A composition for skin anti-inflammatory and moisturizing effects containing meadowsweet extract as an active ingredient.

Abstract

The present invention relates to compositions for anti-inflammatory and moisturizing effects on the skin, and more specifically, provides compositions containing meadowsweet extract as an active ingredient that exhibit anti-inflammatory and moisturizing effects on the skin, as well as pharmaceutical compositions, health functional food compositions, and cosmetic compositions containing the same.

Description

[Technical Field]

[0001] The present invention relates to a composition for skin anti-inflammatory and moisturizing effects, comprising a meadowsweet extract as an active ingredient. [Background technology]

[0002] Inflammation is closely associated with various diseases, particularly those caused by infection and tissue damage in the skin. Disruption of the epidermal barrier and impairment of the immune system are significant causes of inflammatory skin disease (ISD). A damaged skin barrier allows various allergens to easily penetrate the skin, leading to immune system dysregulation and the development and exacerbation of ISD. Overexpressed chemokines in this process act as pro-inflammatory mediators and are major causes of ISD progression.

[0003] Current knowledge indicates that inflammatory chemokines are primarily produced by keratinocytes, Langerhans cells, and leukocytes and infiltrating cells such as neutrophils. Various environmental stimuli resulting from epidermal barrier damage promote the secretion of inflammatory chemokines by keratinocyte lines, stimulation of dendritic and Langerhans cells, and activation of type 2 helper T cells (Th2 cells) to produce Th2 cytokines, leading to impaired barrier function, abnormal keratinocyte differentiation, and persistent itching. Furthermore, damage to the permeability of the stratum corneum leads to dehydration due to increased transepidermal water loss, exacerbating inflammation through increased release of inflammatory cytokines, particularly chemokines. Extracorporeal keratinocyte models are used to investigate the potential of various natural and synthetic substances as anti-inflammatory candidates, as they interact with other cells and play a central role in the progression and onset of ISD.

[0004] Meadowsweet (Filipendula genus), belonging to the Rosaceae family, is a perennial herbaceous plant that prefers moist habitats such as damp grasslands. Due to its pharmacological properties as a diuretic, antiseptic, anti-rheumatic, astringent, stomachic, and antacid, it has been traditionally used in folk medicine.

[0005] The genus Filipendula includes approximately 30 species, but much research has focused on only two major species: F. ulmaria (L.) Maxim (meadowsweet) and F. vulgaris Moench (dropwort). F. palmata (Pall.) Maxim is also known as Siberian meadowsweet and is mainly distributed in North Korea, northeastern China, and eastern Russia. Therefore, the inventors attempted to extract and analyze physiologically active substances from Siberian meadowsweet to clarify its anti-inflammatory and moisturizing effects on the skin. [Overview of the project] [Problems that the invention aims to solve]

[0006] The object of the present invention is to provide a composition for skin anti-inflammatory and moisturizing effects, comprising meadowsweet extract as an active ingredient.

[0007] Other objectives and merits of the present invention will be further clarified by the following detailed description of the invention, claims, and drawings. [Means for solving the problem]

[0008] The present invention provides a composition for skin anti-inflammatory and moisturizing effects, comprising a meadowsweet extract as an active ingredient.

[0009] In the present invention, Spiraea extract means an extract obtained by using water, C1-C4 lower alcohols, or a mixture thereof to extract the leaves, stems, above-ground parts, rhizomes, roots, underground parts, or mixtures thereof of Spiraea. Preferably, it may be an extract obtained by using water, methanol, ethanol, ethyl acetate, acetone, nucleic acids, dichloromethane, or a mixture thereof to extract the extract.

[0010] In the present invention, the meadowsweet extract is preferably a 50-70% ethanol extract.

[0011] In the present invention, conventional methods in the industry such as filtration, hot water extraction, immersion extraction, room temperature extraction, accelerated solvent extraction, reflux condensation extraction, and ultrasonic extraction can be used as methods for extracting the Spiraea extract.

[0012] In the present invention, the Filipendula palmata extract (FPE) may contain a flavonoid compound. The flavonoid compound may include one or more selected from the group consisting of (+)-catechin, gambiriin C, miquelianin, scutellarin, and quercitrin. Preferably, the flavonoid compound contained in the Filipendula palmata extract of the present invention may include miquelianin and quercitrin.

[0013] According to one embodiment of the present invention, the meadowsweet extract exhibits activity to suppress MAPK and NF-κB and is effective in anti-inflammatory effects, and the meadowsweet extract exhibits activity to increase HA production through downregulation of HYAL (HYAL1 and HYAL2) and upregulation of HAS (HAS1, HAS2 and HAS3), and is effective in enhancing skin hydration.

[0014] In this invention, the term "active ingredient" means an ingredient that exhibits the desired activity on its own, or an ingredient that can exhibit activity in combination with a carrier that is inactive on its own.

[0015] In another embodiment of the present invention, the composition may be a pharmaceutical composition.

[0016] The characteristics of the aforementioned meadowsweet extract contained in the pharmaceutical composition can be substituted in the previously mentioned section, and therefore, their description is omitted.

[0017] When the composition according to the present invention is in the form of a pharmaceutical composition, it may contain a pharmaceutically effective amount of meadowsweet extract alone, or one or more pharmaceutically acceptable carriers. In this case, the pharmaceutically acceptable carriers are those commonly used in formulation and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above components, it may further contain lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, and the like.

[0018] The pharmaceutical composition of the present invention can be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) according to the intended method, and the dosage will vary depending on the patient's condition and weight, the severity of the disease, the drug form, the route of administration, and the time, but can be appropriately selected by those skilled in the art.

[0019] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the level of the effective dose can be determined by factors including the type and severity of the patient's disease, the activity and sensitivity of the drug, the time of administration, the route of administration and elimination rate, the duration of treatment, drugs used concurrently, and other factors well known in the medical field. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent, in combination with other therapeutic agents, sequentially or simultaneously with conventional therapeutic agents, and in single or multiple doses. It is important to administer a dose that takes all of the above factors into consideration to obtain the maximum effect with the minimum amount without side effects, which can be easily determined by those skilled in the art.

[0020] Specifically, the effective amount of the pharmaceutical composition of the present invention can vary depending on the patient's age, gender, condition, weight, absorption rate of the active ingredient into the body, inactivation rate and excretion rate, type of disease, and the drugs used in combination. Generally, 1 to 500 mg per kg of body weight may be administered daily or every other day, or it may be administered in 1 to 3 divided doses per day. However, since it can be increased or decreased depending on the administration route, gender, weight, age, etc., the above dosage does not limit the scope of the present invention by any method.

[0021] In still another embodiment of the present invention, the composition may be a health functional food composition.

[0022] The characteristics corresponding to the above-mentioned Siegesbeckia extract contained in the health functional food composition are substitutable in the above-mentioned part, so the description thereof is omitted.

[0023] When the composition according to the present invention is in the form of a health functional food composition, in addition to specific health foods and nutritional supplements, it can be manufactured as a food with high medical and therapeutic effects processed so that the biological regulatory function is efficiently exerted. The above food may, in some cases, be mixed with functional foods, health foods, and health supplements, and can be manufactured in various forms such as tablets, capsules, powders, granules, liquids, pills, etc. to obtain useful effects.

[0024] The health functional food of the present invention may contain additional components that are commonly used in food compositions and can improve odor, taste, vision, etc. For example, it may contain vitamin A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, pantothenic acid, etc. It may also contain minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu), etc. It may also contain amino acids such as lysine, tryptophan, cysteine, valine, etc. In addition, food additives such as preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dihydroacetate, etc.), bactericides (sun-dried powder and highly sun-dried powder, sodium hypochlorite, etc.), antioxidants (butylhydroxyanisole (BHA), butylhydroxytoluene (BHT), etc.), colorants (tar dyes, etc.), color developers (sodium nitrite, sodium acetate, etc.), bleaching agents (sodium sulfite), seasonings (MSG sodium glutamate, etc.), sweeteners (dulcin, cyclamate, saccharin, sodium, etc.), fragrances (vanillin, lactones, etc.), swelling agents (alum, potassium hydrogen D-tartrate, etc.), fortifiers, emulsifiers, thickeners (paste), coating agents, gum bases, defoaming agents, solvents, improvers, etc. can be added. The above additives can be selected according to the type of food and used in appropriate amounts.

[0025] When the health functional food of the present invention is used as a food additive, it can be added as it is or used together with other foods or food components, and can be appropriately used by ordinary methods.

[0026] In the health functional food of the present invention, the content of the extract of Cirsium japonicum DC. is not particularly limited and can be variously changed according to the state of the administration subject, the type of specific disease condition, the degree of progression, etc. If necessary, it may be included as the total content of the food.

[0027] In still other embodiments of the present invention, the composition can be a cosmetic composition.

[0028] The characteristics of the aforementioned cosmetic composition that correspond to the aforementioned Spiraea extract can be substituted in the previously mentioned section, and therefore their description is omitted.

[0029] If the composition according to the present invention is a cosmetic composition, it can be used externally on the skin or taken orally.

[0030] The cosmetic compositions of the present invention contain meadowsweet extract as an active ingredient and can be manufactured together with dermatologically acceptable excipients in the form of basic cosmetic compositions (lotions, creams, essences, facial cleansers such as cleansing foams and cleansing waters, packs, body oils), color cosmetic compositions (foundations, lipsticks, mascaras, makeup bases), hair product compositions (shampoos, rinses, hair conditioners, hair gels), and soaps.

[0031] The excipients are not limited to those mentioned above, but may include, for example, emollients, skin penetration enhancers, colorants, fragrances, emulsifiers, thickeners, and solvents. They may also further contain fragrances, dyes, bactericides, antioxidants, preservatives, and humectants, and may include thickeners, inorganic salts, synthetic polymers, etc., for the purpose of improving physical properties. For example, when manufacturing a facial cleanser and soap using the cosmetic composition of the present invention, they can be easily manufactured by adding the meadowsweet extract to a normal facial cleanser and soap base. When manufacturing a cream, it can be manufactured by adding the meadowsweet extract or a salt thereof to a general oil-in-water (O / W) cream base. In addition, fragrances, chelating agents, dyes, antioxidants, preservatives, etc., and synthetic or natural materials such as proteins, minerals, and vitamins for the purpose of improving physical properties can be further added.

[0032] When the dosage form of the cosmetic composition of the present invention is a paste, cream, or gel, animal oils, vegetable oils, waxes, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc, or zinc oxide can be used as the carrier component.

[0033] When the dosage form of the cosmetic composition of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder can be used as the carrier component, and especially when it is a spray, it may further contain a propellant such as hydrochlorofluorocarbon, propane / butane, or dimethyl ether.

[0034] When the dosage form of the cosmetic composition of the present invention is a solution or emulsion, a solvent, solubilizer, or emulsifier can be used as a carrier component. For example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol, sorbitan fatty acid ester, and the like can be used.

[0035] When the dosage form of the cosmetic composition of the present invention is a suspension, the carrier component can be a liquid diluent such as water, ethanol, or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, or polyoxyethylene sorbitan ester, or microcrystalline cellulose, aluminum methhydroxyl, bentonite, or aga.

[0036] When the dosage form of the cosmetic composition of the present invention is a surfactant-containing cleanser, the carrier component can be an aliphatic alcohol sulfate, an aliphatic alcohol ether sulfate, a sulfosuccinate monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, fatty acid amide ether sulfate, alkylamide betaine, aliphatic alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative, or ethoxylated glycerol fatty acid ester. [Effects of the Invention]

[0037] The present invention provides a composition for skin anti-inflammatory and moisturizing effects containing a meadowsweet extract as an active ingredient, which exhibits activity to suppress MAPK and NF-κB signaling, making it effective for anti-inflammatory effects. It also exhibits activity to increase HA production through the downregulation of HYAL and upregulation of HAS, making it effective for enhancing skin moisturizing effects. [Brief explanation of the drawing]

[0038] [Figure 1] Figure 1 shows the effect of FPE on the viability of normal HaCaT keratinocytes. Viability was evaluated using (A) MTT and (B) live / dead cell staining. Live and dead cells were stained green and red, respectively. "ns" indicates no significant difference between all groups. [Figure 2] Figure 2 shows the effect of FPE on mitochondrial superoxide (Mito-SOX) production and intracellular reactive oxygen species (ROS) production in HaCaT keratinocytes induced by TNF-α + IFN-γ (10 ng / mL each) (T+I). Different superscripts indicate significant differences between groups analyzed using Duncan's multiple range test (p<0.05). DEX represents dexamethasone (20 μg / mL). [Figure 3]Figure 3 shows the effects of FPE on (A) gene expression and (B) protein secretion of inflammatory chemokines in HaCaT keratinocytes induced by TNF-α + IFN-γ (10 ng / mL each) (T+I). Results were obtained using qRT-PCR for mRNA expression and ELISA for protein secretion. Different superscripts indicate significant differences between groups analyzed using Duncan's multiple range test (p<0.05). DEX is dexamethasone (20 μg / mL). [Figure 4] Figure 4 shows the effects of FPE on (A) MAPK and (B) NF-κB signaling pathways in HaCaT keratinocytes induced with TNF-α + IFN-γ (10 ng / mL each) (T+I). Results are shown as Western blot images of each protein and quantified fold changes of phosphorylated morphology / whole morphology. Different superscripts indicate significant differences between groups analyzed using Duncan's multiple range test (p<0.05). DEX is dexamethasone (20 μg / mL). [Figure 5] Figure 5 shows the effects of FPE on (A) hyaluronic acid (HA) secretion and (B) HYAL and HAS gene expression in normal HaCaT keratinocytes. Results were obtained using ELISA for HA secretion and qRT-PCR for HA-related mRNA expression. Different superscripts indicate significant differences between groups analyzed using Duncan's multiple range test (p<0.05). NAG is N-acetyl-D-glucosamine (5 mg / mL). [Figure 6]Figure 6 shows the effects of (A) HAS2 protein expression and (B) FPE on the PI3K / Akt / NF-κB signaling pathway in normal HaCaT keratinocytes. Results are shown as Western blot images of each protein and quantified magnifications of HAS2 / β-actin or phosphorylated form / whole form. Different superscripts indicate significant differences between groups analyzed using Duncan's multiple range test (p<0.05). [Figure 7] Figure 7 shows (A) the photodiode array chromatogram (PDA) and (B) the base peak chromatogram (BPC) results of FPE obtained by UPLC-MS analysis. The estimated identification of five major phytochemicals in FPE is listed in the table below. FPE (10 mg / mL) was introduced into the UPLC-MS system, and the separation and detection conditions are described in Table 3. [Figure 8] Figure 8 shows the MS and (B) MS2 spectra of the five major peaks identified in the (A) base peak chromatogram (BPC, Figure 1B). Each peak in the spectrum was identified using an online natural products database and an in-house MS2 spectral library. [Figure 9] Figure 9 shows the effects of major flavonoids on the viability and secretion of TARC and hyaluronic acid. (A) Viability and (B) TARC secretion were evaluated in HaCaT cells induced with TNF-α + IFN-γ (10 ng / mL each) (T+I), and (C) viability and hyaluronic acid secretion were evaluated in normal HaCaT cells. Results were obtained using MTT (A and C) and ELISA (B and D) methods. Different superscripts indicate significant differences between groups analyzed using Duncan's multiple range test (p<0.05). "ns" indicates no significant difference between any group. [Figure 10] Figure 10 shows the analysis of the content of active substances in FPE. [Modes for carrying out the invention]

[0039] The present invention will be described in detail below with reference to examples to aid in understanding the present invention. However, the following examples are merely illustrative of the content of the present invention, and the scope of the present invention is not limited to these examples. The examples of the present invention are provided to give a more complete explanation of the present invention to a person of average knowledge in the art.

[0040] Example 1. Materials and Methods 1.1. Materials and Methods Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS; F0900-050), West-Q Pico ECL solution (W3652), and AmfiSure qGreen Q-PCR master mix (Q5600-010) were purchased from GenDEPOT (Katy, TX, USA). Antibiotic-Antimycotic solution (100X) (P / S; CA002-010), TRIzol reagent (BIO-38033), and Superscript were also used. TMThe First-Strand cDNA Synthesis Kit (R5600-100) was purchased from Invitrogen Corp. (Carlsbad, CA, USA). Water-soluble 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT; M6494), dimethyl sulfoxide (DMSO; D2650-100ML), dexamethasone (D4902), N-acetyl-D-glucosamine (NAG; A3286), and Grease's Reagent (G4410) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The (+)-catechin (CAS No. 154-23-4), scutellarein 7-O-β-D-glucuronide (scutellarin; CAS No. 27740-01-8), quercetin 3-O-β-D-glucuronide (miquelianin; CAS No. 22688-79-5), and quercetin 3-O-α-L-glucuronide (quercitrin; CAS No. 522-12-3) used as standard materials were provided by the Natural Product Institute of Science and Technology, Anseong, South Korea.

[0041] The live and dead cell viability / cytotoxicity assay kit (L3224) was purchased from Life Technologies Co. (Eugene, OR, USA). The ROS / superoxide detection analysis kit (ab139476) was purchased from Abcam (Cambridge, UK). Recombinant human protein equivalent to tumor necrosis factor-alpha (TNF-α; 210-TA-005) and interferon-gamma (IFN-γ; 285-IF-100) were purchased from R&D Systems (Minneapolis, MN, USA). Enzyme-linked immunosorbent assay (ELISA) kits for human interleukin-8 (IL-8 / CXCL8; 555244), thymic and activating regulatory chemokines (TARC / CCL17; DY364), and hyaluronan (HA; DHYAL0) were supplied by BD Biosciences (Minneapolis, MN, USA) or R&D Systems (Minneapolis, MN, USA).

[0042] RIPA lysis buffer (89900) and bicinchoninic acid (BCA; 23225) protein analysis kit were purchased from Thermo Fisher Scientific (Waltham, MA, USA). All primers for quantitative real-time PCR (qRT-PCR) were designed and supplied by Macrogen (Seoul, South Korea), and their sequences are listed in Table 1. Primary antibodies for Western blotting were supplied by Cell Signaling Technology, Inc. (Danvers, MA, USA), and their Cat. Nos are listed in Table 2.

[0043] [Table 1]

[0044] [Table 2]

[0045] 1.2. Production of Filipendula palmata extract (FPE) Whole wild F. palmata plants were obtained from northern Gyeonggi Province, adjacent to the Demilitarized Zone in South Korea. The collected whole tissue (100g) was dried in sunlight to obtain a dried product (43.5g). All dried tissue was extracted with 70% ethanol (5L) at 20-25°C for 3 days. After filtering with a filter cloth (20μm, Hyundai Micro, Anseong, South Korea), the extract was concentrated in a vacuum concentrator (Buchi Korea Inc., Gwangmyeong, South Korea) and dried using a freeze-dryer (Ilshin Biobase, Daejeon, South Korea) to obtain a 70% ethanol extract (FPE) powder of F. palmata (extraction yield, 11.5 w / w%) relative to the raw material before drying.

[0046] 1.3. Cell culture method for evaluating moisturizing ability HaCaT cells were purchased from CLS Cell Line Service (Eppelheim, Heidelberg, Germany), grown in DMEM containing 10% FBS and 1% P / S, and regulated with 5% carbon dioxide / 95% air. Cells were cultured in 6-well plates (for qRT-PCR and ELISA) and 5 × 10⁶ 5 2 x 10 cells per 60mm dish (for Western blotting) 5Cells were plated at a density of cells / well. After stabilizing the cells for 24 hours, the culture medium was replaced with new DMEM containing 1% P / S and various concentrations of FPE. Instead of FPE treatment, distilled water and N-acetyl-D-glucosamine (5 mg / mL) were applied to the cells as negative and positive controls, respectively. After 24 hours of incubation, hyaluronic acid (HA) secretion, nuclear mRNA expression, and intracellular signaling molecule expression in the culture supernatant were determined using ELISA (Example 1.5), qRT-PCR (Example 1.6), and Western blotting (Example 1.7), respectively.

[0047] 1.4. Cell culture methods for anti-inflammatory evaluation Anti-inflammatory activity was evaluated using HACAT cells induced with TNF-α and IFN-γ (T+I). Briefly, HACAT cells were cultured in 96-well plates (MTT method) and 6-well plates (QRT-PCR and ELISA) at a rate of 2 × 10⁶ 5 cells / well and 5×10 5 2 x 10 cells in a 60mm dish (Western blotting) 4 Cells were plated at a density of cells / well. After stabilizing the cells for 24 hours, the culture medium was replaced with fresh DMEM containing 1% P / S and various concentrations of FPE. Instead of FPE treatment, distilled water and dexamethasone (20 μg / mL) were applied to the cells using negative and positive control groups, respectively.

[0048] After 1 hour of incubation, a recombinant protein mixture containing 10 ng / mL TNF-α and 10 ng / mL IFN-γ (T+I) was added, and the cells were incubated for an additional 24 hours. After two washes with PBS, cell viability was assessed by the MTT method at 570 nm using a microplate reader (Molecular Devices Filter Max F5; San Francisco, USA) according to conventional methods. Furthermore, after 24 hours of T+I treatment, extracellular chemokine secretion, nuclear mRNA expression, and intracellular signaling molecule expression levels were evaluated in the culture supernatant using ELISA (Example 1.5), qRT-PCR (Example 1.6), and Western blotting (Example 1.7).

[0049] 1.5.ELISA (enzyme-linked immunosorbent assay) HACAT cells treated with FPE were cultured for 24 hours, with or without T+I (Examples 1.3 and 1.4), and the culture supernatant was collected. Extracellular chemokine and HA levels were determined using the respective ELISA kits according to the manufacturer's instructions.

[0050] 1.6. qRT-PCR HACAT cells treated with FPE were cultured with T+I for 24 hours or 24 hours (Examples 1.3 and 1.4), and then washed twice with PBS. Total RNA was extracted with Trizole reagent and normalized using nanodrop plate and microplate spectrophotometer (Epoch, Biotek Instruments, Winooski, VT, USA). The same amount of total RNA was then extracted using SuperScript. TMThe first-strand cDNA was reverse transcribed using a first-strand cDNA synthesis kit, and qRT-PCR was performed using AmfiSure qGreen Q-PCR Master Mix and a qRT-PCR detection system (CFX96; Bio-Rad Laboratories, Hercules, CA, USA) along with SYBR Premix Ex TaqTM II (TaKaRa Bio Inc., Kusatsu, Japan). Gene expression levels were normalized to the expression level of the endogenous control gene (glyceraldehyde-3-phosphate dehydrogenase; GAPDH) and calculated using the 2-△△CT method.

[0051] 1.7. Western Blotting HaCaT cells treated with FPE were cultured for 24 hours, regardless of T+I presence (Examples 1.3 and 1.4), and then washed twice with PBS. Total protein was extracted with RIPA lysis buffer and standardized using a BCA protein analysis kit. The same amount (50 μg) of total protein was separated on a 10-12% sodium dodecyl sulfate-polyacrylamide gel, and the separated protein was transferred from the gel to a polyvinylidene fluoride membrane (Thermo Fisher Scientific). The membrane was blocked at 20-25°C for 2 hours with PBS containing 5% skim milk and 0.05% Twin, and then washed three times with PBS containing only 0.05% Twin 20 (PBST). The membrane was then cultured overnight with the primary antibody at 4°C. After washing three times with PBST, the membrane was cultured at 20-25°C for 1 hour with a secondary antibody conjugated to horseradish peroxidase (HRP). Subsequently, the membrane was washed five times with PBST, and the target protein blot was visualized using ECL solution. The expression levels of each target protein were quantified using ImageJ software, which is available from the online website (https: / / imagej.nih.gov / ij / ).

[0052] 1.8. Dyeing method HaCaT cells were cultured in 2 × 10⁶ wells on a 6-well culture plate. 5Cells were smeared at a density of cells / well and stabilized for 24 hours. The culture medium was replaced with fresh DMEM containing 1% P / S and various concentrations of FPE. After 1 hour of incubation, T+I was added and the cells were incubated for an additional 24 hours. After two washes with PBS, two staining methods, live / dead cell staining and ROS / superoxide staining, were used to evaluate cytotoxic effects and the generation of intracellular ROS and mitochondrial superoxide (Mito-SOX). All staining methods were used according to the manufacturer's guidelines. Stained cells were visualized using a fluorescence scanning microscope (Leica, Wetzlar, Germany) and quantified using ImageJ software.

[0053] 1.9. Confirmation of the main active ingredients of FPE The major secondary metabolites present in FPE were analyzed by chromatography and spectroscopy using a Thermo Electron (Waltham, MA, USA) ultra-high performance liquid chromatography-tandem mass spectrometry system (UPLC-MS / MS; LTQ Orbitrap XL) following conventional analytical methods and conditions (Kim et al., 2021). Detailed results are shown in Table 3.

[0054] [Table 3]

[0055] 1.10.Statistical analysis All experiments were performed in triple replication, and results are expressed as mean ± standard deviation. Statistical analysis was performed using IBM SPSS Statistics (Ver. 26; IBM Co., Armonk, NY, USA). Statistical differences were assessed using one-way analysis of variance (ANOVA), and post-hoc analysis was performed using Duncan's multiple comparison test. A p-value less than 0.05 was considered statistically significant.

[0056] Example 2. Experimental Results 2.1. Cytotoxicity and ROS-inhibiting effects of FPE The cytotoxic effects of FPE on HaCaT cells were evaluated using MTT and live / dead cell staining. As shown in Figure 1A, the MTT results showed no significant toxic effects in cells treated with FPE at concentrations of 10–200 μg / mL.

[0057] To further confirm the cytotoxicity of FPE treatment, a live-cell / dead-cell staining assay was performed. Representative images are shown in Figure 1B, where green and red fluorescence images indicate live and dead cells, respectively. The live-cell / dead-cell staining results are consistent with those observed using the MTT method.

[0058] Next, to investigate whether FPE prevents T+I-induced oxidative stress in HaCaT cells, ROS / superoxide analysis was performed using fluorescence microscopy (Figure 2). The results showed two specific fluorescent probes for detecting Mito-SOX (red) and intracellular ROS (green). Levels of Mito-SOX and ROS were significantly increased by T+I treatment compared to the levels of the negative control (NC) group treated with culture medium alone.

[0059] This phenomenon was significantly improved by pretreatment with dexamethasone, which was used as a positive control group (PC). Furthermore, FPE treatment effectively suppressed Mito-SOX and ROS formation in a concentration-dependent manner. In particular, treatment at the highest FPE concentration (100 μg / mL) was shown to reduce both Mito-SOX and ROS formation compared to the PC group.

[0060] 2.2. Suppressive effect of FPE on TNF-α / IFN-γ-induced inflammatory chemokines The effect of FPE on the production of major pro-inflammatory chemokines such as RANTES, TARC, CTACK, and IL-8 was evaluated in T+I-induced HaCaT cells. As shown in Figure 3A, qRT-PCR results showed that T+I stimulation significantly increased the mRNA expression of the above pro-inflammatory chemokines compared to the NC group.

[0061] In contrast, the dexamethasone-treated PC group showed a significantly reduced expression of these inflammatory chemokines compared to the T+I-induced group. FPE pretreatment significantly reduced the expression of all chemokines measured. In particular, treatment with the highest FPE concentration (100 μg / mL) showed a reduction in the expression of all chemokines compared to the PC group. Next, ELISA was introduced to measure the secretion levels of IL-8 and TARC in T+I-stimulated HaCaT cells (Figure 3B).

[0062] Similar to the qRT-PCR results, IL-8 and TARC were excessively secreted from cells after T+I stimulation, but their secretion was significantly suppressed in a concentration-dependent manner by FPE pretreatment. Overall, the qRT-PCR and ELISA results indicate that FPE treatment effectively modulates T+I-induced production of inflammatory chemokines at both the genetic and protein levels.

[0063] 2.3. FPE-mediated inhibitory effect of MAPK and NF-κB signaling in T+I-induced inflammatory response To elucidate the molecular mechanisms associated with the anti-inflammatory effects of FPE, we evaluated the effects of T+I treatment and FPE pretreatment on the MAPK (mitogen-activated protein kinase) and NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) signaling pathways, which are important for regulating pro-inflammatory mediators.

[0064] Figure 4 shows immunoblotting images and quantified results using image processing software. After T+I treatment, the phosphorylation levels of three MAPKs, including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 kinase (p38), were significantly increased without an increase in the total level (Figure 4A).

[0065] Furthermore, in cells stimulated with T+I, the expression levels of complete NF-κB p65 (p65) and phosphorylated NF-κB p65 (p-p65) showed similar trends to those observed with the three MAPKs mentioned above (Figure 4B).

[0066] T+I stimulation not only significantly increased the phosphorylation level of IκBα but also conversely decreased the overall level. Such significant changes in three MAPKs and two NF-κB molecules induced by T+I stimulation were conversely modulated in a concentration-dependent manner by FPE pretreatment.

[0067] 2.4. Effects of FPE on HA production and related gene expression The skin moisturizing effect of FPE was investigated using normal HaCaT cells. As shown in Figure 5A, HA secretion levels were determined in the supernatant of HaCaT cells treated with FPE using ELISA.

[0068] To compare the efficacy of FPE, cells were treated with NAG (5 mg / mL) and used as the PC group. The results showed that FPE treatment significantly increased HA secretion in a concentration-dependent manner. In particular, FPE treatment at a concentration of 100 μg / mL induced HA secretion equivalent to 4.1 times that of the NC group treated with only the culture medium.

[0069] Next, to confirm the mechanism of HA production, we investigated the expression of HA-related genes using qRT-PCR. After FPE treatment, the mRNA expression of two types of hyaluronidase (HYAL; HYAL1 and HYAL2) was significantly downregulated (Figure 5B), while the mRNA expression of three hyaluronic acid synthases (HAS; HAS1, HAS2, and HAS3) was extensively upregulated in HaCaT cells (Figure 5C). These results suggest that FPE can promote HA production by upregulating HA synthase (HAS) and downregulating HA degrading enzyme (HYAL).

[0070] 2.5. FPE-mediated HAS2 synthesis and PI3K / Akt signaling stimulation in normal keratinocytes Based on the above results that FPE treatment promotes HA secretion, we additionally investigated whether FPE treatment activates the synthesis of HAS2 protein, which is the major membrane-bound HA synthase among HASs. As shown in Fig. 6A, HAS2 protein expression increased in a concentration-dependent manner upon FPE treatment.

[0071] Furthermore, we investigated the intracellular signaling pathway underlying FPE-mediated HA production using immunoblotting. Fig. 6B provides the blotting images and quantification results of phosphoinositide 3-kinase (PI3K) / protein kinase B (PKB; Akt) and NF-κB signaling molecules. Such results indicated that FPE treatment significantly and concentration-dependently improved the phosphorylated levels of PI3K, Akt, IκBα, and p65 proteins without changing their corresponding total proteins.

[0072] Collectively, the above results suggest that FPE-mediated HA production is associated with the activation of PI3K / Akt / NF-κB signaling and HAS2 synthesis.

[0073] 2.6. Elucidation of the anti-inflammatory effect of FPE and the active ingredients on hyaluronic acid production To determine the major active compounds that play a role in the inflammatory activity of FPE, UPLC-MS / MS analysis was performed. Figs. 7A and 7B provide the photodiode array chromatogram (PDA) and base peak chromatogram (BPC) of FPE, respectively. In the BPC, mass spectrometry (MS) and tandem MS (MS [[ID=IS]] 2 ) were additionally used to measure the mass-to-charge ratio (m / z), and the internal spectral library and web-based database were investigated with a mass accuracy of less than ±5 ppm to confirm the major peaks.

[0074] Positive ionization mode ([M+H] +MS analysis under the following conditions confirmed that five peaks in BPC at retention times of 3.96, 4.63, 6.64, 7.38, and 7.50 minutes corresponded to the precursor ion at 291.0849, 563.1520, and 479.0792 m / z, respectively (Figure 8A). Such precursor ions were identified by MS 2 Further analysis was performed using [method / tool ​​name], and the generated ions (daughter ions) were produced, as shown in Figure 8B.

[0075] As a result, MS and MS 2 The results suggest that FPE contains five major flavonoids, including (+)-catechin, gambiriin C (epiafzelechin-(4β->8)-catechin), miquelianin (quercetin 3-O-β-D-glucuronide), scutellarin (scutellarein 7-O-β-D-glucuronide), and quercitrin (quercetin 3-O-α-L-rhamnoside). Except for gambirin C, the putative identification of (+)-catechin, mikelianine, scutellarialine, and quercitrin is ultimately determined by MS and MS between FPE and the above reference substances. 2 This was confirmed by comparing the ion spectra obtained using [the specified method].

[0076] Furthermore, the effects of major compounds and FPE on TARC suppression and HA production were evaluated in T+I-induced and normal HaCaT cells, respectively, to investigate their contribution to the bioactivity of HaCaT cells. As shown in Figures 9A and 9B, the MTT results showed that the two major compounds (miceranine and quercitrin) and FPE did not exert significant toxic effects at the same concentrations (40-100 μg / mL) in both T+I-stimulated and normal HaCaT cells. The highest inhibitory activity against T+I-induced TARC production was observed in cells treated with FPE, followed by miceranine and quercitrin (Figure 9C). Conversely, HA secretion from normal HaCaT cells reached its maximum value with quercitrin treatment, followed by miceranine and FPE (Figure 9D).

[0077] 2.7. Analysis of the content of active substances in FPE The content of the active substances micellinin and quercitrin present in FPE was analyzed. For content analysis, an Agilent 1260 Infinity II Quat Pump (CA, USA) and an Agilent Variable Wavelength (VW) UV Detector (CA, USA) were used with a YMC Pack Pro C18 column (4.6 × 250 mm, 5 μm) as the stationary phase. Each column was processed at a temperature of 40°C with a mobile phase (A: 0.1% formic acid / water, B: acetonitrile) injected at a volume of 5 μL and a flow rate of 0.9 mL / min. Detection was performed using an Agilent Variable Wavelength (VW) UV Detector (UV 270 nm). The mobile phase was eluted under the conditions shown in Table 4 below, and the content was calculated using the following formula.

[0078] [Table 4] Content of standard product (mg / g) = C × V × D × P / W (C: Concentration of the standard in the test solution (mg / mL); V: Total volume of the test solution (mL); D: Dilution ratio; P: Purity of the standard; W: Sample size (g))

[0079] As a result, as shown in Figure 10, it was confirmed that the active substances present in FPE, micellianine and quercitrin, were present in amounts of 4.72 ± 0.01 (mg / g) and 13.66 ± 0.01 (mg / g), respectively.

[0080] Having described in detail certain aspects of the present invention, it will be clear to those with ordinary skill in the art that such specific techniques are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A composition for skin anti-inflammatory and moisturizing effects, containing meadowsweet extract as an active ingredient.

2. The composition for promoting anti-inflammatory and moisturizing effects on the skin according to claim 1, wherein the extract is obtained by extraction using water, methanol, ethanol, ethyl acetate, acetone, nucleic acid, dichloromethane, or a mixture thereof.

3. The composition for skin anti-inflammatory and moisturizing according to claim 1, wherein the extract is a 50-70% ethanol extract of Spiraea japonica.

4. The composition for anti-inflammatory and moisturizing the skin according to claim 1, wherein the extract comprises a flavonoid compound.

5. The composition for skin anti-inflammatory and moisturizing according to claim 4, wherein the flavonoid compound comprises one or more selected from the group consisting of (+)-catechin, gambirin C, mikelianin, scutellarin, and quercitrin.

6. The aforementioned Spiraea extract has activity to suppress MAPK and NF-κB, wherein the composition for skin anti-inflammatory and moisturizing according to claim 1.

7. The meadowsweet extract increases HA production through downward regulation of HYAL and upward regulation of HAS, as described in claim 1, for the anti-inflammatory and moisturizing composition for the skin.

8. The composition is a pharmaceutical composition, the composition for anti-inflammatory and moisturizing the skin according to any one of claims 1 to 7.

9. The composition is a functional food composition for the skin, as described in any one of claims 1 to 7.

10. The composition is a cosmetic composition, as described in any one of claims 1 to 7, for anti-inflammatory and moisturizing effects on the skin.

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