Selaginella tamariscina extract, preparation method, application, skin care composition and inhibitor
Through the preparation and application of Cyperium extract, the problem that the prior art failed to effectively reduce the CCN1 expression level of skin cells was solved, and the protection of skin cells UVA stimulation response was achieved.
Patent Information
- Application Number
- CN202510063898.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-15
- Publication Date
- 2025-03-28
AI Technical Summary
The prior art has failed to reveal whether the CCN protein family, especially CCN1, has influenced the expression of the CCN protein family, and has led to the inability to effectively reduce the CCN1 expression level of skin cells.
A method for preparing a cypress extract is provided, including crushing the cypress, extracting, concentrating and purification with ethanol solution at N heating reflux, concentrating and purification, and finally obtaining the cypress extract, used to reduce the CCN1 expression level of skin cells.
Through the use of cypress extract, the CCN1 expression level increased due to UVA irradiation can be significantly reduced, and the skin protection effect can be achieved.
Smart Images
Figure CN119700604A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of Selaginella extraction, and in particular to a Selaginella extract, a preparation method, a use, a skin care composition and an inhibitor. Background Art
[0002] The cellular communication network factor (CCN) protein family is an important regulatory factor for regulating skin conditions. The CCN protein family is a group of extracellular matrix-related proteins, including CCN1 (CYR61), CCN2 (CTGF), CCN3 (NOV), CCN4 (WISP1), CCN5 (WISP2), and CCN6 (WISP3). The CCN protein family is involved in regulating multiple processes such as cell proliferation, differentiation, migration, apoptosis, and matrix remodeling, and is crucial for the homeostasis and repair of skin tissue.
[0003] There have been many reports on CCN1. Specifically, CCN1 activates the p53 signaling pathway to promote cell apoptosis and aging. When cells are subjected to oxidative stress or damage, CCN1 increases the apoptosis rate of cells through the p53 pathway to ensure that damaged cells do not continue to proliferate. CCN1 regulates the response of cells to damage such as ultraviolet rays by activating MAPK pathway molecules such as ERK and JNK, leading to accelerated aging of the skin. Moreover, in inflammatory responses, CCN1 upregulates the activity of the NF-κB pathway and promotes chronic inflammatory responses. Increased expression of CCN1 also activates NF-κB, leading to persistent inflammatory responses in skin tissues and accelerating the aging process. In addition, CCN1 also activates the MMP1 and c-Jun / AP-1 pathways to degrade collagen.
[0004] However, the existing technology has not revealed whether Selaginella has an effect on the expression of the CCN protein family, especially CCN1. Summary of the invention
[0005] One purpose of the embodiments of the present application is to solve at least one problem in the above background and provide corresponding beneficial effects.
[0006] Another object of an embodiment of the present application is to provide a Selaginella extract that can be used to reduce the expression level of CCN1 in skin cells.
[0007] Another object of the embodiments of the present application is to provide a preparation method, use, skin care composition and inhibitor of Selaginella extract.
[0008] In one aspect, the present invention provides a method for preparing a Selaginella extract, comprising:
[0009] crushing Selaginella to obtain Selaginella crushed material;
[0010] Using an ethanol solution to perform N times of heating reflux extraction on the crushed Selaginella, obtaining a filtrate after each extraction as a corresponding extract, combining all the extracts to obtain a Selaginella extract; wherein N is a positive integer greater than or equal to 1;
[0011] Concentrating the Selaginella extract, and then taking the solid part to obtain a Selaginella crude extract;
[0012] Purifying the crude Selaginella extract to obtain a purified Selaginella product;
[0013] The purified Selaginella was dried to obtain a Selaginella extract.
[0014] In another aspect, the embodiments of the present application provide a Selaginella extract prepared according to the preparation method of the Selaginella extract.
[0015] In another aspect, an embodiment of the present application provides a use of the Selaginella extract in preparing a CCN1 expression inhibitor.
[0016] In another aspect, an embodiment of the present application provides a use of the Selaginella extract in preparing a skin care composition.
[0017] In another aspect, the embodiments of the present application provide a skin care composition, comprising: the Selaginella extract.
[0018] In another aspect, the embodiments of the present application provide an inhibitor of CCN1 expression, including: the Selaginella extract.
[0019] The beneficial effects of the embodiments of the present invention include:
[0020] 1. In some embodiments, the embodiments of the present application provide a method for preparing a Selaginella extract. The Selaginella extract prepared according to the preparation method can reduce the expression level of CCN1 in skin cells.
[0021] 2. In some embodiments, the Selaginella extract prepared according to the preparation method of the Selaginella extract of the embodiment of the present application can reduce the expression level of CCN1 in HaCaT cells that is elevated due to UVA stimulation.
[0022] 3. In some embodiments, the embodiments of the present application provide a skin care composition containing a Selaginella extract, which can be used to reduce the expression level of CCN1 in HaCaT cells that is elevated due to UVA stimulation, thereby protecting the skin. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1 This is a schematic diagram of the mRNA expression level of CCN1 in each group in Experimental Example 1 of the present application. DETAILED DESCRIPTION
[0024] The present invention is further described in detail below in conjunction with the accompanying drawings so that those skilled in the art can implement the invention with reference to the description.
[0025] The terms "first", "second", etc. in the embodiments of the present application are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first", "second", etc. may explicitly or implicitly include at least one of the features.
[0026] Furthermore, the terms "comprises", "includes", "contains" and any variations thereof are intended to cover non-exclusive inclusions. For example, a process, method, system, product or device comprising a series of steps or units is not limited to the listed steps or units, but may optionally include steps or units not listed, or may optionally include other steps or units inherent to these processes, methods, products or devices.
[0027] In addition to the above, it is still necessary to emphasize that the reference to "embodiment" in this article means that the specific features, structures or characteristics described in conjunction with the embodiment may be included in at least one embodiment of the present application. The appearance of this phrase in various places in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment that is mutually exclusive with other embodiments. It is explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.
[0028] Unless otherwise expressly specified and limited, the term "mass-to-volume ratio" refers to mixing a certain mass of a substance with a certain volume of another substance in a specific ratio. For example, mixing substance A with substance B at a mass-to-volume ratio of 1:5 (g / mL) means mixing 1g of substance A with 5mL of substance B.
[0029] Unless otherwise expressly specified or limited, the term "μM" refers to micromoles per liter (μmol / L).
[0030] Unless otherwise specified and limited, the term "expression" refers to the formation of the result of protein production. For example, the expression of a protein means that the presence of the protein can be detected in cells or tissues.
[0031] Unless otherwise expressly specified and limited, the term "inhibit" refers to the result that (through various mechanisms) a protein is not produced or the production of a protein is reduced.
[0032] Unless otherwise expressly specified and limited, the term "skin" refers to the skin of the whole body including the face, neck, trunk and limbs.
[0033] <Method for preparing Selaginella extract>
[0034] In the first aspect, the embodiments of the present application provide a method for preparing a Selaginella extract. The Selaginella extract prepared according to the preparation method can be used to reduce the expression level of CCN1, especially to reduce the expression level of CCN1 in skin cells irradiated by UVA, thereby protecting the skin.
[0035] Specifically, the preparation method of the Selaginella extract comprises the following steps S1 to S5:
[0036] S1. Crush Selaginella to obtain Selaginella crushed material.
[0037] The species of Selaginella in S1 is not limited, as long as it belongs to the genus Selaginela (Selaginela P. Beauv.). In some embodiments, the Selaginella is selected from the dried whole herb material of Selaginela tamariscina (P. Beauv.) Spring, a plant of the Selaginaceae family.
[0038] When obtaining the Selaginella crushed material, specifically, the Selaginella medicinal material can be taken and crushed to obtain the Selaginella crushed material.
[0039] S2. Perform N heating reflux extractions on the Selaginella crushed material with an ethanol solution, obtain the filtrate after each extraction as the corresponding extract, combine all the extracts to obtain the Selaginella extract, where N is a positive integer greater than or equal to 1.
[0040] Specifically, the crushed Selaginella material can be mixed with an ethanol solution in a predetermined material-liquid ratio, reflux extracted for a certain period of time under predetermined temperature conditions, and filtered to obtain an extract; when N is greater than 1, the extraction is repeated N times, and the N extracts are combined as the Selaginella extract.
[0041] In some embodiments, when N is equal to 1, the crushed Selaginella is mixed with an ethanol solution, subjected to heating reflux extraction, and filtered to obtain a Selaginella extract.
[0042] In some embodiments, when N is greater than or equal to 2,
[0043] In the first heating reflux extraction, the crushed Selaginella is mixed with the ethanol solution for the first heating reflux extraction, and the first extract and the first filter residue are obtained by filtration;
[0044] In the i-th heating reflux extraction, the i-1-th filter residue is mixed with the ethanol solution to perform the i-th heating reflux extraction, and the i-th extract and the i-th filter residue are obtained by filtration; i is greater than 1 and less than or equal to N;
[0045] After N times of heating reflux extraction, all the extracts are combined to obtain the Selaginella extract.
[0046] In the examples of the present application, there is no particular restriction on the number of times of heating reflux extraction, and the number mainly affects the content of aconitum biflavonoids in the Selaginella extract.
[0047] In step S2, the temperature of each heating reflux extraction is not less than 75°C. When the temperature of the heating reflux extraction is not less than 75°C, the temperature of the heating reflux extraction mainly affects the content of sedum biflavonoids in the Selaginella extract. Optionally, in S2, the temperature of the heating reflux extraction is 75 to 95°C. Further optionally, in S2, the temperature of the heating reflux extraction is 70 to 80°C.
[0048] In step S2, the duration of each heating reflux extraction is not particularly limited, and the duration of the heating reflux extraction mainly affects the content of aconitum biflavonoids in the Selaginella extract. Optionally, in S2, the duration of the heating reflux extraction is 2 to 3 hours.
[0049] In step S2, in each heating reflux extraction, the volume fraction of the ethanol solution used (the volume fraction of ethanol in the ethanol solution) is not less than 50%. When the volume fraction of the ethanol solution is not less than 50%, the volume fraction of the ethanol solution mainly affects the content of the selaginella biflavonoids in the Selaginella extract. Optionally, in S2, in each heating reflux extraction, the volume fraction of the ethanol solution is 50 to 95%. Further optionally, in S2, the volume fraction of the ethanol solution is 50 to 70%.
[0050] In S2, in each heating reflux extraction, the volume of the ethanol solution used is not particularly limited. The volume of the ethanol solution mainly affects the content of aconitum biflavonoids in the Selaginella extract.
[0051] Optionally, the volume of the ethanol solution used in each heating reflux extraction can be limited by the ratio to the mass of Selaginella in step S1. Optionally, in each heating reflux extraction, the volume of the ethanol solution used and the mass of the Selaginella are (5-20) mL:1 g, and further optionally (10-20) mL:1 g; in other words, the mass volume ratio of the Selaginella to the ethanol solution in each heating reflux extraction is 1:5-20 (g / mL), and further optionally 1:10-20 (g / mL).
[0052] S3. Concentrate the Selaginella extract, then take the solid portion to obtain a crude Selaginella extract.
[0053] Specifically, the Selaginella extract is concentrated, and the concentrated solid portion is used as a Selaginella crude extract.
[0054] In some embodiments, the Selaginella extract can be concentrated at 50° C. for 1 h, and the concentrated solid portion can be collected as the Selaginella crude extract.
[0055] S4. Purify the crude extract of Selaginella to obtain a purified Selaginella product.
[0056] The purification method is not particularly limited, and mainly affects the content of aconitum biflavonoids in the Selaginella extract.
[0057] Optionally, in S4, purification is performed by column chromatography, that is, purification is column chromatography purification.
[0058] Specifically, in some embodiments, step S4 includes: purifying the crude Selaginella extract using column chromatography to obtain a purified Selaginella product.
[0059] In some embodiments, the method of purifying the crude extract of Selaginella using column chromatography to obtain a purified Selaginella product further comprises step S41:
[0060] S41. Add the crude extract of Selaginella to a chromatography column and elute it with an ethanol solution to obtain a purified Selaginella product.
[0061] It should be understood that the method of adding the crude Selaginella extract to the chromatography column can be implemented according to the existing technology, such as adding the crude Selaginella extract by dry method, wet method and other loading methods.
[0062] Optionally, in S41, the column filler of the chromatography column is a macroporous resin. Optionally, the macroporous resin is any one of AB-8 macroporous resin, D101 macroporous resin, and DM-130 macroporous resin; that is, the chromatography column is selected from any one of AB-8 macroporous resin chromatography column, D-101 macroporous resin chromatography column, and DM-130 macroporous resin.
[0063] The elution method in S41 is not particularly limited, as long as the eluted target product includes at least aconitum biflavonoids.
[0064] In some embodiments, the column chromatography method sequentially uses 10% ethanol solution, 30% ethanol solution and 70% ethanol solution with a volume fraction as eluents for elution.
[0065] Therefore, it can be understood that, in at least one example, step S41 includes steps S411 to S412:
[0066] S411, adding the crude extract of Selaginella to a chromatography column, and eluting with 10% by volume ethanol solution, 30% by volume ethanol solution and 70% by volume ethanol solution in sequence;
[0067] S412. Collect the eluate obtained by eluting with 70% ethanol solution as the purified Selaginella.
[0068] Step S5, drying the purified Selaginella to obtain a Selaginella extract.
[0069] In at least one embodiment, the purified Selaginella can be dried using a conventional rotary evaporator at a drying temperature of 50° C. for 3 hours to obtain a Selaginella extract.
[0070] <Selaginella Extract>
[0071] In a second aspect, the embodiments of the present application provide a Selaginella extract, which at least includes aconitum biflavonoids.
[0072] In some embodiments, the Selaginella extract is prepared according to the preparation method of the Selaginella extract described in the first aspect.
[0073] The Selaginella extract provided in the examples of the present application can be used to reduce the expression level of CCN1, especially to reduce the expression level of CCN1 in skin cells irradiated by UVA.
[0074] <Composition>
[0075] In a third aspect, the present application provides a composition comprising:
[0076] The first aspect is the Selaginella extract described in the embodiment.
[0077] In some embodiments, the composition includes a solvent. Optionally, the solvent is water.
[0078] In some embodiments, the concentration of the Selaginella extract in the composition is no less than 7.5 μg / mL.
[0079] In some embodiments, the concentration of the Selaginella extract in the composition is 7.5-20 μg / mL; alternatively, the concentration is 7.5 μg / mL; alternatively, the concentration is 15 μg / mL; alternatively, the concentration is 20 μg / mL.
[0080] In some embodiments, the composition is a skin care composition, ie, a skin care composition.
[0081] In some embodiments, the skin care composition can be in the form of any one of an aqueous solution, an oily solution, an aqueous cream, an aqueous gel, and an oily gel.
[0082] In some embodiments, the skin care composition can be any one of a lotion, a microemulsion, a nanoemulsion, a bath gel, a facial mask, an essence, a lotion, a liquid soap, a skin cake, an ointment, a mousse, a spray, a conditioner, and a shampoo.
[0083] In some embodiments, the skin care composition may further include at least one cosmetically acceptable excipient.
[0084] In some embodiments, the excipient may include one or more of a surfactant, an emulsifier, a preservative, a buffer, a chelating agent, a denaturant, an opacifier, a pH adjuster, a reducing agent, a stabilizer, a thickener, a gelling agent, a film-forming polymer, a matting agent, a glossing agent, a pigment, a fragrance, and an essential oil.
[0085] <Inhibitors>
[0086] The present application also provides an inhibitor of CCN1 expression, including:
[0087] The first aspect is the Selaginella extract described in the embodiment.
[0088] <Purpose>
[0089] In addition to the above, the embodiments of the present application also provide a use of a Selaginella extract in preparing a skin care composition.
[0090] Furthermore, the Selaginella extract acts as an inhibitor of CCN1 expression.
[0091] Optionally, the skin care composition is an external skin preparation.
[0092] The present application also provides a use of a Selaginella extract in preparing an inhibitor of CCN1 expression.
[0093] The Selaginella extract contains at least aconitum biflavonoids. The Selaginella extract can be obtained in any manner, and can be purchased from the market, or obtained by a common preparation method in the art, or prepared according to the preparation method described in the embodiment of the first aspect.
[0094] <Preparation Example of Selaginella Extract>
[0095] Preparation Example 1
[0096] The Selaginella extract was prepared according to the preparation method shown in the following steps S101 to S105.
[0097] Step S101, taking 100g of Selaginella medicinal material and crushing it to obtain Selaginella crushed material; the Selaginella medicinal material is selected from the dried whole herb material of Selaginela tamariscina (P.Beauv.) Spring of the Selaginaceae family.
[0098] Step S102, using a 70% by volume ethanol solution at 80°C to perform heating reflux extraction on the Selaginella crushed material three times, each time for 3 hours, filtering after each extraction and using the filtrate as the corresponding extract, combining all the extracts to obtain the Selaginella extract.
[0099] Specifically, step S102 includes steps S1021 to S1024:
[0100] S1021, mixing the crushed Selaginella with an ethanol solution with a volume fraction of 70%, performing a first heating reflux extraction, wherein the temperature of the first heating reflux extraction is 80° C., the time is 3 hours, and filtering to obtain a first extract and a first filter residue; wherein in the first heating reflux extraction, the volume of the ethanol solution with a volume fraction of 70% and the mass of the Selaginella medicinal material are 15 mL: 1 g;
[0101] S1022, mixing the first filter residue with an ethanol solution with a volume fraction of 70% for a second heating reflux extraction, wherein the temperature of the second heating reflux extraction is 80° C., the time is 3 hours, and filtering to obtain a second extract and a second filter residue; wherein in the second heating reflux extraction, the volume of the ethanol solution with a volume fraction of 70% and the mass of the Selaginella medicinal material are in the ratio of 15 mL:1 g;
[0102] S1023, mixing the second filter residue with an ethanol solution with a volume fraction of 70% for a third heating reflux extraction, wherein the temperature of the third heating reflux extraction is 80° C., the time is 3 hours, and filtering to obtain a third extract and a third filter residue; wherein in the third heating reflux extraction, the volume of the ethanol solution with a volume fraction of 70% and the mass of the Selaginella medicinal material are in the ratio of 15 mL:1 g;
[0103] S1024, combining the first extract, the second extract and the third extract to obtain the Selaginella extract.
[0104] S103, concentrating the Selaginella extract at 50°C for 1 hour, and then taking the solid portion as a Selaginella crude extract.
[0105] S104, adding the crude extract of Selaginella to an AB-8 macroporous resin chromatography column, and then eluting it with 10% ethanol solution, 30% ethanol solution, and 70% ethanol solution by volume, collecting the eluate obtained by eluting with 70% ethanol solution as the purified Selaginella.
[0106] S105. Dry the purified Selaginella using a rotary evaporator at a drying temperature of 50° C. for 3 h to obtain a Selaginella extract.
[0107] <Experiment>
[0108] The skin has the function of alleviating external stimuli and controlling the loss of body components such as water. However, when the skin is affected by external factors such as ultraviolet rays, strong air dryness, excessive skin washing, smoking, or as we age, the activity and proliferation of epidermal cells will decrease, and the renewal rate of the epidermis will be delayed, which will cause incomplete differentiation such as thinning of the epidermis or thickening of the stratum corneum, which will lead to reduced moisturizing function or elasticity of the skin and abnormal peeling of keratin. In addition, the skin will age due to external factors, and changes such as wrinkles, dullness, loss of texture, and reduced elasticity will occur.
[0109] Skin aging is related to multiple signaling pathways. At present, the main signaling pathways related to skin aging that have been identified include: 1. Oxidative stress pathway. Oxidative stress is caused by free radicals (such as reactive oxygen and reactive nitrogen), which can damage the DNA, proteins and lipids of skin cells, leading to skin aging. 2. mTOR pathway. mTOR is a protein kinase that is involved in regulating cell growth and metabolism. When the mTOR pathway is overactivated, it promotes the production of aging markers and accelerates skin aging. 3. p53 pathway. p53 is a tumor suppressor gene that promotes cell repair of DNA damage or induces cell apoptosis. Overactivation of p53 can lead to skin cell aging and functional decline. 4. Wnt / β-catenin pathway. This pathway plays an important role in the differentiation and proliferation of skin cells. When the pathway is out of regulation, it will lead to a decrease in skin regeneration ability and accelerated aging. 5. MAPK (Mitogen-Activated Protein Kinase) pathway. This pathway involves subtypes such as ERK, JNK and p38, which promote the aging process of skin cells by responding to external stress (such as ultraviolet rays). 6. NF-κB pathway. NF-κB is an important factor in skin stress and inflammatory responses.
[0110] According to existing studies, extracts from Selaginella tamariscina (P.Beauv.) Spring have a significant effect on delaying skin aging. The antioxidant components in Selaginella can upregulate the expression and activity of antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), which helps to scavenge free radicals and reduce oxidative stress damage to the skin. The antioxidant properties of Selaginella extracts may indirectly affect the expression of p53 and protect cells from damage. Moderate activity of p53 helps repair DNA damage, while excessive activation may lead to cell apoptosis.
[0111] However, the regulation of skin condition is a very complex process. Each signal pathway is affected by a variety of regulatory factors. It is complex but important to continuously discover new regulatory factors to reveal the regulatory mechanism of drugs. The CCN (Cellular Communication Network Factor) protein family is one of the important regulatory factors.
[0112] It has not yet been discovered whether Selaginella has an effect on the CCN protein family, especially the expression of CCN1. Therefore, the relationship between Selaginella and CCN1 expression will be verified through experiments below.
[0113] Experimental cells :
[0114] Keratinocytes HaCaT
[0115] Experimental reagents
[0116] DMEM (dulbecco's modified eagle medium): is a basic culture medium purchased from Gibco Biological Culture Medium of Thermo Fisher Scientific (China) Co., Ltd., with the product number C11995500BT.
[0117] Fetal bovine serum (FBS): purchased from Gibco brand fetal bovine serum of Thermo Fisher Scientific (China) Co., Ltd., product number 10091148.
[0118] Penicillin / streptomycin: Gibco brand penicillin / streptomycin purchased from Thermo Fisher Scientific (China) Co., Ltd., product number 25200114, penicillin / streptomycin solution (10,000 U / mL), which includes penicillin and streptomycin, with a concentration of 10,000 U / mL respectively.
[0119] Phosphate buffered saline (PBS): pH 7.4, 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; Gibco brand PBS purchased from Thermo Fisher Scientific (China) Co., Ltd.
[0120] RNA extraction kit: specifically TransZol Up Plus RNA Kit (Transgen), suitable for extracting total RNA from cells and tissues. After the sample is lysed with TransZol Up and RNA Extraction Agent is added, the solution is divided into a colorless aqueous phase and a pink organic phase, and RNA is in the aqueous phase; the RNA in the aqueous phase is specifically adsorbed using a silica gel membrane centrifugal column. The RNA extraction kit specifically includes the following components: TransZol Up 100ml, RNA Extraction Agent 20ml, Clean Buffer 9 (CB9) 110ml, Wash Buffer 9 (WB9) 24ml, RNase-free Water 40ml, RNase-free Tube (1.5ml) 100each, RNA Spin Columns with Collection Tubes 100each.
[0121] Using the reverse transcription kit of M-MLV reverse transcriptase, the Evo M-MLV reverse transcription premix kit (including gDNA removal reagent, for qPCR) Ver.2 (Agbio, AG11728) was selected, which contains 5X Evo M-MLV RT Reaction Mix, and the cDNA product can be directly used for qPCR detection. The Evo M-MLV reverse transcription premix kit (including gDNA removal reagent, for qPCR) Ver.2 contains all the components required for the reverse transcription reaction.
[0122] 2× real-time quantitative PCR amplification premix solution (English name is 2×qPCR SYBR Green MasterMix, referred to as SYBR Mix), which contains hot start DNA polymerase ( DNA Polymerase), SYBRGreen I, deoxyribonucleoside triphosphates (dNTPs), magnesium ions (Mg 2+ ). qPCR SYBR Green Master Mix can be selected qPCR SYBR Green Master Mix (No Rox) can be purchased from Yeasen, catalog number 11202ES.
[0123] Experimental equipment
[0124] CO2 incubator (Thermo, 150I).
[0125] Ultra-clean workbench (Sujing Antai, SW-CJ-1F).
[0126] Microvolume spectrophotometer (Thermo Scientific NanoDrop).
[0127] ProFlex TM PCR instrument (Applied Biosystems SimpliAmp PCR Thermal Cycler).
[0128] QPCR instrument (Applied Biosystems QuantStudio 1).
[0129] UVA crosslinker (Luyor UCL-3500).
[0130] High-speed refrigerated centrifuge (Eppendorf Centrifuge 5418R).
[0131] Experimental samples and preparation :
[0132] 1. Related solutions used to prepare samples
[0133] Basic culture medium solution: a DMEM culture medium solution containing FBS and penicillin / streptomycin, specifically a DMEM culture medium solution containing 10% FBS and 1% penicillin / streptomycin; wherein the mass fraction of FBS is 10%, and the mass fraction of penicillin / streptomycin is 1%; the basic culture medium solution can be prepared by mixing FBS, penicillin / streptomycin and DMEM culture medium.
[0134] Selaginella extract mother solution: Take the Selaginella extract of Preparation Example 1, dissolve the powder of the Selaginella extract in dimethyl sulfoxide (DMSO) to form a Selaginella extract mother solution expressed by mass fraction, and the concentration can be adjusted as needed.
[0135] 2. Samples
[0136] Blank control group sample: Select the basal culture medium solution as the blank control group sample.
[0137] UVA control group sample: Select the basal culture medium solution as the UVA control group sample.
[0138] Sample 1: DMEM medium solution containing Selaginella extract at a concentration of 7.5 μg / mL. Specifically, the sample can be obtained by diluting the Selaginella extract mother solution with a basal medium solution until the concentration of the Selaginella extract is 7.5 μg / mL.
[0139] Sample 2: DMEM culture medium solution containing Selaginella extract at a concentration of 15 μg / mL. Specifically, the sample can be obtained by diluting the Selaginella extract mother solution with a basal culture medium solution until the concentration of the Selaginella extract is 15 μg / mL.
[0140] Sample 3: DMEM culture medium solution containing Selaginella extract at a concentration of 20 μg / mL. Specifically, the sample can be obtained by diluting the Selaginella extract mother solution with a basal culture medium solution until the concentration of the Selaginella extract is 20 μg / mL.
[0141] <Experimental Example 1, mRNA expression level detection>
[0142] The Selaginella extract provided in the examples of the present application was administered to HaCaT cells that had been irradiated with UVA, and then the mRNA expression level of the CCN1 gene of the HaCaT cells was detected to analyze the anti-UVA efficacy of the Selaginella extract.
[0143] The experimental method includes the following steps S201 to S204.
[0144] S201, cell seeding:
[0145] Specifically, human immortalized keratinocytes (HaCaT) were taken and cultured at 80×10 4 The cells were inoculated into a 6-well plate at a density of 100 μL / well, and 2000 μL complete medium (complete medium is DMEM medium containing 10% FBS and 1% penicillin / streptomycin) was added to each well; then incubated in an incubator (the incubator contains 5% CO2 and the temperature is 37°C) for 24 h.
[0146] S202, drug administration:
[0147] When the cell plating rate in the 6-well plate reached 70% to 80%, the cells were divided into a blank control group (NC), a UVA control group (MOCK) and three experimental groups, namely experimental group 1, experimental group 2 and experimental group 3;
[0148] Except for the blank control group, the UVA control group and all experimental groups were treated with 10 mJ / cm 2 The cells were irradiated with a UVA dose of 100 μg / cm2; the culture medium of the blank control group, UVA control group and each experimental group was then discarded, and the corresponding treatment substances were added to each group for drug administration. Three replicate wells were set up in each group (used as three parallel groups, i.e., parallel groups 1 to 3). After the drug administration was completed, the cells were placed in an incubator (37°C, 5% CO2) and cultured for 24 hours;
[0149] The corresponding treated substances added to the blank control group, UVA control group, experimental group 1, experimental group 2, and experimental group 3 were blank control group sample, UVA control group sample, sample 1, sample 2, and sample 3, respectively.
[0150] S203, RNA extraction:
[0151] After culturing for 24 hours, RNA was extracted from the cells in each group, specifically including the following steps S2031-S20310.
[0152] S2031. After 24 hours of culture, pour out the culture medium and rinse once with PBS. 2 Add 1 ml of TansZol Up to the growing cultured cells, place horizontally for a while to evenly distribute the lysate on the cell surface and lyse the cells, then use a pipette to blow the cells to make them fall off. Transfer the cell lysate to a centrifuge tube, add 0.2 ml of RNA ExtractionAgent, and repeatedly blow and suck with a pipette until there is no obvious precipitation in the lysate, and vortex at room temperature for 5 minutes.
[0153] S2032. Centrifuge at 10,000 g and 2-8°C for 15 minutes. The sample is now separated into three layers: a colorless aqueous phase (upper layer), a middle layer, and a pink organic phase (lower layer); RNA is in the aqueous phase, and the volume of the aqueous phase is about 50% to 60% of the TransZol Up reagent used.
[0154] S2033. Transfer the colorless aqueous phase to a new centrifuge tube, add an equal volume of anhydrous ethanol, and gently invert to mix.
[0155] S2034. Add the obtained solution and precipitate into a centrifugal column, centrifuge at 12,000 g for 30 seconds at room temperature, and discard the effluent.
[0156] S2035, then add 500 μl of CB9 (Clean Buffer 9), centrifuge at 12,000 g for 30 seconds at room temperature, discard the flow-through, and then repeat this step once.
[0157] S2036. Then add 500 μl of WB9 (Wash Buffer 9), centrifuge at 12,000 g for 30 seconds at room temperature, discard the flow-through, and repeat this step once.
[0158] S2037, centrifuge at 12,000g for 2 minutes at room temperature to completely remove residual ethanol.
[0159] S2038. Place the centrifuge column in an RNase-free tube, add 50-200 μl of RNase-free water in the center of the centrifuge column, and let stand at room temperature for 1 minute.
[0160] S2039, centrifuge at 12,000 g for 1 minute at room temperature to elute RNA.
[0161] S20310. Use a micro-spectrophotometer (Thermo Scientific Nano Drop) to measure the concentration of RNA, obtain RNA samples with measured concentrations in each group, and store them at -80°C.
[0162] S204. Perform qPCR on the RNA samples with measured concentrations in each group to determine the relative mRNA expression level (Relative mRNA Level) of the CCN1 gene.
[0163] Step S204 specifically includes steps S2041 to S2043:
[0164] S2041. Reverse transcribe the RNA samples of each group with measured concentration to obtain reverse transcribed samples.
[0165] Specifically, RT-PCR reaction was used for reverse transcription: the RNA sample with measured concentration was used to calculate the corresponding RNA volume based on 2ug, and then the corresponding volume of RNase-free water (RNase Free H2O) and reverse transcriptase mix were added. The total volume of the RT-PCR reaction system was 20μL.
[0166] The specific system of RT-PCR reaction can be found in Table 1, and the specific procedure of RT-PCR reaction can be found in Table 2.
[0167] Table 1. RT-PCR reaction system
[0168] name volume RNA 2ug <![CDATA[RNase Free H2O]]> Up to 20μL 5X Evo M-MLV RT Master Mix 4μL
[0169] Table 2. RT-PCR reaction procedure
[0170] temperature time 37℃ 40min 85℃ 5s 12℃ ∞
[0171] S2042. Then dilute 20 μL of the reverse transcription sample to 400 μL to obtain a cDNA sample with a final concentration of 5 ng / μL.
[0172] S2043. Take cDNA samples for qPCR reaction and analyze the relative expression level of mRNA of the gene to be tested corresponding to each group.
[0173] Specifically, cDNA samples were used as cDNA templates for qPCR reaction, and 3 duplicate wells were made for each gene of each cDNA sample for detection. After adding the samples, the qPCR program was performed on the machine. After the qPCR reaction, the reaction data was processed, and the 2-△△Ct method was used to analyze the relative mRNA expression level (Relative mRNA Level) of the CCN1 gene in each group. The data between the groups were analyzed using T-TEST statistics, and P < 0.05 indicated a significant difference, which was statistically significant.
[0174] Among them, the volume of each well of the qPCR reaction system is 10 μL, as can be seen in Table 3;
[0175] The qPCR reaction program can be found in Table 4.
[0176] Table 3. qPCR reaction system
[0177] name volume cDNA template 4μL 2X qPCR SYBR Green Master Mix 5μL <![CDATA[Double distilled water (ddH2O)]]> 0.4μL 10uM primer (forward + reverse) 0.6μL
[0178] Table 4. qPCR reaction procedure
[0179]
[0180]
[0181] Experimental results and analysis :
[0182] The experimental results are shown in Table 5 and Figure 1 .
[0183] Table 5 records the relative expression levels of CCN1 in three parallel groups, namely, the blank control group, the UVA control group, the experimental group 1, the experimental group 2, and the experimental group 3.
[0184] It can be understood that by calculating the mean of the CCN1 relative expression data of the three parallel groups, an average relative mRNA expression level of each group can be obtained.
[0185] Figure 1 It is a schematic diagram of the relative mRNA expression level (Relative mRNA Level) of the CCN1 gene in the blank control group, UVA control group, experimental group 1, experimental group 2, and experimental group 3.
[0186] Table 5. Relative expression of CCN1 in each group
[0187]
[0188] According to the above results, Selaginella extract can reduce the high expression of CCN1 after UVA irradiation and resist photoaging of skin cells.
[0189] When the concentration of Selaginella extract is 7.5-20 μg / mL, the high expression of CCN1 after UVA irradiation can be significantly reduced, indicating that Selaginella extract can resist photoaging of skin cells by reducing the high expression of CCN1.
[0190] <Application Examples>
[0191] The Selaginella extract of the example of the present application can be used to prepare a skin external preparation. According to this application example, a skin external preparation is provided, and its formula is shown in Table 6.
[0192] The preparation method of the skin external preparation is as follows:
[0193] Step A1, swell phase A and heat it uniformly to 80-85°C, heat phase C to 80-85°C to dissolve, then add the heated phase C to the heated phase A and homogenize for 2 minutes to obtain a first mixture;
[0194] Step A2, heating and dissolving phase B until it becomes clear and transparent to obtain a clear and transparent phase B;
[0195] Step A3, add phase D to the first mixture of step A1 and homogenize for 1 minute, stir and cool to below 40°C, add phase E and the clear and transparent phase B in step A2, stir evenly, and discharge to obtain the skin external preparation.
[0196] Table 6. Formulations of external skin preparations
[0197] Mutually Name of the ingredient in the recipe Component content Component effect A Deionized water margin Solvents A Polyacrylate Crosspolymer-6 0.150 Thickener A Carbomer 0.120 Thickener A Disodium EDTA 0.020 Chelating agents B p-Hydroxyacetophenone 0.500 Antioxidants B Hexanediol 0.500 Moisturizer B Butanediol 2.000 Moisturizer C Isononyl Isononanoate 10.000 Emollients C C14-22 Alcohol (and) C12-20 Alkyl Glucoside 1.300 Emulsifier D Aminomethyl propanol 0.082 pH Adjusters D Deionized water 3.000 Solvents E Selaginella Extract 0.1 Selaginella Extract E Deionized water 2.000 Solvents
[0198] The skin external preparation contains the Selaginella extract of the embodiment of the present application, so it can reduce the high expression of CCN1 after UVA irradiation, and play a role in resisting photoaging of skin cells.
[0199] The above specific embodiments have been described in detail for the present invention, but these do not constitute limitations on the present invention. The protection scope of the present invention is not limited to the above embodiments, and any equivalent modifications or changes made by ordinary technicians in the field based on the content disclosed by the present invention should be included in the protection scope recorded in the claims.
Claims
1. A method for preparing a Selaginella extract, characterized in that: include: crushing Selaginella to obtain Selaginella crushed material; The pulverized Selaginella is subjected to N heating reflux extractions with an ethanol solution, and the filtrate is obtained as the corresponding extract after each extraction, and all the extracts are combined to obtain a Selaginella extract; wherein N is a positive integer greater than or equal to 1; Concentrating the Selaginella extract, and then taking the solid part to obtain a Selaginella crude extract; Purifying the crude Selaginella extract to obtain a purified Selaginella product; The purified Selaginella product is dried to obtain a Selaginella extract.
2. The method for preparing the Selaginella extract according to claim 1, characterized in that: When N is greater than or equal to 2, the Selaginella pulverized material is subjected to N heating reflux extractions with an ethanol solution, a filtrate is obtained after each extraction as a corresponding extract, and all the extracts are combined to obtain a Selaginella extract, comprising: In the first heating reflux extraction, the crushed Selaginella is mixed with the ethanol solution for the first heating reflux extraction, and the first extract and the first filter residue are obtained by filtration; In the i-th heating reflux extraction, the i-1-th filter residue is mixed with the ethanol solution to perform the i-th heating reflux extraction, and the i-th extract and the i-th filter residue are obtained by filtration; i is greater than 1 and less than or equal to N; After N times of heating reflux extraction, all the extracts are combined to obtain the Selaginella extract.
3. The method for preparing the Selaginella extract according to claim 1, characterized in that: The temperature of each heating reflux extraction is not less than 75°C; Each heating reflux extraction lasts 2 to 3 hours; In each heating reflux extraction, the volume fraction of the ethanol solution is 50-95%; In each heating reflux extraction, the ratio of the volume of the ethanol solution used to the mass of the Selaginella is (5-20) mL: 1 g.
4. The method for preparing the Selaginella extract according to claim 1, characterized in that: The method of purifying the crude extract of Selaginella to obtain the purified Selaginella comprises: The crude extract of Selaginella was purified by column chromatography to obtain a purified Selaginella product.
5. The method for preparing the Selaginella extract according to claim 4, characterized in that: The method of purifying the crude Selaginella extract by column chromatography to obtain the purified Selaginella product comprises: adding the crude Selaginella extract to a chromatography column and eluting with an ethanol solution to obtain the purified Selaginella product; The step of adding the crude extract of Selaginella to a chromatography column and eluting with an ethanol solution to obtain a purified Selaginella product comprises: Adding the Selaginella crude extract to a chromatography column, and then eluting with 10% by volume ethanol solution, 30% by volume ethanol solution and 70% by volume ethanol solution in sequence; The eluate obtained by eluting with a 70% by volume ethanol solution was collected as a purified Selaginella.
6. The Selaginella extract prepared according to the method for preparing the Selaginella extract according to any one of claims 1 to 5.
7. Use of the Selaginella extract according to claim 6 in the preparation of a CCN1 expression inhibitor.
8. Use of the Selaginella extract according to claim 6 in preparing a skin care composition.
9. A skin care composition, characterized in that include: The Selaginella extract according to claim 6.
10. An inhibitor of CCN1 expression, characterized in that include: The Selaginella extract according to claim 6.