compound 1), 3'-O-Senecioyl-peonidin (Compound 2), 6'-O-Senecioyl-peonidin (Compound 3) prepared in Example 1; paeonidin.

[0120] 1.2 Instruments

[0121] Full wavelength scanning multifunctional microplate reader (Thermofisher Varioskan LUX, USA); carbon dioxide incubator (Thermo 3111, USA); biological safety cabinet (Thermo 1300series A2, USA), low-temperature centrifuge (Eppendorf centrifuge 5415R, USA).

[0122] 1.3 Experimental process

[0123] 1). Take cells in logarithmic phase, discard old culture medium, wash twice with PBS, and discard PBS;

[0124] 2) Digest the cells with 0.25% trypsin. When the cell outline becomes darker and tends to become round under the microscope, quickly remove the trypsin.

[0125] 3). Use DMEM complete medium containing 10% FBS to terminate digestion and resuspend the cells. Take 10 μL of cell suspension, count with a cell counter, and adjust the cell concentration to 1×10 4 / mL, inoculated in 96-well plates, added 100 μL of cell suspension to each well, and incubated at 37°C, 5% CO 2 Incubate in an incubator for 24 h to allow the cells to adhere;

[0126] 4). Aspirate the culture medium and add the diluted sample (concentration of 10.0μg / mL or 20.0μg / mL, diluent is culture medium, experimental group) to the plate, add 100μL to each well, set up 3 replicates for each concentration, add an equal amount of complete culture medium to the negative group, and continue incubating in the incubator for 48h;

[0127] 5). Aspirate the supernatant culture medium, centrifuge at 4°C, 1000g for 20 min, and take the supernatant for testing;

[0128] 6). Follow the instructions of the HA and ColⅠ detection kits to detect the content of ColⅠ and HA;

[0129] 7). The absorbance was measured at 450 nm using an enzyme-labeled instrument, and the growth rate was calculated using the formula: growth rate = (experimental group - negative group) / negative group × 100%. The statistical software GraphPad prism 5 was used to plot the graph. The experiment was repeated 3 times.

[0130] 2. Results:

[0131] like Fig. 9 and Fig.10 As shown, compounds 1-3 can promote the secretion of hyaluronic acid (HA) and type I collagen (ColⅠ) by HSF cells. At a concentration of 10.0 μg / mL, the growth rates of hyaluronic acid (HA) secretion by compounds 1-3 were 20.0%, 27.0% and 36.0%, respectively, which were significantly higher than the growth rate of paeoniflorin (13.0%). At a concentration of 20.0 μg / mL, the growth rates of type I collagen (ColⅠ) secretion by compounds 1-3 were -16.7%, 22.0% and -6.0%, respectively.

[0132] Experimental Example 3 DPPH free radical scavenging activity of three senecioylpaeoniflorins.

[0133] 1. Materials and Methods

[0134] 1.1 Materials and samples

[0135] 1,1-Diphenyl-2-picrylhydrazyl (DPPH) was purchased from Beijing Solebow Technology Co., Ltd.; anhydrous ethanol (Ethanol) was purchased from Sichuan Xilong Science Co., Ltd.

[0136] Samples: 4'-O-Senecioyl-peonidin (Compound 1), 3'-O-Senecioyl-peonidin (Compound 2), and 6'-O-Senecioyl-peonidin (Compound 3) prepared in Example 1.

[0137] 1.2 Instruments

[0138] Varioskan LUX desktop multifunctional microplate reader (Thermo Scientific, USA); analytical balance (AG135, Metler Toledo, China).

[0139] 1.3 Experimental process

[0140] 1). Prepare DPPH into a 100 μM working solution with anhydrous ethanol;

[0141] 2) In a 96-well plate, add 100 μL of DPPH working solution and 100 μL of the sample to be tested (200 μg / mL, diluent is sterile water) to each well, and set up a control group with an equal volume of diluent, and set up 3 replicate wells for each group;

[0142] 3) Leave at room temperature for 30 minutes;

[0143] 4). The absorbance was measured at a wavelength of 517 nm using an enzyme reader, and the inhibition rate was calculated using the formula: inhibition rate = (control group - experimental group) / control group × 100%, and the statistical software GraphPad prism 6 was used to plot the graph.

[0144] 2. Results

[0145] like Fig.11 As shown, compounds 1-3 have DPPH free radical scavenging activity, and at a concentration of 200 μg / mL, the scavenging rates of DPPH are 7.7%, 1.7% and 10.2%, respectively.

[0146] Experimental Example 4 Tyrosinase inhibitory activity of three types of senecioyl-peonidin.

[0147] 1. Materials and Methods

[0148] 1.1 Materials and samples

[0149] Tyrosinase (TYR) was purchased from Beijing Solebow Technology Co., Ltd.; L-dopa was purchased from MCE Company; PBS was purchased from Wuhan Saiweier Biotechnology Co., Ltd.

[0150] Samples: 4'-O-Senecioyl-peonidin (Compound 1), 3'-O-Senecioyl-peonidin (Compound 2), 6'-O-Senecioyl-peonidin (Compound 3) prepared in Example 1; paeonidin.

[0151] 1.2 Instruments

[0152] Varioskan LUX desktop multifunctional microplate reader (Thermo Scientific, USA); analytical balance (AG135, Metler Toledo, China); constant temperature box (DHP-9082, Shanghai).

[0153] 1.3 Experimental process

[0154] 1). Prepare 100U / mL tyrosinase working solution with PBS;

[0155] 2) Weigh 4 mg of L-DOPA and dissolve it in 10 mL of PBS to make a 2 mM working solution;

[0156] 3) In a 96-well plate, add 50 μL of L-DOPA working solution and 100 μL of the sample to be tested (20 μg / mL or 40 μg / mL, with sterile water as the diluent) to each well, and set up sample test wells and sample control wells for each group;

[0157] 4) After mixing, place at 37°C for 5 min;

[0158] 5). Add 50 μL of tyrosinase working solution to the sample test well and 50 μL of PBS to the sample control well;

[0159] 6) After mixing, place at 37°C for 5 minutes;

[0160] 7) The absorbance was measured at 475 nm using a microplate reader, and the inhibition rate was calculated using the formula: inhibition rate = [(experimental control group wells - background control wells) - (sample test wells - sample control wells)] / (experimental control group wells - background control wells) × 100%, and the statistical software GraphPad prism 6 was used to plot the inhibition rate.

[0161] 2. Results

[0162] like Fig.12As shown, compounds 1-3 have tyrosinase inhibitory activity and show a certain dose-effect relationship. At 20 μg / mL, the inhibition rates are 8.7%, 5.0% and 5.7%, respectively; at 40 μg / mL, the inhibition rates are 10.7%, 5.7% and 10.7%, respectively. Among them, the inhibition rate of compound 1 at 20 μg / mL is higher than that of paeoniflorin (7.3%). The inhibition rates of compounds 1 and 3 at 40 μg / mL are significantly higher than that of paeoniflorin (7.6%).

[0163] in conclusion

[0164] The above results show that the compounds 1-3 prepared by the present invention have an 2 O 2 It has a protective effect on damaged HSF cells and HaCaT cells, promotes the secretion of hyaluronic acid (HA) and type I collagen (ColⅠ) by HSF cells, has DPPH free radical scavenging activity, and has tyrosinase inhibitory activity. It can be used as a cosmetic component for anti-aging, whitening, skin protection, and for supplementing collagen and hyaluronic acid, and is used in various cosmetics.

[0165] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A Senecioyl-peonidin, characterized in that: include: The compound shown in formula (I): Where: When R1=R3=H, R2 is When R1=R2=H, R3 is When R2=R3=H, R1 is 2. The method for preparing the senecioylpaeoniflorin according to claim 1, characterized in that: include: Dissolving compound a in an organic solvent, adding organic tin, a basic catalyst and compound b, and subjecting the mixture to an esterification condensation reaction to obtain a compound represented by formula (I); The synthetic route is as follows: Where: When R1=R3=H, R2 is When R1=R2=H, R3 is When R2=R3=H, R1 is 3. The method for preparing senecioylpaeoniflorin according to claim 2, characterized in that: The molar ratio of compound 1, organotin, alkaline catalyst and compound 2 is 1:1-2:1-2:1-2, preferably 1:1:1.2:1; And / or, the organotin is selected from any one of methyltin dichloride, dibutyltin dichloride, butyltin trichloride, and dibutyltin oxide; And / or, the basic catalyst is selected from any one of triethylamine, trimethylamine, triethylenediamine and 1,8-diazabicyclo[5.4.0]undec-7-ene.

4. The method for preparing senecioylpaeoniflorin according to claim 2 or 3, characterized in that: The reaction conditions of the esterification condensation reaction are room temperature for 0.5 to 5 hours, preferably 1 hour.

5. The method for preparing senecioylpaeoniflorin according to any one of claims 2 to 4, characterized in that: The organic solvent is selected from any one of acetone, dioxane, tetrahydrofuran, ethylene glycol dimethyl ether, N,N-dimethylformamide and dimethyl sulfoxide.

6. The method for preparing senecioylpaeoniflorin according to any one of claims 2 to 5, characterized in that: After the esterification condensation reaction is completed, the product is quenched, extracted, dried and purified.

7. The method for preparing senecioylpaeoniflorin according to claim 6, characterized in that: In the purification step, the column chromatography conditions are as follows: the weight of silica gel used in silica gel column chromatography is 10 to 50 times that of the sample, and methanol-chloroform is eluted at a volume ratio of 2.5 to 5:97.5 to 95.

8. The senecioyl-peonidin according to claim 1 has any of the following uses: (1) Use in soothing, repairing or protecting the skin or in preparing products for soothing, repairing or protecting the skin; (2) Use as an antioxidant or in the preparation of products with antioxidant effects; (3) Use in anti-skin aging and / or skin moisturizing or in the preparation of anti-skin aging and / or skin moisturizing products; (4) Use in promoting the secretion of collagen and / or hyaluronic acid by the skin or in preparing products that secrete collagen and / or hyaluronic acid by the skin; (5) Use in skin whitening, melanin inhibition or in the preparation of skin whitening or melanin inhibition products.

9. The use according to claim 8, characterized in that include: Use in soothing, repairing or protecting skin damaged by H2O2 or in preparing a product for soothing, repairing or protecting skin damaged by H2O2; and / or, use as an antioxidant for scavenging DPPH free radicals or in the preparation of a product with an antioxidant effect for scavenging DPPH free radicals; and / or, use as a tyrosinase inhibitor or in the preparation of a product having the effect of inhibiting tyrosinase; And / or, the product includes cosmetics, medicines or health foods.

10. A cosmetic, characterized in that: It includes the senecioyl-peonidin as described in claim 1.

Images