*4.3. RNA Isolation, cDNA Synthesis and RT-PCR*

RAW264.7 cells were pre-treated with fractions or active components (dissolved in Dimethyl Sulfoxide, DMSO and diluted with DMEM medium) for 1 h and then stimulated with LPS (200 ng/mL) for 6 h, 0.05% DMSO was applied as the parallel solvent control. Total RNA was extracted using TRIzol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol, and subsequently used to obtain cDNA with a reverse transcription polymerase chain reaction following the protocol of PrimeScript reverse transcription reagent kit with genomic DNA (gDNA) Eraser (TaKaRa, Dalian, China) in a 20 μL volume. Levels of IL-1β, IL-6 and TNF-α were determined using specific primers (see Supplementary Table S1) by RT-PCR on a 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The expression of each gene was normalized relative to the GAPDH expression level, and relative expression levels were determined using the 2−ΔΔ*C*<sup>t</sup> method.
