The invention relates to 6apos; -O-acyl paeoniflorin as well as preparation method and application thereof

Abstract

The invention relates to the technical field of cosmetics, in particular to 6 '-O-acyl paeoniflorin and a preparation method and application thereof. Comprising a compound shown in a formula (I), and the compound shown in the formula (I) comprises 6 '-O-p-chlorobenzoyl paeoniflorin (1), 6'-O-p-bromobenzoyl paeoniflorin (2) and 6 '-O-crotonyl paeoniflorin (3). The compound disclosed by the invention has anti-inflammatory activity, effects of relieving and repairing skin, an anti-oxidation effect and capability of increasing secretion of collagen and hyaluronic acid, so that the compound can be applied to various cosmetics.

Description

Technical Field

[0001] The present invention relates to the technical field of cosmetics, and in particular to 6'-O-acyl paeoniflorin, a preparation method and application thereof. Background Art

[0002] Sensitive skin is a skin condition that is sensitive, poorly tolerated, and prone to allergies. It changes with the environment, physical condition, etc. Although sensitive skin is not considered a skin disease in medicine, the symptoms can be alleviated or eliminated through the use of cosmetics. Sensitive skin mainly occurs on the face, scalp, back of the hands, etc., and its main characteristics are dryness, tightness, redness, itching, stinging, scaling, etc., which can affect personal emotions and mental state in severe cases. The causes of sensitive skin are complex, and there is currently no specific treatment. Targeted use of specific functional cosmetics is an effective way to alleviate symptoms.

[0003] The causes of sensitive skin include impaired skin barrier function, neural factors, inflammation, etc., among which impaired skin barrier function is an important cause of skin sensitivity. Skin damage is mainly manifested by intracellular water loss, oxidative free radical damage, reduced cell matrix secretion, and reduced keratinocytes. Therefore, skin sensitivity can be improved by repairing damaged keratinocytes, promoting cell matrix secretion, and reducing inflammatory reactions.

[0004] Skin damage is mainly manifested by intracellular water loss, oxidative free radical damage, and reduced cell matrix secretion. Skin aging reduces the skin's ability to protect and regulate the body, so the skin cannot adapt to changes in the internal and external environment, and changes occur in the overall appearance of color, luster, shape, texture, etc. 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, and sagging skin. 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, and pigmentation. Therefore, ingredients that have both soothing and repairing effects have broad application prospects in the field of cosmetics.

[0005] At present, there is no relevant report on the 6'-O-acylpaeoniflorin of the compound of the present invention and its preparation method and use. Summary of the invention

[0006] Therefore, the technical problem to be solved by the present invention is to provide a 6'-O-acyl paeoniflorin and a preparation method and use thereof. The 6'-O-acyl paeoniflorin provided by the present invention includes 6'-O-p-chlorobenzoyl paeoniflorin (1), 6'-O-p-bromobenzoyl paeoniflorin (2), and 6'-O-crotonyl paeoniflorin (3), which have anti-aging, moisturizing, anti-inflammatory, antioxidant, skin soothing and skin repairing effects, and can be applied to the field of cosmetics. The raw materials of the compound preparation method are easily available, easy to operate, and suitable for industrial production.

[0007] To this end, the present invention provides the following technical solutions:

[0008] The embodiment of the present invention discloses a 6'-O-acyl paeoniflorin, comprising: a compound as shown in formula (I):

[0009]

[0010] R is

[0011] The embodiment of the present invention discloses a method for preparing 6'-O-acylpaeoniflorin, comprising the following steps:

[0012] 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);

[0013] The synthetic route is as follows:

[0014]

[0015] R is

[0016] In some embodiments, the molar ratio of compound 1, organotin, alkaline catalyst and compound 2 is 1:1-2:1-2:1-2, preferably 5:5:6:5.

[0017] In some embodiments, the organotin includes, but is not limited to, any one of dimethyltin dichloride, dibutyltin dichloride, butyltin trichloride, and dibutyltin oxide.

[0018] In some embodiments, the basic catalyst includes, but is not limited to, any one of triethylamine trimethylamine, DABCO, and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU).

[0019] In some embodiments, the reaction conditions of the esterification condensation reaction are room temperature for 0.5 to 5 hours, preferably 1 hour. The room temperature range is 10-30°C, which can be any one of 10, 15, 20, 25, 30°C or a range between any two values.

[0020] 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.

[0021] In some embodiments, after the esterification condensation reaction is completed, the reaction is quenched, extracted, dried, and purified.

[0022] In some embodiments, in the purification step, the column chromatography conditions are as follows: the amount of silica gel in the silica gel column is 10-50 times the weight of the sample, and methanol-chloroform is eluted at a volume ratio of 2.5-5:97.5-95.

[0023] The embodiments of the present invention disclose that the 6'-O-acyl paeoniflorin has any of the following uses:

[0024] (1) Use in soothing, repairing or protecting the skin or in preparing products for soothing, repairing or protecting the skin;

[0025] (2) Use as an antioxidant or in the preparation of products with antioxidant effects;

[0026] (3) Use in anti-skin aging and / or skin moisturizing or in the preparation of anti-skin aging and / or skin moisturizing products;

[0027] (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;

[0028] (5) Use in skin anti-inflammatory or in the preparation of skin anti-inflammatory products;

[0029] In some embodiments, the method comprises: using the method for soothing, repairing or protecting skin damaged by H2O2 or preparing a product for soothing, repairing or protecting skin damaged by H2O2;

[0030] Or, use as an antioxidant for scavenging DPPH free radicals or in the preparation of a product with antioxidant effect for scavenging DPPH free radicals.

[0031] In some embodiments, the product comprises a cosmetic, a pharmaceutical, or a food.

[0032] The embodiment of the present invention discloses a cosmetic comprising the 6'-O-acyl paeoniflorin. The content of the 6'-O-acyl paeoniflorin is 0.001wt% or more, such as any one of 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 a range between any two values.

[0033] In some embodiments, the cosmetic further comprises auxiliary materials, carriers and / or excipients acceptable in the field of cosmetics.

[0034] 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.

[0035] 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 wash, and a facial mask.

[0036] 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.

[0037] The technical solution of the present invention has the following advantages:

[0038] 1. A 6'-O-acyl paeoniflorin provided by the present invention comprises: a compound as shown in formula (I), wherein the compound as shown in formula (I) comprises 6'-O-p-chlorobenzoyl paeoniflorin (1), 6'-O-p-bromobenzoyl paeoniflorin (2), and 6'-O-crotonyl paeoniflorin (3). The present invention has found that: the above-mentioned compound has a protective effect on HSF cells and HaCaT cells damaged by H2O2, so it can be concluded that it can soothe, repair or protect the skin; the compound promotes the secretion of type I collagen (ColⅠ) by HaCaT cells and promotes the secretion of hyaluronic acid (HA) by HSF cells, and it can be concluded that it has the effect of increasing the secretion of collagen and hyaluronic acid in the skin; the compound has DPPH free radical scavenging activity and can be used for skin anti-oxidation; the compound has an anti-inflammatory effect; the above indicates that the compound of the present invention has anti-inflammatory activity, soothing and repairing skin effects, anti-oxidation effects, and increases the secretion of collagen and hyaluronic acid, so the compound can be applied to various cosmetics.

[0039] 2. The present invention provides a method for preparing 6'-O-acylpaeoniflorin. The raw materials of the compound preparation method are readily available, easy to operate, and suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] 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.

[0041] Figure 1 is the 6'-O-p-chlorobenzoylpaeoniflorin in Example 1 of the present invention 1 H NMR spectra;

[0042] Figure 2 is the 6'-O-p-chlorobenzoylpaeoniflorin in Example 1 of the present invention 13 C NMR;

[0043] Figure 3 is the 6'-O-p-bromobenzoylpaeoniflorin in Example 1 of the present invention 1 H NMR spectra;

[0044] Figure 4 is the 6'-O-p-bromobenzoylpaeoniflorin in Example 1 of the present invention 13 C NMR;

[0045] Figure 5 is the 6'-O-crotonyl paeoniflorin in Example 1 of the present invention 1 H NMR spectra;

[0046] Figure 6 is the 6'-O-crotonyl paeoniflorin in Example 1 of the present invention 13 C NMR;

[0047] Figure 7 This is the result of the protective effect of different compounds in Example 1 of the present invention on HaCaT cells damaged by H2O2;

[0048] Figure 8 This is the result of the protective effect of different compounds in Example 1 of the present invention on HSF cells damaged by H2O2;

[0049] 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;

[0050] 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;

[0051] Fig.11 is the result of DPPH radical scavenging activity of different compounds in Example 3 of the present invention;

[0052] Fig.12 It is the result of the anti-inflammatory activity of different compounds in Example 4 of the present invention;

[0053] Above Figure 7-Figure 12 The data statistical method used in this study is the t-test method for statistical analysis. DETAILED DESCRIPTION

[0054] 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.

[0055] 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.

[0056] Example 1

[0057] This embodiment provides a method for preparing 6'-O-acyl paeoniflorin, comprising the following steps: The structural formula of 6'-O-acyl paeoniflorin is as follows:

[0058]

[0059] R is

[0060] The synthetic route is as follows:

[0061]

[0062] R is

[0063] Specific preparation method:

[0064] Paeoniflorin (0.05 mmol, compound a) was dissolved in 1 mL of dry acetone, and dimethyltin dichloride (0.05 mmol), triethylamine (8.3 μL, 0.06 mol) and the corresponding acyl chloride (p-chlorobenzoyl chloride, CAS: 122-01-0; or, p-bromobenzoyl chloride, CAS: 586-75-4; or, trans-2-methyl-2-butenoyl chloride, CAS: 35660-94-7) (0.05 mmol, compound b) were added in sequence under stirring. After reacting at room temperature (25°C) for 1 h, 5 mL of water was added to quench the reaction, and the mixture was extracted with n-butanol (5 mL×3). The organic phases were combined, dried over anhydrous sodium sulfate, and adsorbed on silica gel 1.5 times the weight of the sample. The solution was evaporated to dryness 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 for TLC detection to obtain the following:

[0065] When the acyl chloride is p-chlorobenzoyl chloride, 6'-O-p-chlorobenzoylpaeoniflorin (21 mg, yield 69%) is obtained; or

[0066] When the acyl chloride is p-bromobenzoyl chloride, 6'-O-p-bromobenzoylpaeoniflorin (24 mg, yield 73%) is obtained; or

[0067] When the acid chloride is trans-2-methyl-2-butenoyl chloride, 6'-O-crotonyl paeoniflorin (17 mg, yield 60%) is obtained.

[0068] The compound structural formula of 6'-O-p-chlorobenzoylpaeoniflorin is as follows:

[0069]

[0070] Molecular formula: C 30 H 31 O 12 Cl

[0071] Molecular weight: 619

[0072] Appearance: White powder

[0073] HRESIMS m / z 663.1440 (calculated 663.1486, [M+HCOO] - ).

[0074] like Figure 1 As shown, 1 H NMR (600MHz,pyridine-D5):δ H8.13(2H,d,J=8.4Hz,H-2” / H-6”),8.12(2H,d,J=7.8Hz,H-2”’ / H-6”’),7.48-7.42(m,3H,H-4”,H-3”’ / H-5”’),7.3 2-7.29(m,2H,H-3” / H-5”),5.94(s,1H,H-9),5.20(d,J=12.0Hz,1H,H-8a),5.15(d,J=12.0Hz,1H,H-8b),5.06-4.97 (m,3H,H-6'a,H-6'b,H-1'),4.25-4.22(m,1H,H-5'),4.12-4.03(m,3H,H-2',H-3',H-4'),3.08(m,1H,H-5),2.89-2 .86(m,1H,H-7a),2.45(d,J=12.0Hz,H-3a),2.33(d,J=11.4Hz,H-7b),2.21(d,J=12.0Hz,H-3b),1.68(s,3H,H-10);

[0075] like Figure 2 As shown, 13 C NMR (150MHz, pyridine-D5): δ89.4(C-1),86.5(C-2),45.2(C-3),106.4(C-4),44.2(C-5),71.8(C-6) ,23.4(C-7),61.9(C-8),102.1(C-9),20.3(C-10),100.8(C-1'),75.5(C-2'),78.6(C-3'),72.1(C-4 '),75.3(C-5'),65.8(C-6'),131.0(C-1”),130.4(C-2” / C-6”),129.3(C-3” / C-5”),133.8(C-4”),16 7.1(C-7”’),129.9(C-1”’),131.9(C-2”’ / C-6”’),129.6(C-3”’ / C-5”’),139.9(C-4”’),166.0C-7”’).

[0076] The compound structure of 6'-O-4-bromobenzoylpaeoniflorin is as follows:

[0077]

[0078] Molecular formula: C 30 H 31 B O 15

[0079] Molecular weight: 663

[0080] Appearance: White powder

[0081] HRESIMS m / z 685.0982 (calculated 685.0891, [M+Na] + ).

[0082] like Figure 3 As shown, 1 H NMR (600MHz,pyridine-D5):δ H 8.12(2H,d,J=7.2Hz,H-2” / H-6”),8.07(2H,d,J=8.4Hz,H-2”’ / H-6”’),7.62(2H,d,J=8.4Hz,H-3”’ / H-5”’),7 .48-7.45(m,1H,H-4”),7.32-7.29(m,2H,H-3” / H-5”),5.94(s,1H,H-9),5.21-4.97(m,5H,H-8a,H-8b,H-6'a,H -6'b,H-1'),4.24-4.23(m,1H,H-5'),4.10-4.03(m,3H,H-2',H-3',H-4'),3.08-3.07(m,1H,H-5),2.89-2.86( m,1H,H-7a),2.45(d,J=12.6Hz,H-3a),2.33(d,J=12.0Hz,H-7b),2.20(d,J=12.0Hz,H-3b),1.68(s,3H,H-10);

[0083] like Figure 4 As shown, 13 C NMR (150MHz, pyridine-D5): δ89.4(C-1),86.5(C-2),45.2(C-3),106.4(C-4),44.2(C-5),71.8(C-6) ,23.4(C-7),61.9(C-8),102.1(C-9),20.3(C-10),100.8(C-1'),75.5(C-2'),78.7(C-3'),72.1(C-4' ),75.3(C-5'),65.8(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”’),128.8(C-1”’),132.6(C-2”’ / C-6”’),132.0(C-3”’ / C-5”’),130.3(C-4”’),166.2(C-7”’).

[0084] The compound structural formula of 6'-O-crotonyl paeoniflorin is as follows:

[0085]

[0086] Molecular formula: C 28 H 34 O 12

[0087] Molecular weight: 562

[0088] Appearance: White powder

[0089] HRESIMS m / z 607.2046 (calculated 607.2032, [M+HCOO] - )

[0090] like Figure 5 As shown, 1 H NMR (600MHz,pyridine-D5):δ H 8.13(2H,d,J=7.2Hz,H-2” / H-6”),7.48-7.45(m,1H,H-4”),7.32-7.29(m,2H,H-3” / H-5”),7.03(q,1H,H-3”’),5. 96(s,1H,H-9),5.21-5.04(m,4H,H-8a,H-8b,H-6'a,H-1'),4.82-4.79(m,1H,H-6'b),4.22-4.19(m,1H,H-5'),4. 05-4.02(m,3H,H-2',H-3',H-4'),3.12-3.11(m,1H,H-5),2.90-2.87(m,1H,H-7a),2.57(d,J=12.0Hz,H-3a),2.3 9(d,J=12.0Hz,H-7b),2.30(d,J=12.0Hz,H-3b),1.86(s,3H,H-5"'),1.71(s,3H,H-10),1.62(d,J=7.2Hz,H-4"');

[0091] like Figure 6 As shown, 13C NMR (150MHz, pyridine-D5): δ89.4(C-1),86.5(C-2),45.2(C-3),106.5(C-4),44.3(C-5),71.9(C -6),23.3(C-7),61.9(C-8),102.1(C-9),20.3(C-10),100.7(C-1'),75.3(C-2'),78.7(C-3'),72 .2(C-4'),75.7(C-5'),65.1(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”),168.2(C-1”’),129.4(C-2”’),138.1(C-3”’),12.7(C-4”’),14.6(C-5”’).

[0092] Experimental Example 1 Three 6'-O-acylpaeoniflorins have protective effects on HSF cells and HaCaT cells damaged by H2O2.

[0093] 1. Materials and Methods

[0094] 1.1 Materials and samples

[0095] Materials: 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 (H2O2) was purchased from Xilong Science (Sichuan, China); MTT was purchased from Beyotime Biotechnology Co., Ltd.

[0096] Samples: 6'-O-p-chlorobenzoylpaeoniflorin (Compound 1), 6'-O-p-bromobenzoylpaeoniflorin (Compound 2), 6'-O-crotonylpaeoniflorin (Compound 3) prepared in Example 1; paeoniflorin.

[0097] 1.2 Instruments

[0098] Full-wavelength scanning multifunctional microplate reader (Thermofisher Varioskan LUX, USA); carbon dioxide incubator (Thermo 3111, USA); biological safety cabinet (Thermo 1300 series A2, USA).

[0099] 1.3 Experimental process

[0100] 1). Take cells in logarithmic phase, discard old culture medium, wash twice with PBS buffer, and discard PBS buffer;

[0101] 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.

[0102] 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 on a 96-well plate, added 100 μL of cell suspension to each well, and incubated in a 37°C, 5% CO2 incubator for 24 h to allow the cells to adhere;

[0103] 4). Aspirate and discard the culture medium, add 1mM H2O2 prepared in complete culture medium to the model injury wells (model group), add 100μL to each well, add the same amount of complete culture medium to the control group, and continue incubating in the incubator for 1h;

[0104] 5). Aspirate and discard H2O2, wash twice with PBS buffer, discard PBS buffer, add the diluted sample (concentration of 5μg / mL or 10μg / mL, diluent is culture medium) to the modeling injury wells (experimental group) of the well plate, add 100μL to each well, set 3 replicates for each concentration, and continue incubating in the incubator for 24h;

[0105] 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;

[0106] 7). Aspirate the MTT solution, add dimethyl sulfoxide (DMSO), add 100 μL to each well, and incubate in the incubator for 10 minutes;

[0107] 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.

[0108] 2. Results:

[0109] like Figure 7 and Figure 8As shown, compounds 1-3 have a certain protective effect on HaCaT and HSF cells damaged by H2O2. When the concentration of compounds 1-3 was 5μg / mL, the repair rates of HaCaT cells damaged by H2O2 were 13.4, 37.1 and 38.4%, respectively; when the concentration was 10μg / mL, the repair rates of HaCaT cells damaged by H2O2 were 4.0, 48.3 and 33.5%, respectively. When the concentration of paeoniflorin was 5 and 10μg / mL, the repair rates of HaCaT cells damaged by H2O2 were 25.7 and 41.7%, respectively. It can be seen that at 5μg / mL, the activity of compounds 2 and 3 was higher than that of the control paeoniflorin, and at 10μg / mL, the activity of compound 2 was higher than that of paeoniflorin.

[0110] At a concentration of 5 μg / mL, the repair rates of compounds 1-3 on HSF cells damaged by H2O2 were 8.8, 16.2 and 10.0%, respectively; at a concentration of 10 μg / mL, the repair rates of compounds 1-3 on HSF cells damaged by H2O2 were 6.4, 13.8 and 19.6%, respectively. The repair rates of paeoniflorin on HSF cells damaged by H2O2 at 5 and 10 μg / mL were 21.2 and 11.7%, respectively, among which the repair rates of compounds 2 and 3 at 10 μg / mL were higher than the repair rate of the control paeoniflorin (11.7%).

[0111] Experimental Example 2 Three 6'-O-acyl paeoniflorins promote the secretion of type I collagen (ColⅠ) by HaCaT cells and the secretion of hyaluronic acid (HA) by HSF cells

[0112] 1. Materials and Methods

[0113] 1.1 Materials and samples

[0114] Human immortalized epidermal cells (HaCaT) and 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 (ColⅠELISA Kit) was purchased from Wuhan Huamei Bioengineering Co., Ltd. (Wuhan, China).

[0115] Samples: 6'-O-p-chlorobenzoylpaeoniflorin (Compound 1), 6'-O-p-bromobenzoylpaeoniflorin (Compound 2), 6'-O-crotonylpaeoniflorin (Compound 3) prepared in Example 1; paeoniflorin.

[0116] 1.2 Instruments

[0117] 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).

[0118] 1.3 Experimental process

[0119] 1). Take cells in logarithmic phase, discard old culture medium, wash twice with PBS, and discard PBS;

[0120] 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.

[0121] 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 on a 96-well plate, added 100 μL of cell suspension to each well, and incubated in a 37°C, 5% CO2 incubator for 24 h to allow the cells to adhere;

[0122] 4). Aspirate the culture medium and add the diluted sample (10.0 μg / mL or 20.0 μg / mL, the diluent is the culture medium, which is the experimental group) to the plate, add 100 μL 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 48 hours;

[0123] 5). Aspirate the supernatant culture medium, centrifuge at 4°C, 1000g for 20 min, and take the supernatant for testing;

[0124] 6). Follow the instructions of the HA and ColⅠ detection kits to detect the content of ColⅠ and HA;

[0125] 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) / negative × 100%. The statistical software GraphPad prism 5 was used to plot the graph. The experiment was repeated 3 times.

[0126] 2. Results:

[0127] like Fig. 9 As shown, compounds 1-3 can promote the secretion of type I collagen (ColⅠ) by HaCaT cells. At a concentration of 20.0 μg / mL, the growth rates of type I collagen (ColⅠ) secretion are 3.0, 16.0 and 10.5%, respectively. At a concentration of 10.0 μg / mL, the growth rate of compound 2 on type I collagen (ColⅠ) secretion is 4.4%.

[0128] like Fig.10 As shown, compounds 1-3 can promote the secretion of hyaluronic acid (HA) by HSF cells. At a concentration of 10.0 μg / mL, the growth rates of hyaluronic acid (HA) secretion are 28.3, 6.7 and 24.0%, respectively. At a concentration of 10.0 μg / mL, the activity of compounds 1 and 3 is higher than that of paeoniflorin (13.0%).

[0129] Experimental Example 3 Three 6'-O-acylpaeoniflorins have DPPH free radical scavenging activity.

[0130] 1. Materials and Methods

[0131] 1.1 Materials and samples

[0132] 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.

[0133] Samples: 6'-O-p-chlorobenzoylpaeoniflorin (Compound 1), 6'-O-p-bromobenzoylpaeoniflorin (Compound 2), and 6'-O-crotonylpaeoniflorin (Compound 3) prepared in Example 1.

[0134] 1.2 Instruments

[0135] Varioskan LUX desktop multifunctional microplate reader (Thermo Scientific, USA); analytical balance (AG135, Metler Toledo, China).

[0136] 1.3 Experimental process

[0137] 1). Prepare DPPH into a 100 μM working solution with anhydrous ethanol;

[0138] 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;

[0139] 3) Leave at room temperature for 30 minutes;

[0140] 4). Use an enzyme-labeled instrument to measure the absorbance at a wavelength of 517 nm, and calculate the inhibition rate using the formula: inhibition rate = (control group - experimental group) / control group × 100%, and use the statistical software GraphPad prism 6 to draw a graph

[0141] 2. Results

[0142] like Fig.11 As shown, compound 2 has DPPH free radical scavenging activity, and the inhibition rate of DPPH is 11.0% at a concentration of 200 μg / mL.

[0143] Experimental Example 4 Three 6'-O-acylpaeoniflorins have anti-inflammatory activity.

[0144] 1. Materials and Methods

[0145] 1.1 Materials and Instruments

[0146] RAW264.7 cells (Beijing Beina Chuanglian Biotechnology Co., Ltd.); nitric oxide detection kit (Shanghai Biyuntian Biotechnology Co., Ltd.); lipopolysaccharide (LPS, Beijing Solebow Technology Co., Ltd.); DMEM high glucose medium (Shanghai VivaCell); FBS fetal bovine serum (Moregate, Australia); DMSO dimethyl sulfoxide (Beijing Solebow); 1300series A2 biological safety cabinet (Thermo Corporation, USA); 3111 CO2 incubator (Thermo Corporation, USA); optical inverted microscope (Olympus Corporation, Japan); Varioskan LUX desktop multi-function microplate reader (Thermo Corporation, USA).

[0147] Samples: 6'-O-p-chlorobenzoylpaeoniflorin (Compound 1), 6'-O-p-bromobenzoylpaeoniflorin (Compound 2), and 6'-O-crotonylpaeoniflorin (Compound 3) prepared in Example 1.

[0148] 1.2 Experimental methods

[0149] Cells in logarithmic growth phase were taken and 1×10 5100 μL of the prepared sample (2.5 μg / mL, with the diluent being the culture medium) was inoculated into a 96-well plate, and the cells were incubated in an incubator at 37°C and 5% CO2 overnight. The culture medium was discarded, and the prepared sample (2.5 μg / mL, with the diluent being the culture medium) was added into the wells, and 100 μL of the prepared sample (2.5 μg / mL, with the diluent being the culture medium) was added into the wells, and 3 replicate wells were set for each concentration. At the same time, an equal volume of the diluent was added into a control group, and the cells were incubated in an incubator for 24 h. 50 μL of the cell culture fluid was taken, and the detection reagents were added in sequence according to the instructions of the nitric oxide detection kit. The absorbance was measured at a wavelength of 540 nm using an enzyme reader, and the NO inhibition rate was calculated as (control group - experimental group) / control group × 100%, and the statistical software GraphPad prism 6 was used to draw a graph.

[0150] 2. Results

[0151] like Fig.12 As shown, at 2.5 μg / mL, compounds 1-3 had NO generation inhibition rates of 18.0, 15.3 and 1.3%, respectively, showing anti-inflammatory activity.

[0152] in conclusion

[0153] The above results show that the prepared compounds 1-3 have a protective effect on HSF cells and HaCaT cells damaged by H2O2, promote the secretion of type I collagen (ColⅠ) by HaCaT cells, promote the secretion of hyaluronic acid (HA) by HSF cells, have DPPH free radical scavenging activity, and have anti-inflammatory activity. They can be used as cosmetic components for soothing, repairing, moisturizing, anti-oxidation, anti-aging, anti-inflammatory, and for supplementing collagen and hyaluronic acid, and can be used in various cosmetics.

[0154] 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 6'-O-acyl paeoniflorin, characterized in that: include: The compound shown in formula (I): R is 2. A method for preparing 6'-O-acylpaeoniflorin as claimed in claim 1, characterized in that: The steps 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: R is 3. The method for preparing 6'-O-acylpaeoniflorin according to claim 2, characterized in that: The molar ratio of compound a, organotin, alkaline catalyst and compound b is 1:1-2:1-2:1-2, preferably 5:5:6:5; And / or, the organotin is selected from any one of dimethyltin 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 6'-O-acylpaeoniflorin 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 6'-O-acylpaeoniflorin 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 6'-O-acylpaeoniflorin 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 6'-O-acylpaeoniflorin according to claim 6, characterized in that: In the purification step, the column chromatography conditions are as follows: the amount of silica gel in the silica gel column is 10-50 times the weight of the sample, and methanol-chloroform is eluted at a volume ratio of 2.5-5:97.5-95.

8. The 6'-O-acylpaeoniflorin 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 or anti-aging 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 promote the secretion of collagen and / or hyaluronic acid by the skin; (5) Use in anti-inflammatory treatment of the skin or in the preparation of anti-inflammatory products of the skin.

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; 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; Or, the product includes cosmetics, medicines or health foods.

10. A cosmetic, characterized in that: It includes the 6'-O-acylpaeoniflorin as described in claim 1.

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