J. Cosmet. Sci., 59, 419-430 (September/October 2008) Anti-inflammatory activity of Crinum asiaticum Linne var. japonicum extract and its application as a cosmeceutical ingredient YOUNG HEUI KIM, KI HO KIM, CHANG SUNG HAN, SUN HEE PARK, HONG CHUL YANG, BANG YONG LEE, SANG-YONG EOM, YOUNG-SIL KIM, JONG-HEON KIM, and NAM HO LEE, R&D Center, Bioland Ltd., Songjeong, Byongchon, Cheonan-si, Chungnam, 330-863 (Y.H.K., K.H.K., C.S.H., S.H.P., H.C. Y., B. Y.L.), R&D Center, Charmzone, 1720-4 Taejang 2-dong, Wonju-si, Kangwon-do, 220-962 (S.-Y.E., Y.-S.K., ].-H.K.), and Department of Chemistry, College of Natural Science, Cheju National University, Ara I-dong, Jeju, 690-7 56 (N.H.L.), Republic of Korea Accepted for publication April 9, 2008. Presented at the 2007 ASCS Conference, Singapore, March 7-9, 2007. Synopsis Crinurn asiaticurn Linne var. japonicurn has long been used as a rheumatic remedy, as an anti-pyretic and as an anti-ulcer treatment, and for the alleviation of local pain and fever in Korea and Malaysia. In order to investigate the possibility of Crinurn asiaticurn Linne var. japonicurn extract as a cosmetic ingredient, we measured its anti-inflammatory effect by its inhibition of iNOS (inducible nitric oxide synthase) and the release of PGE2, IL-6, and IL-8. We also measured its anti-allergic effect by its inhibition of 13-hexosamidase release. An HPLC experiment after extraction with 95% EtOH at pH 3.5 showed that Crinurn asiaticurn Linne var. japonicurn was mainly composed of lycorine (up to 1 %), a well-known immunosuppressor. The content of lycorine varied, depending on the type of plant tissue analyzed and the extraction method. In an anti-inflammatory assay for inhibition of nitric oxide formation on lipopolysaccharide (LPS)-activated mouse macrophage RAW 264. 7 cells, the ethanol extract of Crinurn asiaticurn showed an inhibitory activity of NO production in a dose-dependent manner (IC50 = 58.5 µg/ml). Additional study by RT-PCR demonstrated that the extract of Crinurn asiaticurn significantly suppressed the expression of the iNOS gene. Moreover, the extract of Crinurn asiaticurn did not show any cytotoxicity, but did show a cell proliferation effect against LPS (a 10�60% increase in cell viability). In an assay to determine inhibition of the H 2 02-activated release of PGE2, IL-6, and IL-8 in human normal fibroblast cell lines, the release of PGE2 and IL-6 was almost completely inhibited above concentrations of 0.05 % and 1 %, respectively. Moreover, the release of IL-8 was completely inhibited over the entire range of concentration (0.0025%). In order to investigate the Address all correspondence to Young Heui Kim. 419

420 JOURNAL OF COSMETIC SCIENCE skin-sensitizing potentials of the extract of Crinum asiaticum, a human clinical test was performed after repeated epicutaneous 48-h applications under an occlusive patch (RIPT). The repeated and single cutaneous applications of Crinum asiaticum Linne var. japonicum extract under the occlusive patch did not provoke any cumulative irritation and sensitization reactions. The result showed that the extract of Crinttm asiaticum Linne var. japonicum has a sufficient anti-inflammatory effect. Therefore, Crinum asiaticum Linne var. japoni­ cum extract may be useful for development as an ingredient in cosmetic products. INTRODUCTION Crinum asiaticum Linne var. japonicum (Family Amaryllidaceae) is an herbaceous plant of small-to-moderate size. The plant is distributed on South Korea's Jeju Island, and was designated as the 19th of Korea's natural treasures. Crinum asiaticum has been used as a rheumatic remedy, as an anti-pyretic, as an anti-ulcer treatment, and for the alleviation of local pain and fever in Korea and Malaysia. The anti-inflammatory activity of Crinum asiaticztm and its effects on bradykinin-induced contractions in an isolated uterus have been reported (1). The analgesic and anti­ inflammatory activities of an aqueous extract of Crinum glaucumJ also of the Amarylli­ daceae family, have also been reported (2). Amaryllidaceae alkaloids, lycorine and ly­ coricidinol, were isolated from the dried stems of Crinum asiaticum and identified by chromatographic and spectral analysis. It has been reported that lycorine, the active molecule, has an inhibitory effect on tumor cell apoptosis induced by polymorpho­ nuclear leukocyte-derived calprotectin (3 ). In a report by the same authors, lycorine was also reported to have an inhibitory effect on macrophage TNF-a production (4). Although a few pharmacological studies have been reported, as listed above, this plant has not been subjected to a systematic cosmeceutical evaluation to determine its pro­ priety in practical application. In order to investigate the possibility of Crinum asiaticum Linne var. japonicum extract as a cosmetic ingredient in this study, we measured its anti-inflammatory effect by its inhibition of iNOS (inducible nitric oxide synthase) and the release of PGE2, 11-6, and 11-8, cytokines with known associations with inflammatory reactions. Crinum asiaticum Linne var. japonicum extract showed negligible cytotoxicity and good anti-inflammatory activities, suggesting that Crinum asiaticum Linne var. japonicum extract could be useful as a cosmeceutical ingredient. MATERIALS AND METHODS MATERIALS The root of Crinum asiaticum Linne var. japonicum was purchased from Jeju Botanic Garden CT eju, South Korea) and chopped before use. Human fibroblasts were acquired from the ATCC (American Type Culture Collection, CRL-2076, passage number 7-13). The mouse macrophage-like cell line, Raw 264. 7, was purchased from the ATCC (TIB-71, passage number 5-15). Sigma (St. Louis, MO) supplied 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), lycorine · HCl, and Griess soution, and they were used as received. Dulbecco's Modified Eagle's Media (DMEM), fetal bovine serum, and antibiotics were purchased from Life Technologies (Grand Island, NY).