ANTIBACTERIAL ACTIVITY OF SENKYUNOLIDE A 379 DETERMINATION OF ANTIBACTERIAL ACTIVITY PAPER DISK DIFFUS ION METHOD The antibacterial activities of the samples were evaluated using a disc diffusion assay. In brief, Advantec paper discs of 8 mm diameter were impregnated with 50 μL of the solu- tion impregnating ethanol extract and fractions at a concentration of 50 mg ml-1 and evaporated at room temperature for 24 h (6). Then 1 ml of bacteri a (107 cfu ml-1) incubated for 24 h was dispensed into petri dishes, and the medium was poured and solidifi ed. And then discs of COH and SA were placed on the RCM (P. acnes) and mLNB agar plates (M. furfur). The antibacterial activity was evaluated by measuring the zones of inhibition on the agar plate. All experiments were carried out in triplicate. MINIMUM INHIBITORY CON CENTRATION (MIC) The MICs were determin ed using the broth dilution method recommended by the Tenover (7). The cultures were prepared from 48-h and 96-h broth cultures of P. acnes and M. furfur, respectively. A 15-ml conical tube was prepared by dispensing 3 ml of broth media, sample (concentration ranging from 1.00 to 20.00 mg ml-1), and an appropriate amount of cell suspension (107 cfu 3 ml) into each tube. The tubes were incubated at 37°C for 48 h and 27°C for 96 h for P. acnes and M. furfur, respectively. At the end of the incubation period, the tubes were evaluated for the presence or absence of growth. All experiments were performed in triplicate. DETERMINATION OF ANTI-INFL AMMATORY ACTIVITY PANCREATIC LIPASE INHIBITI ON ACTIVITY Porcine pancreatic lipase (PPL, type II) activity was measured using p-nitrophenyl lau- rate (p-NPL) as a substrate. The method used for measuring the PPL activity was modi- fi ed from that previously described by Zheng et al. (8) and Bustanji et al. (9). The reaction mixture consisted of 50 mM Tris-HCl buffer (930 μl, 150 mM NaCl, 1 mM EDTA, and 10 mM MOPS, pH 7.4), PPL 5 mg ml-1 (30 μl), and 5 mM p-NPL (20 μl). The reaction was started by adding p-NPL as a substrate, all in a fi nal volume of 1,000 μL. After incu- bation at 35°C for 15 min, the amount of p-nitrophenol released during the reaction was measured at 405 nm using a UV-visible spectrophotometer. All experiments were carried out in triplicate. LIPOXYGENASE INHIBITION ACTIVITY L ipoxygenase (soybean) enzyme acti vity was measured according to the method of Lycka- nder and Malterud (10) with a minor modifi cation. Tris-HCl buffer (2,800 μl, pH 9.0) was added to 100 μl of sample at different concentrations, nordihydroguaiaretic acid

JOURNAL OF COSMETIC SCIENCE 380 (NDGA), and enzyme (100 μl, 500 U ml-1 in Tris-HCl buffer). Samples and NDGA were added as dimethyl sulphoxide solutions. After incubation of the test solution for 5 min, 100 μl of linoleic acid was added, and the change in the absorbance of the solution was measured after 60 s at 234 nm. CELL VIABILITY AND DETERMINATION OF ANTI -INFLAMMATORY ACTIVITY The cell viability assay was carried out by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphen- yltetrazolium bromide (MTT). RAW 264.7 cells were treated with various doses of S. baicalensis extract (1, 10, 100, 500 μg ml-1). To confi rm the anti-infl ammatory properties, we examined the inhibitory effects on the production of lipopolysaccharide (LPS)-induced NO in RAW 264.7 cells. NO levels were measured by the Griess reaction. After cells (5 × 105 cells ml-1) were stimulated in 24 wells for 24 h, 100 μl of each cultured medium was mixed with the same volume of Griess reagent (1% sulfanilamide/0.1% N-(1-naphthyl)- ethylenediamine dihydrochloride/2.5% H3PO4). NO concentration was determined by measuring the absorbance at 540 nm with a Vmax 96-well microplate spectrophotometer. The NO2 concentration was calculated with reference to a standard curve of NaNO2 gener- ated by known concentrations. All experiments were carried out in triplicate. RESULT ANTIBACTERIAL ACTIVITY The disc diffusio n assay and broth dilution met hod were used to determine the antibacte- rial activity and the MIC of the COH against two bacterial strains. COH exhibited anti- bacterial activities (MIC 15.33 ± 2.11–42.33 ± 2.11 mg ml-1) against P. acnes and M. furfur (Table 1). PANCREATIC LIPASE INHIBITION ACTIVITY Lipase is glyc erol ester hydrolase (E.C. 3.1.1.) tha t acts on acylglycerols to liberate fatty acids and glycerol. Several lipases produced by microbial pathogens play an important role in skin diseases (11). The bacterial lipase is an important factor in the pathogenesis of skin diseases such as acne, dandruff, and atopic dermatitis. They can induce severe infl ammatory reactions (12). The lipase inhibition activity results are shown in Figure 1 Table I Antibacterial Activity of COH Fraction and Senkyunolide A against Tes t Bacteria Sample Paper disk test Bacteria (clear zone size, mm) Bacteria (MIC, mg ml-1) Concentration (mg ml-1) P. acnes M. furfur P. acnes M. furfur COH 50 14.6 13.3 15.33 ± 2.11 30 SA 50 23.5 ND 5.30 ± 0.47 ND ND: not detected .