*2.2. Inhibitory E*ff*ect of Coelonin on LPS-Induced IL-1*β*, IL-6, TNF-*α *Gene Expression and Protein Secretion in RAW264.7 Macrophages*

LPS can induce the expression and secretion of a variety of inflammatory factors, such as IL-1β, IL-6, TNF-α, monocyte chemo-attractant protein-1 (MCP-1), inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX2) et al. Using IL-1β as an index, a highly active ingredient, coelonin, was isolated from *Bletilla striata* by the RAW264.7 cell inflammation model. Here, two other important inflammatory factors, IL-6 and TNF-α were examined as well. We confirmed that coelonin was without a significant cytotoxic at a concentration of up to 5 μg/mL [17] (see Supplementary Figures S3 and S4). LPS markedly elevated IL-1β, IL-6 and TNF-α mRNA expression and protein secretion, but coelonin dose-dependently lowered these levels in macrophages (see Figure 3C,D). These results confirmed the anti-inflammation effect of coelonin. However, there was almost no anti-inflammation report of this compound; therefore, the underlying mechanism is worth further research.

**Figure 3.** (**A**) HPLC characterization of the sub-fractions F40-3, F40-4 and two purified compounds. 10 μL each sample (0.1 mg/mL) was injected and analyzed using a Dionex UltiMateTM 3000 HPLC system with PAD at 193 nm. A Symmetrix ODS-RC18 (25 × 4.6 mm, 5 mm) HPLC column protected with a Phenomenex security guard column (C18, 4 × 3.0 mm) operated at 30 ◦C was used, and the flow rate was maintained at 1 mL/min. The elution solvents were acetonitrile (a) and 0.1% acetic acid (b). Samples were eluted according to the following gradient: 0–5 min 10% to 35% a, 5–12 min 35% a isocratic, 12–16 min 35% to 45% a, 16–22 min 45% a isocratic, 22–35 min 45% to 80% a, and finally washing and recondition of the column. (**B**) The relative expression of IL-1β mRNA treated by active compounds. (**C**) Inhibitory effect of coelonin on LPS-induced gene expression in RAW264.7 macrophages. RAW264.7 cells were pretreated with different concentration of compound I or II for 1 h, then stimulated with 200 ng/mL of LPS for 6 h. Total RNA was extracted and genes expression level were analyzed by RT-PCR in triplicate. (**D**) Inhibitory effect of coelonin on LPS-induced cytokine secretion in RAW264.7 macrophages. RAW264.7 cells were pretreated with coelonin for 1 h and then treated with 200 ng/mL LPS. 12 h after LPS stimulation, culture supernatants was collected for IL-6 and TNF-α detection by cytometric bead array (CBA) method; for IL-1β detection, the remaining cells were following treated by 1 mM adenosine triphosphate (ATP) for an additional 15 min at 37 ◦C [18], then supernatants were collected and analyzed by the CBA method in triplicate. Data are expressed as mean ± SD (*n* = 6). \*\* *p* < 0.01 vs. LPS treatment group.
