582 JOURNAL OF COSMETIC SCIENCE reduced scratching in AD-like skin lesions in the NC/Nga mouse model (31), suggesting that S chinensis may have anti-itching properties. The main objective of this study was to evaluate the efficacy of A houstonianum, B falcatum, and S chinensis as ingredients in skincare products for itchy skin. We observed that combining the three was more beneficial than when the extracts were used individually in modulating the expression of interleukin (IL)–4-induced keratinocyte-derived itching factors, including IL31, thymic stromal lymphopoietin (TSLP), proopiomelanocortin (POMC), and the skin barrier component FLG. MATERIALS AND METHODS REAGENTS IL4, IL13, and type II interferon-γ were procured from ProSpec-Tany Technogene Ltd. (Ness-Ziona, Israel). Anti-IL31 antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA) antiglyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies from Santa Cruz Biotechnology (Dallas, TX, USA) anti-FLG antibodies from BioLegend (San Diego, CA, USA) and anti-TSLP antibodies from Novus Biologicals (Centennial, CO, USA). Anti-POMC antibodies and Alexa Fluor 488- and 555-conjugated secondary antibodies were purchased from Invitrogen (Carlsbad, CA, USA). The firefly luciferase assay system was obtained from Promega (Madison, WI, USA), and Pierce bicinchoninic acid Protein Assay Reagent was obtained from Thermo Scientific (Rockford, IL, USA). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA). PREPARATION OF PLANT EXTRACTS A houstonianum was purchased from the Yangjae Flower Market (Seoul, Republic of Korea). Air-dried leaves and stems of A houstonianum were extracted with ethanol as described previously (19). B falcatum was obtained from the Seoyeon Herbal Medicine Farming Association (Nonsan, Chungcheongnamdo, Republic of Korea) and B falcatum was prepared as described previously (25). S chinensis berries were collected from Mungyeong Miso farm (Gyeongbuk, Republic of Korea), and S chinensis was prepared using dried berries as described previously (32). All ethanolic extracts were freeze-dried and dissolved in dimethyl sulfoxide at a concentration of 20 mg/mL. ANALYTICAL HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY The authentic precocene II, saikosaponin A, gomisin A, gomisin N, and angeloylgomisin H were purchased from Sigma-Aldrich (MilliporeSigma Co, St. Louis, MO, USA). The compounds were identified using analytical high-performance liquid chromatography (HPLC), which was performed as the manufacturer’s procedure. HPLC experiments were carried out on an Agilent 1260 Infinity system (Agilent Technologies., Santa Clara, CA, USA) using Symmetry C18 4.6 × 250 mm (5 μm particle size) column (Waters Corp., Milford, MA, USA). The injection volume and the flow rate were 10 μl and 1.0 mL/ min, respectively. The authentic compounds and sample signals were detected at 230 nm

583 SUPPRESSION OF ITCHING BY THREE HERBAL ETHANOLIC EXTRACTS for A houstonianum, 203 nm for B falcatum, or 220 nm for S chinensis. The mobile phase was acetonitrile/methanol/water (30:40:30) for A houstonianum, 35% acetonitrile aqueous solution for B falcatum, and 65% acetonitrile aqueous solution for S chinensis. The column temperature was set at 35°C. CELL CULTURE The human keratinocyte cell line HaCaT was obtained from the Cell Lines Service (Eppelheim, Germany). HaCaT cells were maintained in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum (HyClone, Logan, UT, USA). CYTOTOXICITY ASSAY HaCaT cells were seeded into 96-well plates at a density of 5 × 103 cells/well. After incubating for 24 h, cells were treated with vehicle (dimethyl sulfoxide) or various concentrations (10, 20, 40, and 80 μg/mL) of A houstonianum, B falcatum, and S chinensis for 24 h or a combination of the three (40 μg/mL A houstonianum, 20 μg/mL, and 40 μg/ mL S chinensis) for various time periods (0–48 h). Cytotoxicity was analyzed using a water- soluble formazan colorimetric assay-based Cell Counting Kit-8 (CCK-8 Dojindo Molecular Technologies, Gaithersburg, MD, USA) according to the manufacturer’s instructions. The absorbance at 450 nm was measured on an EMax Endpoint enzyme-linked immunosorbent assay (ELISA) Microplate Reader (Molecular Devices, Sunnyvale, CA, USA). REVERSE TRANSCRIPTION-POLYMERASE CHAIN REACTION Isolation of total RNA and synthesis of cDNA was performed as previously described (33). The reverse transcription (RT-PCR) primers and thermal cycling conditions were as follows: FLG forward, 5′-CAA ATC CTG AAG AAT CCA GAT GAC-3′, FLG reverse, 5′-TGC TTG AGC CAA CTT GAA TAC C-3′ (5 min at 94°C, followed by 30 s at 94°C, 35 s at 62°C, and 1 min at 72°C for 30 cycles) IL31 forward, 5′-GTC TTG GTA TTT ATG GAA TGC-3′, IL31 reverse, 5′- CCA GGG AGC ATT GAC AAC TCT TAG-3′ (5 min at 94°C, followed by 30 s at 94°C, 35 s at 55°C, and 1 min at 72°C for 30 cycles) POMC forward, 5′-CCT GCC TGG AAG ATG CCG AGA T-3′, POMC reverse, 5′-TGC TGC CGC TGC TGC TGC TGT-3′ (5 min at 94°C, followed by 30 s at 94°C, 35 s at 62°C, and 1 min at 72°C for 30 cycles) GAPDH forward, 5′-CCA AGG AGT AAG AAA CCC TGG AC-3′, GAPDH reverse, 5′-GGG CCG AGT TGG GAT AGG G-3′ (5 min at 94°C, followed by 30 s at 94°C, 30 s at 58°C, and 1 min at 72°C for 30 cycles) TSLP forward, 5’-TAG CAA TCG GCC ACA TTG CCT-3’, TSLP reverse, 5’-GAA GCG ACG CCA CAA TCC TTG -3′ (5 min at 94 followed by 30 s at 94°C, 35 s at 58°C, and 1 min at 72°C for 30 cycles). The amplified products were electrophoresed on a 2% agarose gel and visualized upon staining with ethidium bromide under ultraviolet light. QUANTITATIVE REAL-TIME PCR Validated commercial quantitative real-time PCR (qPCR) primers and SYBR Green-based fluorescent probes specific for FLG (id: qHsaCEP0039328), IL31 (id: qHsaCEP0055722),