580 Address all correspondence to Soon Young Shin, shinsy@konkuk.ac.kr Ethanolic Extracts of Ageratum houstonium, Bupleurum falcatum, and Schisandra chinensis Inhibit Interleukin-4-Induced Expression of Itching Factors in HaCaT Keratinocytes HYOUNG SEOK SHIN, SUNG SHIN AHN, HYUNJIN YEO, YOU JEONG JEONG AND SOON YOUNG SHIN Hansol Bio Inc., Seongnam 13487, Republic of Korea (H.S.S.) Department of Biological Sciences, Sanghuh College of Lifesciences, Konkuk University, Seoul 05029, Republic of Korea (S.S.A., H.Y., S.Y.S.) Agera BioTech, Seoul 05029, Republic of Korea (Y.J.J.) Accepted for publication August 14, 2021. Synopsis Chronic itch is the most frequently observed symptom of inflammatory skin disorders. It causes sleep depriva- tion and mental distress and eventually leads to reduced quality of life. Various herbs have been widely used as the main ingredients in functional cosmetics to soothe itchy skin. This study evaluates the effect of the ethanolic extracts of Ageratum houstonianum, Bupleurum falcatum, and Schisandra chinensis on the modulation of the skin barrier component filaggrin (FLG) and keratinocyte-derived itch-inducing factors, including IL31, thymic stromal lymphopoietin (TSLP), and β-endorphin. Gene expression profiles were analyzed using reverse transcription-polymerase chain reaction and quantitative real-time polymerase chain reaction. Protein levels were measured using immunoblotting and enzyme-linked immunosorbent assays. The cellular localization of IL31 and FLG was examined by double immunofluorescence staining. Treatment with A houstonianum, B falcatum, or S chinensis significantly (p 0.05) restored the reduction in FLG expression by IL4 plus IL13 stimulation. The IL4-induced expression of IL31, TSLP, and β-endorphin was also significantly (p 0.05) suppressed at the mRNA and protein levels following treatment with the abovementioned extracts. We also evaluated the efficacy of the A houstonianum, B falcatum, and S chinensis combination for improving the skin barrier function and soothing itchy skin in a clinical study. In conclusion, A houstonianum, B falcatum, and S chinensis (either alone or in combination) can be used as beneficial ingredients for cosmetic applications against chronic inflammatory itch. INTRODUCTION Itch is a cutaneous sensation that triggers the urge to scratch. Itch perception is transmitted by intrinsic and extrinsic stimuli that activate peripheral sensory nerve endings of the nonmyelinated C-fibers branched from dorsal root ganglion neurons at the epidermis and J. Cosmet. Sci., 72, 580–599 (November/December 2021)

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