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).

ANTI-INFLAMMATORY ACTIVITY OF C. ASIATICUM 421 PREPARATION OF THE ETHANOL EXTRACT OF CRINUM ASIATICUM The dried root of Crinum asiaticum Linne var. japonicum (0.34 kg) was extracted with 95% ethanol (1 kg) at 75°C for 3 h. Its pH was adjusted to 3.5 by adding 12 N HCl. After filtration through a 400-mesh filter cloth, the filtrate was filtered again through filter paper (Whatman, No. 5). The ethanol solution was evaporated to remove the solvent under 50°C. Lyophilization over one day yielded a yellowish powder (28.3 g, yield 8.32%). ANALYSIS OF LYCORINE CONTENT BY HPLC The ethanol extract powder of Crinum asiaticum Linne var. japonicum and lycorine · HCl (Sigma, used as a standard) were dissolved in 50% methanol. The lycorine content was investigated and calculated by HPLC (conditions: Capcell Pak Cl8 column [ 4.6 x 250 mm, Shiseido} mobile phase, 0.05 M KH 2 PO 4 and acetonitrile (95:5) flow rate, 1.0 ml/min detector, UV A. = 290 nm injection volume, 20 µl). CELL CULTURE Cells were maintained in DMEM (Dulbecco's Modified Eagle's Medium) with 10% FBS (fetal bovine serum), 50 U/ml penicillin, and 50 µg/ml streptomycin. The medium was changed every two or three days. Cells were cultured at 3 7°C in a humidified atmosphere of 95% air and 5% CO2 . CYTOTOXICITY ASSAY Raw 264.7 was seeded into 24-well plates at a density of 8 x 105 cells and cultured at 37°C in 5% CO 2 . After one day, fresh medium containing 10% serum was added to the cells, which were then treated with LPS (lipopolysaccharide, 1 µg/ml), followed by treatment with the ethanol extract of Crinum asiaticum for 24 h. Cytotoxicity was evaluated by an MTT ,(3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyl tetrazolium bromide [Sigma}) assay (5). After 24 h, 100 µl of 2.5 mg/ml MTT was inserted into each well, and the plates were incubated at 3 7°C for an additional 4 h. Then, the media containing MTT was discarded and the MTT formazan product was extracted with 1 ml DMSO. The amount of formazan in the culture medium was determined by the absorbance measured at 5 70 nm by an ELISA reader. Cell viability was calculated as: Cell viability(%)=(OD57o(sam p le/OD57o(control)) X 100 where OD 57 o(sample) is the absorbance at 5 70 nm of the cells treated with the sample and OD 57 o(conrrol) is the absorbance at 5 70 nm of the negative control (non-treated cells). DETERMINATION OF NITRITE SYNTHESIS Nitrite in the media was measured by the Griess assay (6) and was used as an indicator of NO synthesis in the cells. In brief, an equal volume of the culture supernatants in the Raw cell line 264.7 and the Griess solution (1:1 mixture [v/vJ of 1 % sulfanilamide and 0.1 % N-[naphthyl} ethylenlide diamine dihydrochloride in 5% H3PO4) was added into