Use of a composition for the preparation of a retinoic x receptor gamma activator, and topical skin care product containing the same

Abstract

Embodiments of the present disclosure disclose a use of a composition in the preparation of a retinoic acid X receptor gamma activator, the composition comprising phytol, teprenone, and silybin. Embodiments of the present disclosure also disclose a use of the composition in the preparation of an extracellular matrix metalloproteinase inhibitor and an interleukin-6 inhibitor at a specific ratio.

Description

TECHNICAL FIELD

[0001] Embodiments of the present disclosure relate to the field of cosmetic technology, in particular to use of a composition in the preparation of a retinoid X receptor gamma activator, and a topical skin care product containing the same. BACKGROUND

[0002] Retinoid X receptor gamma (RXRγ) is a subtype of retinol receptors belonging to the nuclear receptor family of retinoid X receptors (RXRs). When the RXRγ receptor functions normally, it can bind to ligands such as retinoic acid, activating the expression of a series of genes, ensuring that keratinocytes differentiate according to the normal program. With age, the expression level or function of RXRγ receptor may change. For example, in aging skin, the activity of RXRγ receptor is reduced, which can lead to abnormal differentiation of keratinocytes, impairing the barrier function of the skin, and changing the structure and composition of the stratum corneum, which cannot effectively prevent water loss and resist the invasion of harmful substances from the outside world, making the skin dry and rough.

[0003] It is known in the art that retinoic acid and retinol can be used as retinoid X receptor gamma activators. The inventors have sought to provide new retinoid X receptor gamma activators.

[0004] The above information disclosed in this Background section is only for the purpose of enhancing the understanding of the background of the present inventive concepts, and therefore, it can contain information that does not form the prior art that is already known to those of ordinary skill in the art. SUMMARY

[0005] The Summary section is provided to introduce a selection of concepts in a simplified form that are further described in the detailed description section. The Summary section is not intended to identify key features or essential features of the claimed technology, nor is it intended to be used to determine the scope of the claimed technology.

[0006] Some embodiments of the present disclosure provide use of a composition in the preparation of a retinoid X receptor gamma activator, and a topical skin care product containing the same, to solve one or more of the technical problems mentioned in the Background section.

[0007] In a first aspect, some embodiments of the present disclosure provide use of a composition in the preparation of a retinoid X receptor gamma activator; the composition comprises phytol, teprenone, and silybin.

[0008] In a second aspect, some embodiments of the present disclosure provide use of a composition in preparation of an extracellular matrix metalloproteinase inhibitor; the composition comprises phytol, teprenone, and silybin; the mass ratio of the phytol, the teprenone, and the silybin is 1:0.5-1.5:1-5.

[0009] In a third aspect, some embodiments of the present disclosure provide use of a composition in preparation of an interleukin-6 inhibitor; the composition comprises phytol, teprenone, and silybin; the ratio of the phytol, the teprenone, and the silybin is 1:0.5-1.5:1-5.

[0010] In a fourth aspect, some embodiments of the present disclosure provide use of a transdermal penetration enhancer in preparation of a transdermal penetration enhancer for silybin; the transdermal penetration enhancer is phytol and / or teprenone.

[0011] In a fifth aspect, some embodiments of the present disclosure provide a skin care composition for skin care, comprising silybin and a transdermal penetration enhancer; the transdermal penetration enhancer is phytol and teprenone; the mass ratio of the phytol, the teprenone, and the silybin is 1:0.5-1.5:0.2-5.

[0012] Some embodiments of the present disclosure provide that the ratio of the phytol, the teprenone, and the silybin is 1:0.5-1.5:1-5.

[0013] In a sixth aspect, some embodiments of the present disclosure provide an external skin care product, comprising the skin care composition of the fifth aspect.

[0014] Some embodiments of the present disclosure provide the external skin care product, wherein,

[0015] The phytol is added in an amount of 0.05%-2%;

[0016] The teprenone is added in an amount of 0.05%-2%;

[0017] The silybin is added in an amount of 0.05%-5%.

[0018] Some embodiments of the present disclosure provide that the external skin care product comprises one or more of a water agent, a milk agent, a cream agent, a paste agent, a gel agent, a cleanser, and a mask.

[0019] The various embodiments of the present disclosure have the following technical effects:

[0020] The present disclosure verifies a new retinoic acid X receptor gamma activator through a molecular docking experiment screening.

[0021] The present disclosure verifies that the phytanol, the tetraplerin and the silymarin composition can synergistically inhibit the matrix metalloproteinase-3 and interleukin-6 protein expression in the aging HaCaT cells through the cell experiment. It is illustrated that the skin care composition provided by the present disclosure including the phytanol, the tetraplerin and the silymarin can synergistically inhibit the generation and sagging of the skin wrinkles, can relieve the skin inflammation, maintain the barrier function of the skin, and has the effect of synergistically resisting the light damage and improving the photoaging.

[0022] Surprisingly, through further research, it is found that the phytanol and the tetraplerin alone or in combination with the silymarin can improve the penetration and absorption amount of the silymarin in the skin, and improve the intradermal retention amount of the silymarin, and improve the bioavailability of the silymarin. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 It is a structural schematic diagram of the phytanol;

[0024] Figure 2 It is a structural schematic diagram of the tetraplerin;

[0025] Figure 3 It is a structural schematic diagram of the silymarin;

[0026] Figure 4 It is a docking schematic diagram of the target molecule and RARγ;

[0027] Figure 5 It is a docking schematic diagram of the target molecule and RXRγ;

[0028] Figure 6 It is a CCK8 cell viability test result;

[0029] Figure 7 It is a schematic diagram of the mRNA relative expression level of MMP-3;

[0030] Figure 8 It is a schematic diagram of the mRNA relative expression level of IL-6. DETAILED DESCRIPTION

[0031] Embodiments of the present disclosure will be described in more detail by making reference to the accompanying drawings. Although certain embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure can be implemented in various forms, and should not be interpreted as being limited to the embodiments set forth herein. On the contrary, these embodiments are provided in order to more thoroughly and completely understand the present disclosure. It should be understood that the drawings and embodiments of the present disclosure are only for exemplary purposes, and are not intended to limit the scope of protection of the present disclosure.

[0032] Unless otherwise indicated, all technical and scientific terms used herein have the meanings that are commonly understood by one of ordinary skill in the art in the field of the application. Generally, the terms used herein have their normal and customary meanings as used in the field of the application and are consistent with the normal and customary meanings as used in the field of the application. When amounts, concentrations, or other values or parameters are expressed in the context of a range, a preferred range, or a range having an upper preferred limit and a lower preferred limit, it is understood that all ranges formed with any of the range upper limit or upper preferred limit and any of the range lower limit or lower preferred limit are specifically disclosed. Whether the range is expressly disclosed or not, when a numerical range is described herein, unless otherwise stated, the range is intended to include the end values and all the integers and fractions within the range.

[0033] In addition, it needs to be further pointed out that, for the convenience of description, only the parts related to the application are shown in the drawings. The embodiments in the present disclosure and the features in the embodiments can be combined with each other without conflict.

[0034] It should be noted that the modification of "one" and "multiple" mentioned in the present disclosure is illustrative but not restrictive, and those skilled in the art should understand that "one or more" should be understood unless otherwise explicitly indicated in the context.

[0035] The main terms used in the present disclosure are defined as follows:

[0036] The term "phytol" is a kind of isoprenoid alcohol, and the chemical structural formula is as shown in Figure 1 The molecular formula of "phytol" is C 20 H 40 O, molecular weight: 296.53, English name: Phytol. It has anti-cancer and immune enhancing effects. Phytol has certain antioxidant properties and can scavenge free radicals in the skin, reducing the damage of free radicals to skin cells.

[0037] The term "Teprenone" has the chemical structural formula as shown in Figure 2 The compound name of "Teprenone" is 6,10,14,18-tetramethyl-5,9,13,17-nonadecatetraen-2-one. The molecular formula of "Teprenone" is C 23 H 38 O, molecular weight: 330.55, English name: Teprenone. Teprenone has certain antioxidant capacity and can neutralize free radicals in the skin to prevent oxidative stress from causing damage to the skin.

[0038] The term "Silybin" is a natural active substance extracted from the dried fruits of the plant Silybum marianum of the family Asteraceae. The molecular formula of Silybin is C 25 H 22 O 10Silybin, molecular weight: 482.44, is an extract from the fruit and seeds of silybum marianum L. Gaertn, and its structure is shown in Figure 3 Silybin is often used as an antioxidant. Due to its complex molecular structure and water insolubility, the transdermal penetration effect of silybin on the skin is not ideal. The common solution is to use it together with a penetration enhancer or to use it after improving the dosage form.

[0039] The term "retinoic acid X receptor gamma" is a type of protein receptor present in the nucleus. RXRγ can participate in the regulation of keratinocyte differentiation, which is essential for maintaining the barrier function of the skin. Specifically, under normal physiological conditions, retinoic acid can bind to RARγ, and activated RARγ can form a heterodimer with RXRγ, bind to specific DNA sequences, and regulate the expression of a series of genes. When the skin is photoaged, retinoic acid can play a role through its receptor pathway, and RARγ agonists can significantly inhibit the expression of MMP3. Since RXRγ needs to work with RARγ, it can indirectly affect the expression of MMP3. RXRγ receptor also plays a role in anti-inflammation. In aged skin, when the RXRγ receptor function is disordered, the response of skin cells to inflammatory stimuli may be enhanced, and the release of inflammatory mediators is increased, further exacerbating skin aging.

[0040] The term "retinoic acid receptor gamma" (Retinoic Acid Receptor Gamma, RARγ) is one of the important members of the nuclear receptor superfamily, and plays an important role in the processes of growth, development, metabolism, immunity, differentiation, proliferation, inflammation, and apoptosis. RARγ generally forms a heterodimer with nuclear receptor RXRα, and plays a role in regulating gene transcription in the nucleus.

[0041] The term "matrix metalloproteinase-3" is a type of enzyme protein, and the chemical name of matrix metalloproteinase-3 is Matrix Metalloproteinase, abbreviated as MMP3. Matrix metalloproteinase-3 belongs to one of the members of the matrix metalloproteinase (MMP) family. Increased expression of MMP3 can lead to degradation of dermal collagen, resulting in wrinkles and other signs of aging in the skin.

[0042] The term "interleukin-6" is a type of pleiotropic cytokine, and its English name is Interleukin-6, abbreviated as IL-6. It can control and regulate the inflammatory response in the immune system.

[0043] In a first aspect, the present application provides a use of a composition in the preparation of a retinoic acid receptor gamma activator; the composition comprises one or more of phytol, teprenone and silybin. The present application verifies new retinoic acid receptor gamma activators, phytol, teprenone and silybin, through molecular docking experiment screening.

[0044] In a second aspect, some embodiments of the present disclosure provide a use of a composition in the preparation of an extracellular matrix metalloproteinase inhibitor; the composition comprises phytol, teprenone and silybin; the mass ratio of the phytol, the teprenone and the silybin is 1:0.5-1.5:1-5; preferably, the mass ratio is 1:1-1.11:1-5.

[0045] In some embodiments, the cells are HaCaT cells.

[0046] The present disclosure verifies that the phytol, teprenone and silybin composition can inhibit matrix metalloproteinase-3 through an aging HaCaT cell experiment. According to the experimental results, it is found that phytol, teprenone and silybin cannot inhibit the increase of MMP3 expression in UVB-induced HaCaT cells when used alone, and phytol, teprenone and silybin can inhibit the increase of MMP3 expression in UVB-induced HaCaT cells when used in combination, and have a synergistic effect of resisting photodamage and improving photoaging.

[0047] In some embodiments, the mass ratio of the phytol, the teprenone and the silybin is 1:1:1; or 1:1:5; or 1:1:4; or 1:1:2; or 1:1:1-4.

[0048] In a third aspect, some embodiments of the present disclosure provide a use of a composition in the preparation of an interleukin-6 inhibitor; the composition comprises phytol, teprenone and silybin; the mass ratio of the phytol, the teprenone and the silybin is 1:0.5-1.5:1-5; preferably, the mass ratio is 1:1-1.11:1-5.

[0049] In some embodiments, the cells are HaCaT cells.

[0050] The present disclosure verifies that the phytol, teprenone and silybin composition can inhibit interleukin-6 through an aging HaCaT cell experiment. According to the experimental results, it is found that phytol, teprenone and silybin cannot inhibit the increase of interleukin-6 expression in UVB-induced HaCaT cells when used alone, and phytol, teprenone and silybin can inhibit the increase of interleukin-6 expression in UVB-induced HaCaT cells when used in combination, and have a synergistic effect of resisting photodamage and improving photoaging.

[0051] In some embodiments, the mass ratio of the phytol, the teprenone and the silymarin is 1:1:1; or 1:1:5; or 1:1:4; or 1:1:2; or 1:1:1-4.

[0052] In the fourth aspect, some embodiments of the present disclosure provide an application of a transdermal penetration enhancing composition in a silymarin transdermal penetration enhancer; the transdermal penetration enhancing composition is phytol and / or teprenone.

[0053] In the fifth aspect, some embodiments of the present disclosure provide a skin care composition for skin care, comprising silymarin and a transdermal penetration enhancer; the transdermal penetration enhancer is phytol and teprenone, and the mass ratio of phytol, teprenone and silymarin is 1:0.5-1.5:0.2-5. Preferably, the ratio of phytol, teprenone and silymarin is 1:0.5-1.5:1-5; more preferably, the mass ratio is 1:1-1.11:1-5.

[0054] In some embodiments, the mass ratio of phytol, teprenone and silymarin is 1:0.5:0.2-1; or 1:0.5:1-2.5; or 1:0.5:2.5-5; or 1:1:0.2-1; or 1:1:1-2.5; or 1:1:2.5-5.

[0055] In some embodiments, the ratio of phytol, teprenone and silymarin is 1:1-1.11:1-5. The specific composition of the specific ratio can synergistically inhibit the expression of matrix metalloproteinase-3 and interleukin-6 proteins, while also improving the penetration and absorption of silymarin in the skin, and improving the intradermal retention of silymarin, improving the bioavailability of silymarin, and improving the antioxidant properties.

[0056] In the sixth aspect, some embodiments of the present disclosure provide an external skin care product comprising the skin care composition of the fifth aspect.

[0057] In some embodiments, the external skin care product described above, wherein,

[0058] The addition amount of phytol is 0.05%-2%.

[0059] The addition amount of teprenone is 0.05%-2%.

[0060] The addition amount of silymarin is 0.05%-5%.

[0061] The external skin care product can refer to a skin care product that acts on the surface of the skin by smearing, covering and the like.

[0062] Optionally, the external skin care product comprises one or more of: a water agent, a milk agent, a cream agent, a paste agent, a gel agent, a cleanser, a mask. In practice, the water agent can comprise one or more of: a skin care water, a toner, an essence. The milk agent can comprise: a milk. The cream agent can comprise: a face cream. The face cream can be a face cream with transparent texture, or a face cream with non-transparent texture. The paste agent can comprise: a skin care paste. The cleanser can comprise one or more of: a cleansing foam, a cleansing water, a cleansing liquid. When the external skin care product is a mask, a substrate commonly used in the art and a skin care composition of the present disclosure applied or soaked on the substrate can be used. The substrate can include but is not limited to: silk mask paper, cotton mask paper, polyester mask paper or blended mask paper. The selection of the substrate can be selected by a person skilled in the art according to actual needs. The form of the mask can include but is not limited to: a gel type mask, a cream type mask and an essential oil mask.

[0063] The present disclosure will be described in detail below with reference to examples.

[0064]

Experiment 1 Molecular docking experiment

[0065] Previous studies have determined that retinoic acid can bind to RARγ, and activated RARγ can form a heterodimer with RXRγ, bind to specific DNA sequences, and regulate the expression of a series of genes. The following compares the binding ability of retinoic acid and retinol to RAR and retinoic acid X receptor γ through a molecular docking experiment to verify the use of phytol, teprenone and silymarin as RXRγ receptor agonists, to further illustrate the technical solutions and beneficial effects provided by the embodiments of the present application.

[0066] The name of the molecular docking calculation software is Hermite platform (Shenli Science and Technology).

[0067] The steps of the molecular docking test of phytol, teprenone and silymarin on retinoic acid X receptor γ (RXRγ) are as follows:

[0068] Step 1: Pretreatment of macromolecular receptor:

[0069] The human retinoic acid receptor (RARγ) crystal complex 3LBD (https: / / www.rcsb.org / structure / 3LBD) and the human retinoid X receptor γ (RXRγ) crystal complex 2GL8 (https: / / www.rcsb.org / structure / 2GL8) were downloaded from the PDB database (http: / / rcsb.org), and the protein was pretreated with molecular docking calculation software, dehydrated, ligand deleted, hydrogen added, charge calculated, and the structure of the protein optimized to form a pretreated protein file 3LBD-pre and 2GL8-pre.

[0070] Second step, small molecule ligand pretreatment:

[0071] Search the smiles number of teprenone, phytol, silymarin in the organic small molecule bioactivity database, and use the above molecular docking calculation software to pretreat the small molecule structure before docking to form three pretreatment files teprenone-pre, phytol-pre, silymarin-pre, retinoic acid-pre, retinol-pre. The above organic small molecule bioactivity database is pubchem (https: / / pubchem.ncbi.nlm.nih.gov / ).

[0072] Teprenone (https: / / pubchem.ncbi.nlm.nih.gov / #query=Teprenone).

[0073] Phytol (https: / / pubchem.ncbi.nlm.nih.gov / #query=phytol).

[0074] Silymarin (https: / / pubchem.ncbi.nlm.nih.gov / #query=Silymarin).

[0075] Retinoic acid (https: / / pubchem.ncbi.nlm.nih.gov / compound / 444795).

[0076] Retinol (https: / / pubchem.ncbi.nlm.nih.gov / compound / 445354).

[0077] Third step, RARγ docking analysis:

[0078] According to the docking tutorial of the above molecular docking calculation software (Hermite platform), it is selected not to dock based on conditional constraints, according to the binding form of retinoic acid and retinoic acid receptor (RARγ) crystal complex 3LBD, the active pocket center coordinates are set as (X 41.807 Y 13.3525 Z 85.1495), the pocket size is X 16.178 Y 25.645 Z 27.935, and the rest of the parameters adopts the system default value.

[0079] The results of molecular calculation are shown in Table 1 below.

[0080] Table 1 RARγ docking results

[0081]

[0082]

[0083] From the docking results shown in Table 1 above and the docking schematic diagram of the target molecules with RARγ shown in Figure 1, it can be seen that the binding ability of teprenone with RARγ is similar to that of retinoic acid and retinol. The binding ability of phytol and silybin with RARγ is close to that of retinol. Teprenone, phytol and silybin can bind to RARγ, and the activated RARγ forms a heterodimer with RXRγ, binds to a specific DNA sequence, and regulates the expression of a series of genes. Figure 4

[0084] Step 4: RXRγ docking analysis:

[0085] According to the docking tutorial of the above-mentioned molecular docking calculation software (Hermite platform), docking without condition-based constraints is selected, and according to the binding form of retinoic acid and human retinoid X receptor γ (RXRγ) crystal complex 2GL8, the active pocket center coordinates are set as (X-4.3015Y16.1055Z 8.2005), the pocket size is X 14.081Y 21.453Z15.139, and the remaining parameters are the system default values.

[0086] The results of molecular calculation are shown in Table 2 below.

[0087] Table 2: RXRγ docking results

[0088]

[0089] From the docking results shown in Table 2 above and the docking schematic diagram of the target molecules with RXRγ shown in Figure 2, it can be seen that the binding ability of teprenone with RXRγ is close to that of retinoic acid, and has a stronger binding ability relative to retinol. The binding ability of phytol and silybin with RXRγ is close to that of retinol. Teprenone, phytol and silybin can bind to RXRγ. Figure 5 From the above, it can be seen that teprenone, phytol and silybin can directly or indirectly activate the activity of retinoic acid X receptor γ, and further affect the expression of MMP3 and IL-6.

[0090]

Experiment 2: Verification experiment of inhibition of expression of MMP-3 and IL-6 in HaCaT cells

[0091] The following verifies the inhibition of expression of MMP-3 and IL-6 in HaCaT cells by phytol, teprenone and silybin combination through HaCaT cell experiment, to further illustrate the technical solutions and beneficial effects provided in the embodiments of the present application.

[0092] 2.1 Drugs and their sources:

[0093] 2.1 Drugs and their sources:

[0094] ​Tianeptine: Puyi Biomedicine Technology Co., Ltd.

[0095] Scopoletin: Shanghai Meiyun Biotechnology Co., Ltd.

[0096] Phytol: Puyi Biomedicine Technology Co., Ltd.

[0097] DMEM (dulbecco's modified eagle medium) basal medium, purchased from Gibco biological culture medium of Thermo Fisher Scientific (China) Co., Ltd., product number C11995500BT.

[0098] Fetal bovine serum (FBS), purchased from Gibco fetal bovine serum of Thermo Fisher Scientific (China) Co., Ltd., product number 10091148.

[0099] Penicillin / streptomycin solution (10,000 U / mL), purchased from Gibco penicillin / streptomycin of Thermo Fisher Scientific (China) Co., Ltd., number 25200114, wherein the penicillin / streptomycin solution includes penicillin and streptomycin, and the concentrations are 10,000 U / mL respectively.

[0100] CCK-8 kit, C0040 Cell Counting Kit-8, Beyotime / Biyundian. CCK8 working solution, 1ml CCK8 mother liquor is added into 9ml DMEM, which can be configured into 10ml CCK8 working solution.

[0101] PBS (phosphate buffered saline), pH 7.4, purchased from Thermo Fisher Scientific (China) Co., Ltd.

[0102] 2.2 Use device:

[0103] CO2 incubator (Thermo, 150I),

[0104] Clean bench (Suqing Antai, SW-CJ-1F),

[0105] Microplate reader (BioTek, Epoch),

[0106] Microspectrophotometer (Thermo Scientific NanoDrop),

[0107] ProFlex TM PCR instrument (Applied Biosystems SimpliAmp PCR thermal cycler),

[0108] QPCR instrument (Applied Biosystems QuantStudio 1),

[0109] UV crosslinker (UVB crosslinker, Luyor UCL-3500),

[0110] High-speed refrigerated centrifuge (Eppendorf Centrifuge 5418R).

[0111] 2.3 HaCaT cell survival rate detection

[0112] The CCK8 (Cell Counting Kit CCK 8) kit was used to test the survival rate of HaCaT cells to detect the effect of the combination of phytanol, teprenone and silymarin on cells.

[0113] The test method includes the following steps:

[0114] Step 1, cell inoculation.

[0115] Specifically, human immortalized keratinocytes (HaCaT for short) were inoculated into a 96-well plate at a seeding density of 1×10 4 cells / well, 100 μL of complete culture medium (complete culture medium is DMEM culture medium containing 10% FBS and 1% penicillin / streptomycin) was added to each well, and then incubated in an incubator (incubator containing 5% CO2, temperature 37°C) for 24 h.

[0116] Step 2, drug treatment.

[0117] Specifically, after 24 hours, the culture medium was removed, and the prepared complete culture medium sample containing phytanol, teprenone and silymarin was added, 100 μL of corresponding sample was added to each well of each group; after the addition was completed, it was placed in an incubator (37°C, 5% CO2) for 24 h. The sample is complete culture medium containing phytanol, teprenone and silymarin (complete culture medium is DMEM culture medium containing 10% FBS and 1% penicillin / streptomycin), and the concentrations of phytanol, teprenone and silymarin in the sample are shown in Table 3.

[0118] Table 3 Sample composition of toxicity experiment

[0119]

[0120]

[0121] Step 3, CCK8 detection.

[0122] After the drug treatment, the cells were incubated for 24 h, and then the supernatant was discarded. 100 μL of CCK8 working solution was added, and the mixture was incubated at 37°C in the dark for 2 h. After incubation, the OD values were read at 450 nm and 650 nm. The corrected OD value was calculated according to the following formula, and the corrected OD value was taken as the relative activity of the cells in each group.

[0123] Corrected OD value = OD value at 450 nm - OD value at 630 nm

[0124] OD value: the optical density value, which was determined by an enzyme marker. It reflects the amount of formazan dye produced by cell metabolism, and thus indirectly reflects the number or activity of cells.

[0125] Step 4, calculate the cell survival rate of each experimental group.

[0126] The cell survival rate of the control group (sample 1) was set to 100% as a reference to further determine the cell survival rate of each experimental group. The formula for calculating the cell survival rate of each experimental group is as follows:

[0127] Cell survival rate = [(OD sample group - OD blank group) / (OD control well - OD blank group)] x 100%

[0128] Blank group: CCK-8 working solution without cells, used to correct the background value of the enzyme marker detection and exclude the interference of culture medium and other factors on the OD value.

[0129] The results of the CCK8 cell viability test are shown in Table 1. Figure 5

[0130] 2.4 Detection of MMP-3 and IL-6 expression in HaCaT cells

[0131] Real-time quantitative gene amplification fluorescence detection (qPCR detection) of matrix metalloproteinase-3 and interleukin-6 was performed by the following steps to obtain the detection result information:

[0132] Step 1, in HACAT cells, according to the cytotoxicity results, the control group, UVB control group and drug treatment group were prepared according to the component concentrations in Table 4, and QPCR of MMP-3 and IL-6 was performed.

[0133] The drug compositions to be added to the control group and experimental group were prepared according to Table 4:

[0134] Table 4 Composition of drug compositions to be added to the control group and experimental group

[0135]

[0136] ​

[0137] The DMEM complete medium containing phytol, 20 μM of tirproprate and 10 μM of silybin in the administration experimental group 5 has a concentration of 5.93 μg / ml of phytol, 6.61 μg / ml of tirproprate and 4.82 μg / ml of silybin by conversion [molar concentration x relative molecular mass value (molar mass) = mass concentration]; the mass ratio is 1:1.11:0.81.

[0138] The DMEM complete medium containing phytol, 20 μM of tirproprate and 50 μM of silybin in the administration experimental group 6 has a concentration of 5.93 μg / ml of phytol, 6.61 μg / ml of tirproprate and 24.12 μg / ml of silybin by conversion; the mass ratio is 1:1.11:4.07.

[0139] Second step, cell inoculation:

[0140] First, take human immortalized keratinocytes (HaCaT for short) and inoculate them into a 6-well plate at a seeding density of 8 x 10 5 cells / well. Add 100 μL of complete medium (the complete medium can be DMEM medium containing 10% FBS and 1% penicillin / streptomycin) to each well in the 6-well plate. Finally, incubate the 6-well plate in an incubator (containing 5% CO2, temperature 37°C) for 24 hours.

[0141] Third step, administration treatment:

[0142] First, when the cell plating rate in the 6-well plate reaches 70%-80%, set up a blank control group and 7 experimental groups (as shown in Table 4 above) on the 6-well plate, 5 experimental groups are: UVB control experimental group, administration experimental group 1, administration experimental group 2, administration experimental group 3, administration experimental group 4. Then, except for the blank control group, the 5 experimental groups are irradiated with a UVB dose of 35 mJ / cm 2 . After that, discard the culture medium of the blank control group and the 5 experimental groups, and add 100 μL of the corresponding drug composition to each group for administration treatment, with 3 replicate wells for each group. After the administration treatment is completed, place the 6-well plate in an incubator (37°C, 5% CO2) for 24 hours.

[0143] Fourth step, first, after 24 hours of culture, extract RNA from the cells in each group. Then, use a microspectrophotometer (Thermo Scientific NanoDrop) to determine the concentration of the RNA, and obtain the RNA samples with measured concentrations in each group.

[0144] Fifth step, reverse transcription of each group of measured concentration of RNA sample to obtain cDNA sample.

[0145] Sixth step, take the cDNA sample to perform qPCR reaction, analyze the mRNA relative expression level of each group of related genes (MMP3 gene and IL-6 gene), and obtain the detection result information.

[0146] As shown in Figure 6 , it is a schematic diagram of the mRNA relative expression level of MMP-3 of the above blank control group, the above UVB control experiment group, and the above drug experiment groups 1-6. As shown in Figure 6 , by comparing the blank control group and the UVB control experiment group, it can be known that the expression of MMP-3 in HaCaT cells increases after UVB irradiation, so the cell model is successfully established. By comparing the UVB control experiment group and the drug experiment groups 1-6, it can be known that when phytol, teprenone and silybin are used alone, they cannot significantly inhibit the increase of the expression of MMP-3 in HaCaT cells after UVB irradiation, and the mixture of phytol, teprenone and silybin can significantly inhibit the increase of the expression of MMP-3. Figure 6 As shown in

[0147] , it is a schematic diagram of the mRNA relative expression level of IL-6 of the above blank control group, the above UVB control experiment group, and the above drug experiment groups 1-6. As shown in Figure 7 , by comparing the blank control group and the UVB control experiment group, it can be known that the expression of IL-6 in HaCaT cells increases after UVB irradiation, so the cell model is successfully established. By comparing the UVB control experiment group and the drug experiment groups 1-6, it can be known that when phytol, teprenone and silybin are used alone, they cannot significantly inhibit the increase of the expression of IL-6 in HaCaT cells after UVB irradiation, and the mixture of phytol, teprenone and silybin can significantly inhibit the increase of the expression of IL-6. Figure 7 Figure 7

[0148]

Experiment 3 Transdermal Penetration Experiment

[0149] The transdermal penetration experiment is used to verify the use of phytol and teprenone to promote the transdermal penetration of silybin, so as to further illustrate the technical solutions and beneficial effects provided by the embodiments of the present application.

[0150] 3.1 Drug and source

[0151] 1. Teprenone: Pu Rui Biomedicine Technology Co., Ltd.

[0152] 2. Silybin: Shanghai Meiyan Biotechnology Co., Ltd.

[0153] 3. Phytol: Pu Rui Biomedicine Technology Co., Ltd. ​​

[0154] 4.4% paraformaldehyde, Beijing Lanjike Science and Technology Co., Ltd.

[0155] 5. PBS (Phosphate Buffered Salts), pH 7.4, purchased from Gibco, a brand of Thermo Fisher Scientific (China) Co., Ltd., is a balanced salt solution suitable for a variety of cell culture applications, such as washing cells before dissociation, transporting cells or tissues, diluting cells for counting, and preparing reagents.

[0156] 6. Bama miniature pigskin (thickness: 800-1000 μm), Shenzhen University of Technology.

[0157] 3.2 Instruments

[0158] C18 chromatographic column

[0159] Ten-thousandth analytical balance

[0160] High-performance liquid chromatograph

[0161] Franz transdermal diffusion cell, effective diffusion area 1.54 cm 2 , receiving liquid volume 8 mL.

[0162] Freezing microtome

[0163] 3.3 Preparation of pharmaceutical composition

[0164] Prepare the pharmaceutical composition according to the composition of Table 5 below, and the preparation steps of the pharmaceutical composition are as follows:

[0165] Step 1, put shugan sini and solvent into a beaker, turn on the heating and stirring (30-50 r / min) to heat to 60-80℃, ultrasonic dissolution to no particulate clear liquid, cool to room temperature for standby;

[0166] Step 2, add penetration enhancer to the above beaker, stir until uniform, then transfer to a light-proof container for storage, for the next transdermal experiment.

[0167] Table 5 Specific composition of pharmaceutical composition for transdermal penetration experiment

[0168]

[0169]

[0170] 3.4 Transdermal penetration experiment

[0171] The transdermal penetration amount experiment can be carried out by a method conventional in the art. A specific exemplary method is given below for observing the in vitro transdermal penetration of shugan-sijunzi decoction and the retention of shugan-sijunzi decoction in different skin layers using Franz transdermal diffusion cells. The transdermal penetration experiment comprises the following steps:

[0172] Firstly, a fresh skin sample (Bama miniature pig skin, about 1 mm thick) is fixed between the receiving cell and the supply cell.

[0173] The stratum corneum of the skin sample is directed upwards.

[0174] Secondly, 0.5 mL of the pharmaceutical composition is placed in the supply cell, and the solution is prevented from volatilizing by sealing with plastic wrap.

[0175] It should be noted that PBS / anhydrous methanol (v / v, 70 / 30) is used as the diffusion medium of the receiving cell. The diffusion medium is stirred at 32°C and 350 rpm during the experiment. Four parallel samples are set up for each composition.

[0176] Thirdly, after 24 hours, the skin tissue corresponding to each composition is fixed in 4% paraformaldehyde for 24 hours, and then dehydrated in 15% and 30% sucrose solutions, respectively, after 24 hours. The skin tissue is then washed with PBS and embedded in 10% CMC-Na for cryopreservation.

[0177] Fourthly, the transdermal part of the skin is cut into the stratum corneum (0-20 μm) and the epidermis + dermis (transdermal part without stratum corneum) by a cryostat microtome.

[0178] Fifthly, the stratum corneum and the epidermis + dermis are cut into pieces, and the pieces are weighed after being cut.

[0179] Sixthly, after weighing, the stratum corneum pieces and the epidermis + dermis pieces are subjected to 1 hour of water ultrasonic treatment.

[0180] Seventhly, the remaining sample solution in the supply cell is recovered, the drug content in the receiving cell and the intradermal drug content are determined by HPLC (high performance liquid chromatograph), the transdermal penetration curve is drawn, and the retention amount of shugan-sijunzi decoction in different skin layers is calculated, and the recovery rate is calculated.

[0181] The liquid phase method comprises: mobile phase: water:methanol=50:50, flow rate 1.0 mL / min; detection wavelength: 275 nm; column temperature: 30°C.

[0182] 24-hour epidermis + dermis intradermal retention rate = (24-hour epidermis + dermis intradermal retention amount) / total recovery amount

[0183] The calculation method of recovery rate is: recovery rate (%) = actual total recovery amount / actual sample amount.

[0184] The transdermal penetration test results of the above pharmaceutical compositions 1-4 are shown in Table 6 below:

[0185] Table 6 Transdermal penetration test results of pharmaceutical compositions 1-4

[0186]

[0187] It should be noted that the calculation method of penetration efficiency shown in Table 6 above is:

[0188] Penetration efficiency = (24-hour experimental group epidermis + dermis skin retention rate - 24-hour control group epidermis + dermis skin retention rate) / 24-hour control group skin retention rate.

[0189] The control group corresponding to pharmaceutical compositions 2-4 is pharmaceutical composition 1.

[0190] Comparing the transdermal penetration test results of pharmaceutical compositions 1-4, phytol and tianeptine can both promote the transdermal penetration of shuganjiabin, and increase the epidermis + dermis skin retention amount.

[0191] From pharmaceutical compositions 2-4, it can be seen that the combination of phytol and tianeptine has a better effect of promoting the transdermal penetration of shuganjiabin than phytol and tianeptine added respectively, further increasing the epidermis + dermis skin retention amount. Collagen and elastic fibers are located in the dermis, and the occurrence of skin inflammation involves various cells and inflammatory mediators in the epidermis and dermis. Therefore, the increase of shuganjiabin in the epidermis + dermis skin retention amount can greatly promote the efficacy of shuganjiabin.

[0192] The penetration test results of the above compositions 5-compositions 13 are shown in Table 7 below:

[0193] Table 7 Transdermal penetration test results of compositions 5-compositions 13

[0194]

[0195] It should be noted that the calculation method of penetration efficiency shown in Table 7 above is:

[0196] Penetration efficiency = (24-hour experimental group epidermis + dermis skin retention rate - 24-hour control group epidermis + dermis skin retention rate) / 24-hour control group skin retention rate.

[0197] The control group corresponding to pharmaceutical compositions 6-10 is pharmaceutical composition 5; the control group corresponding to pharmaceutical compositions 12-13 is pharmaceutical composition 11.

[0198] From the test results of pharmaceutical compositions 4-13, it can be seen that when the mass ratio of phytol, teprenone and silymarin is in the range of 1:0.5-1:0.2-5, the transdermal penetration of silymarin can be promoted, and the intracutaneous retention amount of epidermis+dermis layer can be increased. Further, when the mass ratio of phytol, teprenone and silymarin is 1:1:1-5, the combination of phytol, teprenone and silymarin has a significant synergistic effect on inhibiting the increase of expression of MMP-3 and IL-6.

[0199] Application example:

[0200] The following Table 8 shows the components of emulsions and creams provided for the application example:

[0201] Table 8 Composition of emulsions and creams

[0202] Phase Ingredient Emulsion Cream A Deionized water to 100 to 100 A 981 0.2 0.4 A EDTA-2Na 0.02 0.02 B Frescon 0.5 0.5 B Hexylene glycol 0.5 0.5 B Butylene glycol 3 3 C Cetearyl alcohol 1 3 C Coco-caprylate / caprate 2 5 C Cetyl alcohol ethylhexanoate 2 5 C A170 1 3 D Phytol 0.1 0.1 D Teprenone 0.1 0.1 E Butylene glycol 2 2 E Silybin 0.25 0.1 F Deionized water 2 3 F Arginine 0.14 0.28

[0203] Preparation method:

[0204] Step 1, phase A: bubble to no white powder, stir at 75-80℃ until no large particles, homogenize for 1-2 minutes.

[0205] Step 2, phase B: dissolve at 65-75℃ until transparent and no particles, add phase A and homogenize for 1 minute, warm back to 80-90℃, add pre-processed phase C and phase D, phase E, homogenize for 2-3 minutes, stir and cool to 45℃.

[0206] Step 3: add pre-processed phase F, stir for 8-15 minutes.

[0207] Step 4: stir and cool, check and pass, discharge.

[0208] The above various embodiments of the present disclosure have the following technical effects: the skin care composition for skin care of the present disclosure improves the penetration and absorption amount of silybin in the skin, improves the intradermal retention amount of silybin and improves the bioavailability of silybin. And further reduces the phenomenon of skin allergy, thereby improving the stability and safety of the skin care composition. Specifically, the cause of the phenomenon of skin allergy is that when the penetration enhancer is used in combination, the high concentration or long use time of the penetration enhancer causes greater irritation to the skin, resulting in the phenomenon of skin allergy; when used in combination with external anti-inflammatory drugs, the external anti-inflammatory drugs have a certain irritating effect on the skin, causing the skin to be prone to allergic reactions. Based on this, the skin care composition for skin care of the present disclosure includes, by mass fraction: phytol, 0.8-1.5; teprenone, 0.4-1.5; silybin, 0.3-4. Also because the skin care composition for skin care includes phytol, which can significantly increase the retention amount of silybin in the skin, and the combination of phytol and teprenone can further enhance the retention effect of silybin in the skin, thereby improving the bioavailability of silybin. Also because the phytol included in the above skin care composition for skin care has the effects of resisting photodamage and improving photoaging, and can inhibit the generation and sagging of skin wrinkles, thereby having an anti-aging effect. And phytol can also increase the elasticity and brightness of the skin, and has good oil control effect, thereby achieving good skin care effect. And the anti-inflammatory effect on the skin is not achieved by directly using external anti-inflammatory drugs, but by the antioxidant and anti-inflammatory effects of silybin and teprenone included in the above skin care composition for skin care. Therefore, the above skin care composition for skin care also has an anti-inflammatory effect. And teprenone also has the effects of maintaining the barrier function of the skin and anti-aging. Therefore, the above skin care composition for skin care improves the penetration and absorption amount of silybin in the skin, thereby improving the intradermal retention amount of silybin and improving the bioavailability of silybin. And further reduces the phenomenon of skin allergy, thereby improving the stability and safety of the skin care composition.

[0209] The above description is only some of the preferred embodiments of the present disclosure and an explanation of the technical principles applied. Those skilled in the art should understand that the scope of the application involved in the embodiments of the present disclosure is not limited to the technical solutions formed by the specific combinations of the above technical features, and should also cover other technical solutions formed by any combination of the above technical features or their equivalent features without departing from the above inventive concept. For example, the above features are replaced with each other to form a technical solution with similar functions to the technical solutions disclosed in the embodiments of the present disclosure (but not limited to).

Claims

1. The use of a composition in the preparation of an anti-skin aging product, the composition comprising phytol, teprenone, and silymarin, wherein the mass ratio of phytol, teprenone, and silymarin is 1:0.5~1.5:1~5.

2. The use of the composition according to claim 1 in the preparation of anti-skin aging products, wherein, The composition is used to prepare inhibitors of extracellular matrix metalloproteinases.

3. The use of the composition according to claim 1 in the preparation of anti-skin aging products, wherein, The composition is used to prepare an interleukin-6 inhibitor.

4. The use of a transdermal permeation enhancing composition in the preparation of silymarin transdermal permeation enhancer; wherein the transdermal permeation enhancing composition is phytol and / or teprenone.

5. A skin care composition for skin care, comprising silymarin and a transdermal penetration enhancer; wherein the transdermal penetration enhancer is phytol and teprenone, and the mass ratio of phytol, teprenone and silymarin is 1:0.5~1.5:0.2~5.

6. The skincare composition according to claim 5, wherein, The ratio of phytol, teprenone and silymarin is 1:0.5~1.5:1~5.

7. A topical skin care product comprising silymarin and a transdermal penetration enhancer; wherein the transdermal penetration enhancer is phytol and teprenone, and the mass ratio of phytol, teprenone and silymarin is 1:0.5~1.5:0.2~5.

8. The topical skin care product according to claim 7, wherein, The amount of phytol added is 0.05%~2%; The amount of teprenone added is 0.05%~2%; The amount of silymarin added is 0.05% to 5%.

9. The topical skin care product according to claim 7, wherein, The topical skin care products include one or more of the following: aqueous solutions, emulsions, creams, ointments, gels, cleansers, and masks.

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