CN114028296A - Multi-dimensional skin sensitivity repairing cosmetic component and application - Google Patents
Multi-dimensional skin sensitivity repairing cosmetic component and application Download PDFInfo
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- CN114028296A CN114028296A CN202111521355.1A CN202111521355A CN114028296A CN 114028296 A CN114028296 A CN 114028296A CN 202111521355 A CN202111521355 A CN 202111521355A CN 114028296 A CN114028296 A CN 114028296A
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- peony root
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- keratinocytes
- root extract
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
- A61K8/9783—Angiosperms [Magnoliophyta]
- A61K8/9789—Magnoliopsida [dicotyledons]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/005—Preparations for sensitive skin
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
- C12N5/0629—Keratinocytes; Whole skin
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/70—Undefined extracts
- C12N2500/76—Undefined extracts from plants
Abstract
The invention discloses a multi-dimensional cosmetic component for repairing sensitive skin and application thereof.
Description
Technical Field
The invention relates to the field of daily chemical industry, in particular to application of a peony root extract in improving skin sensitivity.
Background
Skin sensitization (sensitive muscle) is a common skin reaction that occurs in women in china at a rate of about 36%. Skin sensitivity is different from skin sensitivity, and allergic reaction is mainly caused by sensitizers, such as coumarin, imidazolidinyl urea, farnesol and the like. Skin sensitivity is primarily the symptoms of skin discomfort caused by non-sensitizers, including stinging, itching, and sometimes redness. The mechanism of sensitive muscle formation is very complex and is currently believed to be due to incomplete skin barrier, excessive neural responses, inflammatory signals, etc.
Although the prior art has been studied for each mechanism of skin sensitivity, attempts have been made to solve the problem by combining ingredients that require complex factors such as compatibility, cooperativity, etc. among the ingredients. Not all active ingredients should be used together, for example, niacinamide and vitamin C, both having good skin care effects, are used together, but rather, niacinamide accelerates the degradation of vitamin C.
There is therefore a great need in the art for substances that can act from multiple mechanical dimensions, improving the problem of skin sensitivity.
Disclosure of Invention
The invention aims to provide a method for repairing sensitive skin in multiple dimensions.
The invention provides application of a peony root extract in preparing a composition for strengthening and/or repairing skin barriers.
In another embodiment, the strengthening and/or repairing the skin barrier comprises promoting the production of filaggrin, loricrin, ceramide synthase3, and/or ultralong chain fatty acid elongase 4 in keratinocytes.
The invention provides application of a peony root extract in preparing a composition for improving skin discomfort.
In another embodiment, the ameliorating skin discomfort comprises reducing inflammatory factors in the skin.
In another embodiment, the improvement in skin discomfort comprises thymic stromal lymphopoietin that inhibits keratinocyte production.
In another embodiment, the ameliorating skin discomfort comprises inhibiting tumor necrosis factor α produced by macrophages.
In another embodiment, the improving skin discomfort comprises inhibiting nerve growth factor production in keratinocytes.
The invention provides application of a peony root extract in preparing a composition for improving skin sensitivity.
In another embodiment, the improvement in skin sensitivity comprises strengthening and/or repairing the skin barrier, reducing inflammatory factors, and having a soothing effect.
In another embodiment, the exerting soothing effect comprises inhibiting nerve growth factor production in keratinocytes.
In another embodiment, the composition comprises 0.01-100 wt% of the extract of peony root, based on the total weight of the composition.
In another embodiment, the composition comprises a peony root extract and a cosmetically acceptable carrier.
In another embodiment, the composition is a dermatological product.
In another embodiment, the product form is selected from a solution, gel, cream, liniment, microemulsion spray, suspension or emulsion.
In another embodiment, the concentration of the peony root extract in the liquid form of the composition is 0.01-3.0 mg/mL.
The present invention provides a method for promoting in vitro production of filaggrin, loricrin, ceramide synthase3, and/or ultralong chain fatty acid elongase 4 in keratinocytes, the method comprising the steps of: mixing the keratinocytes with the extract of peony root.
The present invention provides a method for inhibiting in vitro production of keratinocyte thymic stromal lymphopoietin, and/or inhibiting nerve growth factor produced in keratinocytes, comprising the steps of: mixing the keratinocytes with the extract of peony root.
The invention provides a method for inhibiting tumor necrosis factor alpha generated by macrophages in vitro, which is characterized by comprising the following steps: mixing macrophage with peony root extract.
The peony root extract provided by the invention can play a role in multiple mechanism dimensions and improve the problem of skin sensitivity.
Drawings
FIG. 1 shows how peony root extract of example 1 promotes Filaggrin (Filaggrin), Loricrin (Loricrin), Ceramide Synthase 3(Ceramide synthsase 3) and very long chain fatty acid-extended protein 4(ELOVL4) in keratinocytes.
FIG. 2 shows the suppression of keratinocyte-produced Thymic Stromal Lymphopoietin (TSLP) by the extract of peony root in example 2.
FIG. 3 shows the inhibition of tumor necrosis factor alpha (TNF-. alpha.) production by macrophages by the extract of peony root of example 3.
FIG. 4 shows the inhibition of Nerve Growth Factor (NGF) produced in keratinocytes by the extract of peony root of example 4.
Detailed Description
The inventor finds that the peony root extract can play a role in repairing skin barriers, reducing cell inflammatory factors, playing a role in relieving and the like on the skin so as to improve sensitive muscles through intensive research. On the basis of this, the present invention has been completed.
As used herein, the "peony root extract" contains phytochemicals such as phenols and phenolic glycosides, monoterpenes and monoterpene glycosides, sterols and their glycosides, and flavones.
As used herein, "skin barrier" refers to a physical barrier, which is a "brick wall structure" formed by a sebum membrane, a cornified corneous envelope, lipids, etc., and has the function of preventing the entry of external harmful substances, irritants, and sunlight, and simultaneously preventing the loss of water. Impaired physical barrier of the skin will cause skin dryness, skin aging, pigmentation and skin sensitivity.
As used herein, "keratinocytes (keratinocytes)" also known as keratinocytes or keratin-forming cells, are the major cellular components that make up the epidermal layer. Keratinocytes at different differentiation stages form the four-layer structure of the epidermal layer of the skin, which is the stratum corneum, the stratum granulosum, the stratum spinosum and the stratum basale from the outside to the inside. The epidermal layer of keratinocytes serves a barrier function.
As used herein, "inflammatory factor" refers to various cytokines involved in inflammatory responses. Mainly comprises tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6) and the like.
As used herein, "soothing" or "reducing a nerve hyperresponse" are used interchangeably and refer to the inhibition and/or reduction of skin discomfort caused by external stimuli, including, but not limited to, itching, redness, warmth, stinging, etc.
The term "effective amount" is intended to be used for the purpose of strengthening and/or repairing the skin barrier, improving skin sensitivity, etc., and such an amount is capable of strengthening and/or repairing the skin barrier, reducing cytokines and/or providing soothing effects after a suitable period of use.
"composition" refers to a composition that, when applied to an individual (typically a human), is capable of penetrating the skin to induce the desired improvement in skin sensitivity.
As used herein, the term "cosmetically acceptable carrier" refers to a carrier that allows a cosmetic or personal care product to be applied, including various excipients and diluents, which are not themselves essential active ingredients, and which do not have undue toxicity after application. Suitable carriers are well known to those of ordinary skill in the art. A sufficient discussion of cosmetically acceptable excipients can be found in the cosmetic hygiene specifications 2015 edition. Such carriers may include humectants, emulsifiers, thickeners, chelating agents, emollients, and the like in the composition. Such as, but not limited to, water, potassium hydroxide, 1, 2-hexanediol, p-hydroxyacetophenone, ethylhexylglycerin, butylene glycol, panthenol, dipotassium glycyrrhizinate, arginine, glycerin, sodium hyaluronate, propylene glycol, hexylene glycol, glyceryl stearate/PEG-100 stearate, glyceryl caprylate, xanthan gum, betaine, hydroxyethylcellulose, carbomer, disodium EDTA, isomeric hexadecane, isooctyl palmitate, ethylhexyl palmitate, cetostearyl alcohol, cetearyl alcohol, polydimethylsiloxane, citric acid or a salt thereof, behenyl alcohol, beheneth, ceteth, pentaerythritol tetrakis (ethylhexanoate), squalane, cetyl ethylhexanoate, and the like.
The term "administering" as used herein means directly administering an equivalent amount of the extract of the root of peony.
The terms "individual" or "personal" are used herein to refer to a person who can receive the described moutan root extract and/or method for application to the skin.
As used herein, "room temperature" means 15-45 deg.C, preferably 20-35 deg.C.
The present invention provides a method of improving skin sensitivity comprising repairing the skin barrier, reducing inflammatory factors, and/or providing soothing benefits. The method comprises administering a peony root extract and/or a composition comprising a peony root extract.
In one embodiment of the invention, repairing the skin barrier comprises promoting expression of filaggrin, expression of keratinized envelope protein, and expression of long-chain lipid synthesis-related enzymes in keratinocytes; for example, promote the production (including gene expression) of Filaggrin (Filaggrin), Loricrin (Loricrin), Ceramide Synthase 3(Ceramide synthsase 3) and/or ultralong chain fatty acid elongase 4(ELOVL4) in keratinocytes.
In one embodiment of the invention, reducing the inflammatory factor comprises reducing the expression of tumor necrosis factor alpha (TNF- α) and Thymic Stromal Lymphopoietin (TSLP) in skin cells following stimulation.
In one embodiment of the invention, exerting soothing efficacy comprises reducing substance P-induced overexpression of Nerve Growth Factor (NGF).
The peony root extract administered in the present invention is a liquid, and its concentration is between 0.01-3.0mg/mL, such as, but not limited to, 0.01-0.5mg/mL, 0.02-0.7mg/mL, 0.3-2.0mg/mL, 0.05-1.5mg/mL, etc.
In one embodiment of the present invention, the administered peony root extract is the only active ingredient in the composition.
In one embodiment of the present invention, the extract of Paeonia suffruticosa root is obtained from Shanghai Jincheng chemical Co., LtdMaterials of PAE.
The present invention provides a composition comprising a peony root extract and a cosmetically acceptable carrier, thereby providing various cosmetic or personal care products for application to human skin, including, but not limited to, a makeup cream, a sunscreen cream, a facial cream, an eye cream, an emulsion, a serum, a lotion, a gel, etc. In one embodiment of the present invention, the peony root extract is the only active ingredient in the composition.
The cosmetic or personal care product provided by the present invention contains 0.005-1 wt% of the extract of peony root, such as, but not limited to, 0.02-0.1 wt%, 0.007-0.3 wt%, 0.04-0.9 wt%, 0.06-0.7 wt%, 0.01-0.4 wt%, etc., based on the total weight of the product.
In some embodiments of the present invention, the cosmetic or personal care product is obtained by combining the compositions provided herein with a cosmetically acceptable carrier that can be used to form the aqueous phase. The cosmetically acceptable carrier that may be used to form the aqueous phase includes, but is not limited to, one or more of ethylhexyl glycerin, butylene glycol, glycerin, sodium hyaluronate, and water.
In one embodiment of the present invention, the composition provided by the present invention is mixed with water to form an aqueous phase, and then mixed with a cosmetically acceptable carrier for the oil phase and emulsified to obtain the cosmetic or personal care product. Preferably, the cosmetic or personal care product may be formed by adding preservatives, perfumes, etc. after emulsification and cooling to room temperature.
In another embodiment of the present invention, the cosmetic or personal care product is obtained by adding the composition provided by the present invention after mixing and emulsifying the cosmetically acceptable carrier of the oil phase and the cosmetically acceptable carrier of the water phase. Preferably, the cosmetic or personal care product may be formed by adding the composition provided by the present invention after emulsification and cooling to room temperature.
In another embodiment of the invention, the cosmetic or personal care product may also be obtained by adding the composition provided by the present invention after homogenization of the aqueous cosmetically acceptable carrier. Preferably, the cosmetic or personal care product may be formed by adding the composition provided by the present invention after homogenization and cooling to room temperature.
In some embodiments of the invention, the cosmetically acceptable carrier used to form the oil phase includes, but is not limited to, one or more of glyceryl stearate, glyceryl monostearate, glyceryl stearate/PEG-100 stearate complex, isomeric hexadecanes, isooctyl palmitate, ethylhexyl palmitate, cetearyl alcohol, behenyl alcohol, cetostearyl alcohol, ceteth, dimethicone, pentaerythritol tetrakis (ethylhexanoate), squalane, cetyl ethylhexanoate petrolatum, shea butter, squalane, lecithin.
Although numerical ranges and parameters setting forth the broad scope of the invention are approximate, the values set forth in the specific examples are presented as precisely as possible. Any numerical value, however, inherently contains certain standard deviations found in their respective testing measurements. As used herein, "about" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a particular value or range. Alternatively, the term "about" means that the actual value falls within the acceptable standard error of the mean, as considered by those skilled in the art. Except in the experimental examples, or where otherwise expressly indicated, it is to be understood that all ranges, amounts, values and percentages herein used (e.g., to describe amounts of materials, length of time, temperature, operating conditions, quantitative ratios, and the like) are to be modified by the word "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, these numerical parameters are to be understood as meaning the number of significant digits recited and the number resulting from applying ordinary carry notation.
Unless defined otherwise herein, the scientific and technical terms used herein have the same meaning as is commonly understood and used by one of ordinary skill in the art. Furthermore, as used herein, the singular tense of a noun, unless otherwise conflicting with context, encompasses the plural form of that noun; the use of plural nouns also covers the singular form of such nouns.
The features mentioned above with reference to the invention, or the features mentioned with reference to the embodiments, can be combined arbitrarily. All features disclosed in this specification may be combined in any combination, provided that there is no conflict between such features and the combination, and all possible combinations are to be considered within the scope of the present specification. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, the features disclosed are merely generic examples of equivalent or similar features.
The main advantages of the invention are: the peony root extract or the composition taking the extract as the only active ingredient provided by the invention can play a role in repairing skin barriers, reducing cell inflammatory factors and reducing excessive reaction of nerves at the same time and improve sensitive muscles.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers. All percentages, ratios, proportions, or parts are by weight unless otherwise specified. The weight volume percentage units in the present invention are well known to those skilled in the art and refer to, for example, the weight of solute in a 100 ml solution. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.
Example 1
The peony root extract promotes the production of Filaggrin (FLG), Loricrin (LOR), Ceramide Synthase 3(Ceramide Synthase3, CerS3) and very long chain fatty acid elongase 4 (aggregation of chain fatty acids protein 4, ELOVL4) in keratinocytes
Material
The test system comprises:
the cells used in this test were keratinocytes, lot No.: EP21012001, obtained by cell primary, subculture, Guangdong Boxi Biotech limited.
The main reagents are as follows:
KC2500 broth (guangdong boxi), mtt (sigma), dmso (sigma), PBS (bosch de), rnaioso Plus (Takara), reverse transcription kit (Takara), fluorescent dye (Takara).
The main equipment is as follows:
CO2incubator (Thermo), clean bench (Sujing Antai), inverted microscope (Olympus), micro-oscillator (Linbel), enzyme labeling instrument (BioTek), incubator (Tester), fluorescent quantitative PCR instrument (BioRad), common PCRPCR instruments (Bori).
The test substance:
the control group is cell + KC2500 culture solution
Method
Inoculation:
by 2.8X 105Cell/well inoculation Density cells were plated onto 6 well plates, incubators (37 ℃, 5% CO)2) And incubated overnight.
Preparing liquid:
control group: inoculating cells (blank without any other components, to show the expression level of certain molecules in cells under normal conditions)
Peony root extract: 0.03mg/mL (peony root extract dissolved in DMSO and added into KC2500 culture solution)
Administration:
when the cell plating rate in the 6-hole plate reaches 40% -60%, the medicine is administered in groups, the dosage of each hole is 2mL, each group is provided with 3 multiple holes, and the incubator (37 ℃ and 5% CO)2) Middle incubation for 24 hours
Collecting a sample:
after 24 hours of incubation, the solution was discarded, washed twice with 1 mL/well PBS, 1mL of RNAioso Plus was added to each well, lysed cells were aspirated, and samples were collected
And (3) gene expression detection:
extracting RNA, reverse transcribing to cDNA, and fluorescent quantitative PCR detection with 2-△△CT method carries out result calculation
And (4) result statistical analysis:
the graph is drawn by using GraphPad Prism Program software, t-test statistical analysis is adopted among groups, p <0.05 indicates that the difference is obvious, and p <0.01 indicates that the difference is extremely obvious
Results
See figure 1.
In keratinocytes, the peony root extract had the effect of promoting the expression of filaggrin (+ 28%), loricrin (+ 14%), ultralong-chain fatty acid elongase 4 (23%) and ceramide synthase 3(+ 11%) genes (p < 0.05).
Filaggrin and loricrin play a positive regulation role in the maturation process of horny layer cells and have the effect of strengthening and toughening the horny cell barrier; the ultralong chain fatty acid protractor protein 4 is responsible for synthesizing long-chain free fatty acid, and the ceramide synthetase 3 is responsible for synthesizing long-chain ceramide from the long-chain free fatty acid. Long chain ceramides are an important component of stratum corneum lipids, responsible for maintaining the function and integrity of the skin barrier.
The results indicate that the peony root extract has the effect of strengthening the skin barrier.
Example 2
Thymic Stromal Lymphopoietin (TSLP) that peony root extract inhibits keratinocyte production
Material
The test system comprises:
the cells used in this test were keratinocytes, lot No.: EP21012001, obtained by cell primary, subculture, Guangdong Boxi Biotech limited.
The main reagents are as follows:
KC2500 broth (guangdong boxi), mtt (sigma), dmso (sigma), PBS (bosch de), rnaioso Plus (Takara), reverse transcription kit (Takara), fluorescent dye (Takara).
The main equipment is as follows:
CO2incubator (Thermo), clean bench (Sujing Antai), inverted microscope (Olympus), micro-oscillator (Linbel), enzyme labeling instrument (BioTek), incubator (Tester), fluorescent quantitative PCR instrument (BioRad), general PCR instrument (Bori).
The test substance:
the control group is cell + KC2500 culture solution
Polyinosinic acid was purchased from Guangdong Boxi Biotech Ltd
Lipopolysaccharide was purchased from Sigma
Dexamethasone Bulganling organism
Experimental methods
Inoculation:
by 2.8X 105Cell/well inoculation Density cells were plated onto 6 well plates, incubators (37 ℃, 5% CO)2) And incubated overnight.
Preparing liquid:
control group: inoculating cells (blank without any other components, to show the expression level of certain molecules in cells under normal conditions)
Stimulus: poly I C + LPS (24. mu.g/mL + 20. mu.g/mL) (formed in combination with DMSO + KC2500 medium) wherein Poly I C (Poly myo-inositol) is an agonist for Toll-like receptor (TLR 3); lipopolysaccharide (LPS) is an agonist of the Toll-like receptor (TLR 4). Reports from PAR2 media nature TRPV3 signalling in keratoctytes. journal of Investigative detail, 140(8), 1524. 1532) and k.h. Lee et al (Lee, k.h., choo, k.a., Kim, j.y., Baek, j.h., Woo, s.y., & Kim, J.W (2011.) cosmetic receptor activator induced by inflammatory cell receptor activator, inflammatory cell receptor activator, tissue receptor activator, tissue receptor activator, tissue receptor, tissue.
Dexamethasone: 0.01 wt% (dissolved in DMSO + KC2500 culture solution)
Peony root extract: 0.06mg/mL (dissolved in DMSO first and then KC2500 culture)
Administration induction:
respectively according to the test scheme, when the cell plating rate in the 6-well plate reaches 40% -60%, carrying out administration induction operation, wherein the administration amount of each well is 2mL, each group is provided with 3 multiple wells, and an incubator (37 ℃, 5% CO)2) Middle incubation for 24 hours
Collecting a sample:
after 24 hours of incubation, the solution was discarded, washed twice with 1 mL/well PBS, 1mL of RNAioso Plus was added to each well, lysed cells were aspirated, and samples were collected
And (3) gene expression detection:
extracting RNA, reverse transcribing to cDNA, and fluorescent quantitative PCR detection with 2-△△CT method carries out result calculation
And (4) result statistical analysis:
the graph is drawn by using GraphPad Prism Program software, t-test statistical analysis is adopted among groups, p <0.05 indicates that the difference is obvious, and p <0.01 indicates that the difference is extremely obvious
Results
See figure 2.
In keratinocytes, a stimulus induces expression of Thymic Stromal Lymphopoietin (TSLP) through Toll-like receptors on keratinocytes; both the anti-inflammatory drug dexamethasone and the peony root extract significantly inhibited thymic stromal lymphopoietin expression (p < 0.05).
The results indicate that the stimulus increased TSLP expression, inducing neuroinflammation. The peony root extract reduces the effect of irritants, and restores the intracellular neuroinflammation to a level close to that of the placebo (i.e., the level in the normal state).
Example 3
Peony root extract can inhibit tumor necrosis factor alpha (TNF-alpha) produced by macrophage
Material
The test system comprises:
the cells used in this experiment were mouse mononuclear macrophage leukemia cells (RAW264.7) obtained by laboratory subculture.
The main reagents are as follows:
DMEM high-glucose medium (Biosharp), fetal bovine serum (Biosharp), dpbs (Biosharp), mtt (Biosharp), dmso (Biosharp), 0.25% pancreatin (Biosharp).
The main equipment is as follows:
CO2 incubator (Shanghai Boxun, BC-J160), biological safety cabinet (Shanghai, BHC-1300IIA2), enzyme-linked immunosorbent assay (TECAN, Infinite F50), and centrifuge (Hunan Xiang apparatus, H1850R).
The test substance:
the control group was DMEM (high-glucose) containing 10% fetal bovine serum, which is a culture medium described below
Lipopolysaccharide (LPS) from Sigma
Dexamethasone Bulganling organism
Experimental methods
Inoculation:
cells were plated at 100000 cells/well in 96-well plates, 3 replicates per assay concentration, 37 ℃, 5% CO2The incubator is used for 24 hours plus or minus 2.
Preparing liquid:
control group: inoculating cells (blank without any other components, to show the expression level of certain molecules in cells under normal conditions)
Negative control group: cell culture medium containing LPS (5. mu.g/ml)
Positive control group: medium containing dexamethasone (100. mu.g/mL) and LPS
Test group 1: culture medium containing peony root extract (0.8mg/mL) and LPS (lipopolysaccharide)
Test group 2: culture medium containing peony root extract (1.6mg/mL) and LPS (lipopolysaccharide)
Administration:
after 24 hours +/-2 hours, the culture medium in the 96-well plate is removed, the DPBS is washed once, a test object group 1 and a test object group 2 are added into a test object hole, a positive control group is added into a positive control hole, and a negative control group is added into a negative control hole, wherein each hole is 200 mu l. After the administration, the 96-well plate was placed in CO2Continuously culturing for 6h in the incubator
Collecting a supernatant:
after 24 hours of incubation, the solution was discarded, washed twice with 1 mL/well PBS, 1mL of RNAioso Plus was added to each well, lysed cells were aspirated, and samples were collected
Detecting the content of TNF-alpha by an ELISA kit:
ELISA detection needs to be carried out according to the instruction of the mouse tumor necrosis factor alpha (TNF-alpha) enzyme-linked immunoassay kit
Results
See figure 3.
On macrophages, Lipopolysaccharide (LPS) induces upregulation of tumor necrosis factor α (TNF α) protein; the anti-inflammatory drug dexamethasone obviously reduces the quantity of tumor necrosis factor alpha; the peony root extract also reduced the amount of this inflammatory factor (by 10% -13%).
The result shows that the expression of TNF-alpha is greatly increased under the stimulation condition, the hormone anti-inflammatory drug dexamethasone pulls the TNF-alpha back to the normal level (but the hormone drug cannot be used for a long time and has side effect), and the peony root extract has no obvious effect of the hormone drug, but also obviously reduces the inflammation.
Example 4
Peony root extract inhibits Nerve Growth Factor (NGF) produced in keratinocytes
Material
The test system comprises:
the cells used in this test were keratinocytes, lot No.: EP21012001, obtained by cell primary, subculture, Guangdong Boxi Biotech limited.
The main reagents are as follows:
KC2500 broth (guangdong boxi), mtt (sigma), dmso (sigma), PBS (bosch de), rnaioso Plus (Takara), reverse transcription kit (Takara), fluorescent dye (Takara).
The main equipment is as follows:
CO2incubator (Thermo), clean bench (Sujing Antai), inverted microscope (Olympus), micro-oscillator (Linbel), enzyme labeling instrument (BioTek), incubator (Tester), fluorescent quantitative PCR instrument (BioRad), general PCR instrument (Bori).
The test substance:
the control group is cell + KC2500 culture solution
Substance P was purchased from Guangdong Boxi Biotech Ltd
Experimental methods
Inoculation:
by 2.8X 105Cell/well inoculation Density cells were plated onto 6 well plates, incubators (37 ℃, 5% CO)2) And incubated overnight.
Preparing liquid:
control group: inoculating cells (blank without any other components, to show the expression level of certain molecules in cells under normal conditions)
Substance P: at a concentration of 20nM
Test group 1: culture medium containing peony root extract (0.03mg/mL) and substance P
Test group 2: culture medium containing peony root extract (0.06mg/mL) and substance P
Administration induction:
respectively according to the test scheme, when the cell plating rate in the 6-well plate reaches 40% -60%, carrying out administration induction operation, wherein the administration amount of each well is 2mL, each group is provided with 3 multiple wells, and an incubator (37 ℃, 5% CO)2) Middle incubation for 24 hours
Collecting a sample:
after 24 hours of incubation, the solution was discarded, washed twice with 1 mL/well PBS, 1mL of RNAioso Plus was added to each well, lysed cells were aspirated, and samples were collected
And (3) gene expression detection:
extracting RNA, reverse transcribing to cDNA, and fluorescent quantitative PCR detection with 2-△△CT method carries out result calculation
And (4) result statistical analysis:
the graph is drawn by using GraphPad Prism Program software, t-test statistical analysis is adopted among groups, p <0.05 indicates that the difference is obvious, and p <0.01 indicates that the difference is extremely obvious
Results
See figure 4.
Substance P is a nerve signal peptide that induces vasodilation (redness of the skin) and expression of Nerve Growth Factor (NGF). In the experiment, the nerve signal peptide P substance induces the expression of nerve growth factor in keratinocyte, and the peony root extract obviously inhibits the expression of the nerve growth factor under the stimulation condition (P is less than 0.01) and has concentration effect.
The results indicate that the increase of NGF is involved in the path of skin itching, so the peony root extract has a soothing effect.
Example 4
Skin product
Toner
Name of raw materials | Content (wt%) |
Water (W) | To 100 |
Peony root extract | 0.05 |
Panthenol | 1 |
1, 3- |
3 |
1, 2-hexanediol | 0.3 |
Octanoyl hydroximic acid | 0.2 |
Glycerol | 5 |
PPG-26-Butaneth-26 | 0.2 |
Hyaluronic acid | 0.1 |
Emulsion and method of making
Name of raw materials | Content (wt%) |
Water (W) | to100 |
Ethyl hexyl palmitate | 5 |
Glycerol | 5 |
Polydimethylsiloxane | 2 |
|
1 |
|
1 |
Hydrogenated polyisobutenes | 4 |
|
1 |
Peony root extract | 0.5 |
|
1 |
Trimethylpentanediol/adipic acid/ |
1 |
|
1 |
1, 2-hexanediol | 0.5 |
P-hydroxyacetophenone | 0.3 |
PEG-40 stearate | 0.5 |
Polysorbate-60 | 0.5 |
Triethanolamine | 0.2 |
Carbomer | 0.2 |
Xanthan gum | 0.1 |
EDTA disodium salt | 0.1 |
The foregoing is merely a preferred embodiment of the invention and is not intended to limit the scope of the invention, which is defined by the claims appended hereto, and any other technical entity or method that is encompassed by the claims as broadly defined herein, or equivalent variations thereof, is contemplated as being encompassed by the claims.
Claims (12)
1. Use of extract of peony root for preparing a composition for strengthening and/or repairing skin barrier is provided.
2. Use of peony root extract in preparing composition for improving skin discomfort is provided.
3. Use of peony root extract in preparing composition for improving skin sensitivity is provided.
4. Use according to claim 3, wherein improving skin sensitivity comprises strengthening and/or repairing the skin barrier, reducing inflammatory factors, and having a soothing effect.
5. The use of any one of claims 1 to 4, which comprises 0.01 to 100 wt% of the extract of peony root, based on the total weight of the composition.
6. The use of claim 5, wherein said composition comprises peony root extract and a cosmetically acceptable carrier.
7. The use of claim 5, wherein the composition is a dermatological product.
8. The use according to claim 7, wherein the product form is selected from the group consisting of a solution, a gel, a cream, a liniment, a microemulsion spray, a suspension or an emulsion.
9. The use as claimed in claim 8, wherein the concentration of the peony root extract in the liquid form of the composition is 0.01-3.0 mg/mL.
10. A method for promoting production of filaggrin, loricrin, ceramide synthase3, and/or ultralong chain fatty acid elongase 4 in keratinocytes in vitro, comprising the steps of: mixing the keratinocytes with the extract of peony root.
11. A method of inhibiting in vitro thymic stromal lymphopoietin produced by keratinocytes and/or inhibiting nerve growth factor produced in keratinocytes, comprising the steps of: mixing the keratinocytes with the extract of peony root.
12. A method of inhibiting tumor necrosis factor α produced by macrophages in vitro, comprising the steps of: mixing macrophage with peony root extract.
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