Brazilin

Powdered CS heartwood was purchased from Chakkrawatherb Co., Ltd. (Bangkok, Thailand). The report of the Integrated Taxonomic Information System (ITIS) contains taxonomic data about sappan (taxonomic serial number: 506349). The powder (500 g) was macerated in 95% ethanol (3 × 28 L) at room temperature for 72 h, and the extraction process was repeated twice. The combined crude ethanolic extracts were filtered through Whatman No. 1 filter paper. At a temperature of 40 °C, the solvent was completely evaporated using a rotary evaporator. The extracts were kept at 20 °C until further usage. Brazilin, catechin, and epicatechin were purchased from Sigma-Aldrich Co (St Louis, MO, USA).

The material samples used in this study include sappan wood (C. sappan L.) obtained from Magelang in the Central Region of Java, and cinnamon bark (C. burmanii Blume) from Padang, West Sumatera Indonesia. Both plants were authenticated at the Herbarium Bogoriense, Botanical Gardens, Bogor, West Java, Indonesia. Choline chloride was purchased from Xi’am Rongsheng, Hangzhou, China; glycerol was purchased from PT Molex Ayus, Tangerang, Indonesia; demineralized water and 96% v/v ethanol were purchased from Brataco, Bogor, Indonesia; methanol (HPLC grade), acetonitrile (HPLC grade), glacial acetic acid, brazilin, coumarin, and trans-cinnamaldehyde standard were purchased from Sigma Aldrich through PT. Elo Karsa Pratama, South Jakarta, Indonesia; double-distilled water (ddH₂O) was purchased from PT. Ikapharmmindo Putramas, East Jakarta, Indonesia; and DPP IV kits were purchased from Cayman Chemical, Ann Arbor, USA.

Primary murine BMDMs were seeded in 96-well plates overnight before use (1x10⁶ cells/ml). Cells were primed with lipopolysaccharide (LPS; E.coli O26:B6) (1 μg mL⁻¹) (Sigma, L2654) in DMEM (10% v/v FBS, 1% v/v penicillin–streptomycin, 1% v/v pyruvate) for 4 h, before the media was replaced with ‘treatment media’ constituting DMSO (1% v/v) (Sigma, D2650) or brazilin (≥98% purity; HPLC) (Merck, SML2132; Rahway, NJ, USA) (30, 10, 3, 1, 0.3, 0.1, 0.03 or 0.01 μM) in serum-free DMEM (1% v/v penicillin–streptomycin, 1% v/v pyruvate). After 15 min treatment incubation, NLRP3 was activated by adding nigericin (10 μM) (Sigma, N7143) into the wells, with ethanol (0.5% v/v) used as a vehicle control, and incubated for 2 h. Alternatively, to assess the direct effect of brazilin treatment on AIM2 or NLRC4 inflammasome activation, cells were transfected with either 1 μg mL⁻¹ poly(deoxyadenylic-deoxythymidylic) acid sodium salt (poly(dA:dT), Sigma, P0883) or 1 μg mL⁻¹ flagellin from Salmonella typhimurium (Invivogen, tlrl-stfla), respectively, using Lipofectamine 3000 (Thermofisher, L3000001) diluted in OptiMEM (Thermofisher, 11058021) per manufacturer’s instructions, for 2 h. Following 2-h inflammasome activation, supernatants were collected and used for cell death analysis and Enzyme-Linked Immunosorbent Assays (ELISA).