84 Address all correspondence to Bingliang Wang, wangbl@usx.edu.cn Junxiang Li, lijunxiang@acrdc.cn Application of Euglena gracilis-Derived Peptides as a Cosmetic Ingredient to Prevent Allergic Skin Inflammation XUHUI LI, CHUNXIN XIA, DANNI KONG, MING XU, JIN ZHU, CONGFEN HE, BINGLIANG WANG AND JUNXIANG LI School of Life Science, Shaoxing University, Shaoxing, Zhejiang, 312000, China (D.K., B.W.) Zhejiang Provincial Key Laboratory of Applied Enzymology, Yangtze Delta Region Institute of Tsinghua University, Zhejiang, China (X.L.) AgeCode R&D Center, Yangtze Delta Region Institute of Tsinghua University, Zhejiang, China (X.L., C.X., M.X., J.Z., C.H., J.L.) TUS-HARVEST biotech. Co. Ltd., China (C.X., M.X., J.Z., C.H., J.L.) Beijing Key Laboratory of Plant Resources Research and Development, School of Science, Beijing Technology and Business University, Beijing, China (C.H.) Accepted for publication January 18, 2022. Synopsis Bioactive peptides have a wide range of therapeutic activities. They have emerged as interesting candidates for cosmetic ingredients. Euglena gracilis contains high amounts of proteins consisting of all 20 proteinogenic amino acids. As a result, it is thus proposed that enzymatic peptides from E gracilis proteins would have multiple biological activities. To verify their potential application in cosmetics, the therapeutic effects of ­ E gracilis-derived peptides (BPE) on allergic inflammation were evaluated in this study. In 2D skin models, HaCaT cells were used to assess cell viability, a fluorescence of 2’,7’-dichlorofluorescein diacetate was used to evaluate reactive oxygen species (ROS) contents, and an enzyme linked immunosorbent assay (ELISA) analysis of tumor necrosis factor α (TNF-α) reflected inflammatory response. Meanwhile, transcriptome analysis was performed to reveal the changes in gene expression. In 3D skin models, hematoxylin-eosin staining revealed the barrier-repairing ability of BPE. Filaggrin (FLG) and loricrin (LOR) levels were measured by their immunofluorescence. ELISA analysis of thymic stromal lymphopoietin (TSLP) also reflected the inflammatory response. A double-blind clinical trial was conducted to evaluate the erythema and itch, which reflects the degree of inflammation. Sodium lauryl sulfate, lipopolysaccharide, and histamine were used to induce allergic responses in vitro and in vivo. In 2D skin models, BPE increased cell viability from 41 to 55%. It also decreased intracellular ROS content from 100% to 61%. Combined with the decrease in TNF-α expression, it showed that BPE exerted multiple biological activities, including antioxidant and anti- inflammatory activities. According to the transcriptome analysis, BPE significantly changed the amounts of inflammation– and immune–response-related genes and enhanced the anti-inflammatory ability of cells. In 3D skin models, hematoxylin-eosin staining showed that BPE repaired the damages induced by Poly I:C and lipopolysaccharides. After BPE treatment, filaggrin and LOR expressions reached 124 and 127% J. Cosmet. Sci., 73, 84–95 (March/April 2022)

85 APPLICATION OF E GRACILIS-DERIVED PEPTIDES of the negative control, respectively, and the TSLP content significantly increased. It suggested that BPE treatment significantly alleviated inflammatory response and repaired the inflammation-induced damages. Furthermore, clinical trials in vivo also showed that BPE significantly suppressed allergic inflammation. The simulative experiments in 2D and 3D skin models as well as clinical trials verified that BPE had therapeutic effects on allergic inflammation. Our results propose BPE as a novel cosmetic ingredient to prevent allergic responses. INTRODUCTION Bioactive peptides are specific protein fragments, usually ranging from 2 to 20 units, with a molecular weight under 3 kDa. They can interact with a large number of biological macromolecules and biochemical compounds, showing a wide range of therapeutic antimicrobial, antihypertensive, antioxidant, antitumoral, antiproliferative, hypocholesterolemic, and anti-inflammatory activities (1,2). Bioactive peptides are generally obtained from diverse natural protein sources, such as food protein, vertebrates, bacteria, and microalgae (3). Among these protein sources, the microalgae-derived peptides are advantageously employed as bioactive compounds in cosmetics as they are selective, effective, safe, and well-tolerated once consumed (1). Euglena gracilis is a group of free-living, single-celled flagellates (4). It belongs to the green plant line, but in evolutionary terms it is most closely related to the protozoa (5). Nowadays, it is seen as one of the most attractive algal species for bioproducts. The most important aspect of E gracilis is that it contains high amounts of proteins, vitamins, and polyunsaturated fatty acids (6,7). Euglena gracilis has a relatively high amino acid content and higher nutritional quality compared to Chlorella and Spirulina (8). E gracilis contains all 20 proteinogenic amino acids (9). Although E gracilis has been exploited for decades (7), no studies focus on the application of bioactive BPE. According to previous studies of other microalgae-derived bioactive peptides, E gracilis are effective in promoting skin hydration and in fighting skin osmotic stress damages. Moreover, they can abolish reactive oxygen species (ROS) induction caused by oxidative stress agents (10). Because of this, it is interesting to investigate the skin care-related activity of bioactive peptides derived from E gracilis. The skin is one of the largest immunologic organs and is often a target for allergic and immunologic responses (11). Both external and internal factors can induce immune- mediated responses including dermatitis, urticarial, and angioedema, among others. Due to occupational exposures, face and hands suffer from a high incidence of dermatitis (12). Except for the clinical practice, cosmetic interventions are required to avoid the deterioration of quality of life (13). Some topical agents, such as zinc salts, taurine, and D-panthenol, can create a protective barrier over the skin and reduce the impact of external irritants, resulting in an effective treatment for rashes (14). However, it is still unclear whether bioactive peptides, especially the E gracilis-derived peptides (BPE), can be a promising alternative as therapeutic agents against allergic skin inflammation. To verify this previously mentioned hypothesis, we performed a variety of biological experiments based on 2D skin models, transcriptome analysis, and 3D skin models, which demonstrated that BPE reduced allergic inflammation.