Solvents (methanol, ethanol, and acetone) were obtained from Samchun Pure Chemical Co., Ltd. (Pyeongtaek, Korea). Cynanchi wilfordii Radix was purchased from an Oriental drug store in Seoul, Korea. Folin-Ciocalteu reagent, 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) reagent, Griess reagent, lipopolysaccharide (LPS), gallic acid, α-tocopherol, and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco's modified Eagle's medium (DMEM), fetal bovine albumin (FBS), and gentamycin were purchased from GIBCO BRL (Grand Island, NY, USA). Enzyme-linked immunosorbent assay (ELISA) kit and Mueller-Hinton agar were purchased from R&D Systems (Minneapolis, MN, USA) and Difco (Detroit, MI, USA), respectively.

Dried Cynanchi wilfordii Radix (20 g) was extracted with hot water and 80% methanol, 80% ethanol, or 80% acetone under reflux for 24 h, and filtered using Whatman filter paper (Macherey-Nagel; Düren, Germany). The filtered supernatants were evaporated using a rotary evaporator (Eyela; Tokyo, Japan) under reduced pressure, yielding a viscous solution. The concentrated extracts were freeze-dried and stored in the dark at 4℃ until the time of the experiments.

Total polyphenol content (TPC) of the extracts was measured using the Folin-Denis method [21]. Folin-Ciocalteu reagent (750 µl) was added to 150 µl of the sample and the reaction mixture was incubated for 5 min. After the incubation, 600 µl of 10% Na₂CO₃ was added and the reaction mixture was incubated at room temperature for 60 min. Absorbance of the reaction mixture was subsequently measured at 760 nm using Infinite® F200 plate reader (Tecan, Männedorf, Switzerland). Gallic acid was used to prepare the standard curve.

Murine RAW 264.7 cells were obtained from the Korea Cell Bank (Seoul, Korea) and cultured in DMEM containing 10% FBS, 20 µg/µL of gentamycin at 37℃ in a CO₂ incubator (Model 3111, Thermo Fisher Scientific; Waltham, MA, USA). The cells were seeded at 5×10⁵ cells/well in 24-well plates. Cell viability was tested following incubation with 0, 1, 5, 10, 50, 100, and 250 µg/µl concentrations of each extract. After 24 h of incubation, the cells were subjected to an MTT assay. Medium was replaced with 500 µl of serum-free media with MTT (0.5 mg/µl). After incubation for 1 h, the medium was removed and solubilized by adding 500 µl of DMSO to each well. The absorbance was monitored at a 590 nm wavelength using a microplate reader.

RAW 264.7 cells were plated in 24-well cell culture plates with 1 ml of culture medium and incubated for 24 h. The cells were then treated with 0, 1, 5, 10, 50, 100, and 250 µg/µl of each extract with 0.1 µg/µl of LPS, and incubated for 24 h. The nitrite generated was quantitated using the Griess reagent system. The supernatant was mixed with an equal volume of Griess reagent and incubated for 10 min at room temperature. The absorbance was measured at 540 nm using a microplate reader and nitrite concentration was determined.

The PGE₂, IL-1β, IL-6, IL-10, and TNF-α levels were determined in the supernatants from RAW 264.7 cells cultures using an ELISA kit according to the manufacturer's instructions. The cells were incubated with LPS (0.1 µg/µl) in the presence of a range of concentrations of Cynanchi wilfordii Radix extracts for 24 h. The absorbance was measured at 450 nm using a microplate reader and a standard curve was used to determine the levels of each cytokine.

RAW 264.7 cells were incubated with LPS (0.1 µg/µl) in the presence of a range of concentrations of Cynanchi wilfordii Radix extracts for 24 h. Total protein was extracted from cultured cells using the freeze-thaw lysis method. Equal amounts of protein from each sample were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted onto polyvinylidene fluoride membranes (Bio-Rad; Hercules, CA, USA). After blotting, the membrane was incubated with specific primary antibody overnight at 4℃, and subsequently incubated for 1 h with a secondary antibody. Protein signals on the membrane were quantified using a luminescent image analyzer LAS-4000 mini (Fujifilm Life Sciences; Tokyo, Japan).

Antioxidant activity was evaluated using 2,2-diphenyl-1-picrylhydrazyl hydrate (DPPH) free radical scavenging activity according to the method described by Hatano and Kagawa [22]. α-Tocopherol reagent (Sigma-Aldrich; St. Louis, MO, USA) was used as a positive control. Each extract was mixed with 100 µl of DPPH solution to a final concentration of 0.2 mM and allowed to stand for 30 min. The absorbance of the mixture was measured at 517 nm using a microplate reader. Scavenging activity was calculated using the following equation. where A_test represents the absorbance of the test solution, and A_control represents the absorbance of the α-tocopherol solution.

Anti-bacterial activity against 5 pathogens commonly involved in food poisoning was determined by the agar disc diffusion method using the measurement of the diameter of the clear zone. Bacillus cereus KCTC 3624^T was obtained from the Korean Collection for Type Cultures (KCTC; Daejeon, Korea). Bacillus anthracis ATCC 14578^T and Escherichia coli O157:H7 ATCC 43894 were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). Salmonella typhimurium NCCP 14774 was obtained from the National Culture Collection for Pathogens (NCCP; Cheongwon, Korea). Vibrio cholerae O139 was obtained from the Department of Microbiology, College of Medicine, Chonnam National University (Gwangju, Korea). The strains were grown aerobically on Mueller-Hinton agar at 37℃ for 18 h. Cultured media from test microorganisms were placed in sterilized paper discs (10 mm diameter) with each extract and placed on the inoculated agar surface. A broth micro-dilution susceptibility assay was conducted in accordance with NCCLS 2009 [23] guidelines for the minimal inhibitory concentration (MIC) determinations. MIC values were defined as the lowest concentrations of Cynanchi wilfordii Radix extracts where no visible growth can be observed.

All measurements were performed in triplicate and the results are expressed as means±standard error (SE). Statistical analyses of the differences between samples were carried out using one-way analysis of variance (ANOVA) followed by a post-hoc correction for multiple comparisons using the Duncan test and the Student's t-test using the Predictive Analytics Software (PASW) statistics package for Windows. p<0.01 was considered statistically significant.

The amount of TPC determined with 1,000 µg/ml of each of Cynanchi wilfordii Radix extract is shown in Table 1. TPC concentrations in water, methanol, ethanol, and acetone extracts were determined to be 5.6±0.6, 10.0±0.3, 19.8±6.4, and 10.7±0.6 µg gallic acid equivalents (GAE)/g, respectively. The highest TPC was detected in the ethanol extract.

The cytotoxic effects of polyphenols in Cynanchi wilfordii Radix extracts were evaluated in RAW 264.7 cells using the MTT assay. Polyphenols were measured at treatment concentrations in the cell culture system. Cells were treated for 24 h with 0, 1, 5, 10, 50, 100, or 250 µg/mL of each Cynanchi wilfordii Radix extract. Control group cells were treated with media only. As shown in Fig. 1, no cytotoxic effects of polyphenols (up to a concentration of 250 µg/ml) were noted after a 24-h incubation.

The inhibitory effects of 4 extracts of Cynanchi wilfordii Radix on the production of nitrite, a stable metabolite of NO, and PGE₂ were evaluated and compared in LPS-challenged RAW 264.7 cells. Cells were treated for 24 h with 0, 1, 5, 10, 50, 100, or 250 µg/ml of each extract in the presence of 0.1 µg/ml LPS. As shown in Fig. 2, Cynanchi wilfordii Radix extracts effectively suppressed LPS-induced nitrite production in a dose-dependent manner. Consistent with the observed nitrite accumulation, the extracts profoundly inhibited PGE₂ production. NO production in cells treated with the ethanol extract was significantly lower than that in cells treated with the water, methanol, or acetone extracts at 50 and 100 µg/ml concentrations. NO levels in LPS-stimulated RAW 264.7 cells exposed to 100 µg/ml of water, methanol, ethanol, or acetone extract were determined to be 91.1±2.7%, 81.3±1.3%, 24.0±4.6%, and 40.9±3.3%, respectively, relative to the untreated control. PGE₂ levels following exposure to 100 µg/ml of the water, methanol, ethanol, or acetone extracts were determined to be 80.5±8.7%, 92.5±3.4%, 47.9±6.4%, and 64.6±2.8% of the control values. These results showed that only methanol, ethanol, and acetone extracts exerted an anti-inflammatory effect by ameliorating the production of the inflammatory mediators NO and PGE₂. In contrast, equal concentration of water extract did not elicit anti-inflammatory effects. The water extract exhibited anti-inflammatory effects at 250 µ g/ml. In addition, PGE₂ was dramatically reduced (82.5%) following treatment with 250 µg/ml of the water extract. The 250 µg/ml concentration in the acetone extract and water extract was reduced dramatically than the ethanol extract. These results indicates that the water and acetone extract was more related to pain. In particular, levels of NO were dramatically reduced (76.0%) compared to those of PGE₂ and other inflammatory factors following incubation of RAW 264.7 cells with 100 µg/ml of the ethanol extract. Based on the comparison of the 4 Cynanchi wilfordii Radix extracts, the highest anti- inflammatory effects were observed with the ethanol extract.

The effect of Cynanchi wilfordii Radix extracts on the production of IL-1β, IL-6, IL-10, and TNF-α were evaluated in LPS-stimulated RAW 264.7 cells. As shown in Fig. 3, production of IL-1β, IL-6, IL-10, and TNF-α was dramatically diminished following treatment with the extracts. Treatment with ethanol extract in particular was observed to reduce IL-1β and IL-6 production to lower levels than treatments with the water, methanol, and acetone extracts. However, no inhibition of IL-10 production was observed following incubation with the water extract and no inhibition of TNF-α was observed following treatment with the water, methanol, or acetone extracts. TNF-α production was not significantly reduced, compared to other cytokines. Incubation with the highest concentration of the ethanol extract (250 µg/ml) was the only treatment that inhibited TNF-α.

Expression of iNOS and COX-2 enzymes was evaluated in murine macrophage RAW 264.7 cells using immunoblotting. As shown in Fig. 4, the ethanol extract was found to inhibit the expression levels of iNOS and COX-2 proteins in a concentration-dependent manner, with highest inhibitory effects observed following treatment with the 100 µg/ml concentration. Methanol and acetone extracts inhibited the protein expression levels of iNOS and COX-2 in a concentration-dependent manner, with highest inhibitory effects observed at 250 µg/ml. Conversely, water extract inhibited the protein expression levels of COX-2, with highest inhibition observed at 250 µg/ml.

The antioxidant activity of polyphenols was rapidly evaluated. As shown in Table 2, DPPH radical scavenging activity of Cynanchi wilfordii Radix extracts significantly increased with increasing extract concentration. Relatively high DPPH radical scavenging activities were found in water, methanol, ethanol, and acetone extracts (56.8%, 85.2%, 85.3%, and 83.2%, respectively) at 10.0 mg/ml. The highest DPPH radical scavenging activity (91.0%) was observed in the ethanol extract.

As shown in Table 3, ethanol extract elicited relatively higher anti-microbial activity against B. cereus and B. anthracis (12.3 mm and 12.7 mm, respectively) than the other extracts. However, no anti-microbial activity was observed against E. coli O157:H7, S. typhimurium, and V. cholerae O139. The disc diffusion values of the ethanol extract at 50 mg/ml concentration demonstrated its effectiveness against the bacteria involved in food poisoning. In contrast, the water, methanol, and acetone extracts were observed not to be effective in inhibiting bacteria involved in food poisoning. The MIC value of the ethanol extract was 6.25 mg/ml against B. cereus and B. anthracis (Table 4).