*4.1. Active Component Separation, Purification and Identification*

A modified polyamide adsorption separation method was used [51]. An amount of 100.0 g tuber powder of *Bletilla striata* (collected from Meichuan, Wuxue, Hubei province, China) was reflux extracted by 1 L 80% ethanol, and the process was repeated three times. The filtrate was concentrated under vacuum to the volume of 600 mL, and an equal volume of distilled water and 30.0 g polyamide (100–200 mesh, Taizhou Luqiao Sijia Biochemical Plastics Factory, Taizhou, China) was added, then the suspension was concentrated under vacuum to the volume of 600 mL. Finally, the mix suspension was packed, and successively eluted with water, 20%, 40%, 60% and 80% ethanol, and each elution was concentrated and dried under vacuum to obtain the fractions F0, F20, F40, F60 and F80, respectively. The IL-1β mRNA expression level, detected by a RT-PCR as the index of anti-inflammation activity on the LPS-induced RAW264.7 cell model was carried out to evaluate the anti-inflammatory effect of the fractions. Then, the active fraction was further separated using silica gel (200–300 mesh, Qingdao Haiyang Chemical Co., Ltd., Qingdao, China) column chromatography, the subfractions were collected after monitoring by TLC (Qingdao Haiyang Chemical Co., Ltd., Qingdao, China), and the anti-inflammation effect was analyzed. The final active compound was purified by Dionex UltiMate™ 3000 semi-prepared HPLC system (Dionex, Sunnyvale, CA, USA) following the guidance of anti-inflammation assay. Furthermore, the molecular weight of the active compounds were determined on an ACQUITY UPLC system coupled to a SYNAPT-G2-Si high-definition mass spectrometer (Waters,

Milford, MA USA), and their NMR spectra were measured on a Bruker DRX-600 spectrometer (Bruker, Rheinstetten, Germany) with CD3OD as the solvent.

### *4.2. Cell Culture*

RAW264.7 cells (ATCC) were cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% heat inactivated fetal bovine serum (Gibco, Waltham, MA, USA), 100 units/mL penicillin and 100 μg/mL streptomycin. The cells were grown at 37 ◦C in a 5% CO2 incubator.
