*2.1. Separation, Purification and Identification of Active Components from Bletilla striata*

The ethanol extraction of *Bletilla striata* tuber was separated into five fractions using the polyamide adsorption method, then they were characterized by the high performance liquid chromatography (HPLC) method (see Figure 1A). The results indicated that there were few common peaks in each fraction, which shows the effective enrichment effect of the polyamide column. The anti-inflammation activity of the five fractions was screened on the LPS-induced RAW264.7 cell model, and the real-time polymerase chain reaction (RT-PCR) results indicate that except F0 and F80, the fractions dose-dependently inhibited IL-1β expression, whereas F80 showed inhibition activity at low dosage, but the messenger RNA (mRNA) expression level of IL-1β was dose-dependently increased to even higher than the LPS-treated group at 30 μg/mL (see Supplementary Figure S1). F40 showed remarkable inhibition activity and 83.07% of IL-1β mRNA expression was inhibited at a concentration of 10 μg/mL (see Figure 1B).

**Figure 1.** (**A**) HPLC characterization of the five fractions. A total of 10 μL each sample (1 mg/mL) was injected and analyzed using a Dionex UltiMateTM 3000 HPLC system with photodiode array detection (PAD) at 259 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: 0–35 min 30% a isocratic, 35–45 min 30% to 40% a, 45–55 min 40% a isocratic, and finally washing and recondition of the column. (**B**) Relative expression of IL-1β mRNA after treatment with F40. RAW264.7 cells were pretreated with different concentration of F40 for 1 h and then treated with 200 ng/mL LPS for 6 h. Total RNA was extracted and genes expression level were analyzed by RT-PCR in triplicate. The expression level of each gene was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. Data are expressed as mean ± SD (*n* = 6). \*\* *p* < 0.01 vs. LPS treatment group.

The F40 fraction was further separated by silica gel chromatography. Dry silica gel was packed into a glass column (diameter via height ratio 1:10), then dry sample (sample silica gel ratio 1:3) was loaded and eluted with chloroform-methanol (50:1, V/V) at 2 mL/min. Fractions of 10 mL were collected and monitored by thin-layer chromatography (TLC), visualized by iodine vapor and those possessing similar Rf values were combined and six sub-fractions of F40 were obtained (see Figure 2A). Anti-inflammation assay manifested that all sub-fractions inhibited the expression of IL-1β in a dose-dependent manner (see Supplementary Figure S2), and the sub-fractions of F40-3 and F40-4 showed significant inhibition ratio at 20 μg/mL (see Figure 2B). HPLC analysis revealed that F40-3 and F40-4 were mainly composed of two identical compounds (see Figure 3A), and the two compounds were purified by Dionex UltiMateTM 3000 semi-prepared HPLC system. A Welch Ultimate® XB-C18 (10 × 250 mm, 10 μm) HPLC column operated at 30 ◦C was used, and the flow rate was maintained at 5 mL/min. Samples were isocratic eluted with acetonitrile (30%) and 0.1% acetic acid (70%).

The results of mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectra are as follows: Compound I (HPLC > 98%) electrosprary ionization-mass spectrometry (ESI-MS) *m*/*z*: 245.1108 (M + H)+. 1H-NMR (CD3OD) δ: 2.79 (4H, m, CH2), 3.71 (3H, s, OCH3), 6.19 (1H, dd, *J* = 1.8, 2.4 Hz, H-4), 6.25 (2H, dd, *J* = 1.8, 1.8 Hz, H-2, 6), 6.63 (3H, m, H-2', 4', 6'), 7.08 (1H, dd, *J* = 7.8, 8.4 Hz, H-5'); 13C-NMR (CD3OD) δ: 160.83 (C-5), 157.98 (C-3), 156.93 (C-3'), 144.06 (C-1), 143.29 (C-1'), 128.84 (C-5'), 119.43 (C-6'), 114.93 (C-2'), 112.36 (C-4'), 107.63 (C-2), 105.12 (C-6), 98.54 (C-4), 54.10 (OCH3), 37.80 (CH2-a'), 37.47 (CH2-a). Compared with the data shown in literature [15], the compound was identified as batatasin III. Compound II (HPLC > 98%) ESI-MS *m*/*z*: 243.1006 (M + H)+. 1H-NMR (CD3OD) δ: 2.64 (4H, s, H-9, 10), 3.83 (3H, s, 4-OCH3), 6.32 (1H, d, *J* = 2.4 Hz, H-1), 6.41 (1H, d, *J* = 2.4 Hz, H-3), 6.64 (1H, d, *J* = 1.8 Hz, H-6), 6.63 (1H, dd, *J* = 6, 2.4 Hz, H-8), 8.02 (H, d, *J* = 9 Hz, H-5). 13C-NMR (CD3OD) δ: 157.68 (C-2), 154.63 (C-7), 156.04 (C-4), 140.42 (C-10a), 139.08 (C-8a), 128.63 (C-5), 113.61 (C-4a), 124.77 (C-5a), 112.17 (C-6), 115.37 (C-8), 106.90 (C-1), 97.83 (C-3), 54.45 (-OCH3), 29.80 (C-9), 30.38 (C-10). Comparison with the data shown in literature [16], led to the compound being identified as coelonin.

**Figure 2.** (**A**) Sub-fractions separated from F40 by silica gel chromatography. TLC was performed on precoated silica gel 60 F254 plates (Qingdao Haiyang Chemical Co., Ltd., Qingdao, China), developed with chloroform-methanol (4:0.1, V/V) and then exposed to the iodine vapor in a dark enclosed chamber for 10 min. (**B**) The relative expression of IL-1β mRNA after treatment with sub-fractions of F40-3 and F40-4. RAW264.7 cells were pretreated with different concentration of different sub-fractions of F40 for 1 h, then treated with 200 ng/mL LPS for 6 h. Total RNA was extracted and genes expression levels were analyzed by RT-PCR in triplicate. The expression level of each gene was normalized to GAPDH mRNA. Data are expressed as mean ±SD (*n* = 6). \*\* *p* < 0.01 vs. LPS treatment group.

The anti-inflammation activity was verified (see Figure 3B) and both coelonin and batatasin III showed dose-dependent inhibition activity. A total of 93.1% of IL-1β mRNA expression was inhibited by coelonin at 2.5 μg/mL, which was significantly better than that of batatasin III at 10 μg/mL (62.3%). This result implies that coelonin is probably the main anti-inflammatory component of *Bletilla striata*.
