581 SUPPRESSION OF ITCHING BY THREE HERBAL ETHANOLIC EXTRACTS dermis–epidermal junction (1). Many factors are associated with itching, including dry skin, eczema, insect bites, and hives (2). Itch is classified as either histaminergic acute or nonhistaminergic chronic (3). The former is temporary and can be quickly relieved by scratching the affected area or applying antihistamine agents however, the latter, which is defined as a symptom that persists for longer than 3 mo., is usually induced by a nonhistaminergic pathway (4). Chronic itch is the most frequently observed symptom of inflammatory skin disorders, such as atopic dermatitis (AD), allergic contact dermatitis, and psoriasis (5), and leads to reduced quality of life due to the associated sleep deprivation and mental distress (6). Currently, there are a wide variety of cosmetic products (e.g., moisturizers, lotions, and body creams) used to relieve chronic itching. Moisturizers are commonly used to relieve pruritic symptoms (7). Although some moisturizers can improve certain aspects of dry skin and abnormal skin barrier function, their usage cannot normalize the gene expression of itch mediators in inflammatory skin disorders (8). Various systemic and topical medications have been developed for the treatment of chronic itch (3). In general, immunosuppressants, (e.g., steroids and calcineurin inhibitors) and Janus kinase inhibitors are effective for many forms of chronic itch however, their potentially serious side effects limit their long-term applicability (9). Alternatively, various herbal formulations are widely used as active ingredients in functional cosmetics to improve skin health (10,11). Herbal cosmetic ingredients are adventitious over harmful chemicals because they include fewer side effects, have higher availability, and strong modulation of the skin immune system. Ageratum houstonianum, a member of the Asteraceae family commonly known as floss flower, blue mink, and blueweed, has wound-healing, antifungal, and antibacterial properties (12– 17). Aquaporin-3 (AQP3) is a pore protein expressed in the basal layer of the epidermis that transports water and glycerol from the dermis to the epidermis to maintain proper hydration in the epidermis (18). Previously, we demonstrated that the ethanolic extract of A houstonium restored AQP3 expression in keratinocytes, which was reduced by ultraviolet-B exposure (19). In addition, agerarin in A houstonianum restored decreased filaggrin (FLG) expression in AD-like skin lesions in mice (20), suggesting that A houstonianum may help strengthen the skin barrier by retaining moisture and restoring skin barrier integrity. We hypothesized that A houstonianum with different herbal extracts showing anti-inflammatory properties might have beneficial effects when used as an anti-itch ingredient in functional cosmetics. To identify functional cosmetic ingredients that help relieve itching, we further selected the medicinal herbs Bupleurum falcatum and Schisandra chinensis. The dried roots of B falcatum are widely used as a component of Asian folk medicine for treating inflammatory disorders and infectious diseases (21). The ethanolic extract of B falcatum exerts multiple pharmacological activities, (i.e., antiviral, anticancer, immunomodulatory, and anti-inflammatory effects) (21–26). Saikosaponin A, an active ingredient in B falcatum, exerts anti-inflammatory effects by inhibiting the nuclear factor kappa B (NF-κB) signaling pathway (22) and ameliorates nasal inflammation in ovalbumin-induced allergic rhinitis (27). S chinensis is a tonic medicinal herb belonging to the Schisandraceae family. The ethanolic extract of S chinensis demonstrates various pharmacological activities, including anti-inflammatory, antioxidant, antimicrobial, antiallergic, and hepatoprotective effects (28,29). Schizandrin, a bioactive component of S chinensis, inhibits passive cutaneous anaphylaxis and suppresses the urge to scratch by preventing mast cell degranulation and the expression of inflammatory cytokines, such as TNFα and IL4 (30). Moreover, topical application of S chinensis led to

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