Traditional Chinese medicine Salvia miltiorrhiza Bunge (Danshen) has been prescribed in clinica in China for centuries while its material basis for action remains unclear. The lipophilic tanshinones isolated from this herb such as tanshinone IIA, cryptotanshinone, etc. demonstrate potent cardiovascular protective and anti-cancer effect. However, the isolation and bioactivities of trace compounds from Danshen have not been unraveled. Specifically, tanshinones with hydroxyl group are rarely reported due to their low contents in Danshen. In this study, the isolation and purification of compounds in Danshen and their pharmacological activity evaluation and mechanism exploration were illustrated. Our results showed that four novel tanshinones (1-4) as well as twenty three reported compounds (5-27) were isolated and purified from Danshen. The structures of novel compounds were determined by analyzing 1D and 2D NMR, HRESIMS data, etc.. The anti-inflammatory properties of all the compounds towards LPS stimulated RAW264.7 macrophages and their anti-cancer effects on H1299 and Bel-7402 cell lines coupled with a structureactivity relationship (SAR) were investigated. The novel compound 4 demonstrated the best anti-inflammatory activity and was chosen for further research. Compound 4 greatly suppressed secretion of nitric oxide (NO), tumor necrosis factor (TNF)-α and interleukin-6 (IL-6) in RAW264.7 macrophages stimulated by LPS. The mechanism exploration indicated that compound 4 was able to dramatically disrupt TLR4 dimerization to inhibit LPS-induced activation of JNK1/2 and ERK1/2, suggesting that compound 4 targets TLR4 dimerization to exert anti-inflammatory effects. The anti-cancer activity investigation indicated that most of tanshinones exhibited cytotoxicity and the SAR analysis showed that the intact D ring was indispensable and unsaturated D ring played a vital role. Of these compounds, Hydroxymenthylene iv tanshinone (HMT) was chosen for further study. Our results indicated that HMT induced non-small cell lung cancer cells A549 and H1299 cells death through necroptosis. HMT’s IC50s on A549 and H1299 for 24 h were calculated at 5.34 and 2.45 μM, respectively. HMT induced MLKL trimerization to translocate to cell plasma membrane during necroptosis both in A549 and H1299 cells. By constructing different MLKL wide type and mutant plasmids, we demonstrated that HMT induced MLKL trimerization through coiled-coil domain. Specifically, we found that membrane translocation of MLKL is indispensable for Ca2+ influx during HMTinduced necroptosis. Furthermore, HMT easily induced drug resistance A549 death through necroptosis, which provides us with an alternative way to overcome cancer cells drug resistance.