4.1 General experimental procedures

XtaLAB Pro diffractometer was used to determine X-ray crystallography. HR-ESI–MS, IR, UV, NMR spectra were determined as described previously [30].

4.2 Plant material

The whole plants of T. quadrifarium were collected in Pingtang County, Guizhou Province, China, in August, 2021, and identified by Prof. Ni Zhang. A specimen voucher (No. TZC20210806) is stored in the Natural Products Research Center of Guizhou Province.

4.3 Extraction and isolation

The air-dried material (20.0 kg) was smashed and extracted with 95% aqueous EtOH (3 × 70 L, each 2 h) under reflux. The crude EtOH extract was obtained by evaporation under vacuum conditions. The crude samples were suspended in H2O and then leached with petroleum ether (PE), and ethyl acetate (EA), respectively. The PE part (1047.4 g) and EA part (819.9 g) were evaporated to dryness. The PE layer was purified with silica gel chromatography, employing stepwise gradient elution with PE/EA mixture ranging from.

100:0 to 0:100, to afford seven fractions (Fr. A–G), according to TLC plates. Fr. D (74.5 g) was isolated on CC (MeOH/H2O, from 50:50 to 95:5) to yield eight fractions (Fr. D1–D8). Fr. D5 (13.0 g) was further purified via Sephadex LH-20 (CH2Cl2/MeOH, 1:1) to yield compound 5 (32.3 mg), 6 (18.4 mg). Compound 1 (MeOH/H2O, 57:43, tR 19.5 min, 9.3 mg), and 9 (MeCN/H2O, 49:51, tR 26.0 min, 9.1 mg) were obtained from Fr. D6 via semi-preparative HPLC. And compounds 4 (MeCN/H2O, 45:55, tR 53.0 min, 5.6 mg), 10 (MeCN/H2O, 45:55, tR 47.0 min, 3.6 mg) isolated from Fr. D6 by semi–preparative HPLC. Fr. D4 was isolated through Sephadex LH-20 (CH2Cl2/MeOH, 1:1) and semi-preparative HPLC to give compounds 11 (MeOH/H2O, 64:36, tR 27.5 min, 9.6 mg), and 12 (MeOH/H2O, 48:52, tR 31.0 min, 1.9 mg). Fr. D3 was repeatedly separated on CC, and subsequently purified to have 7 (13.2 mg) and 8 (6.3 mg) via Sephadex LH-20 (CH2Cl2/MeOH, 1:1). Fr. F (87.5 g) was chromatographed on CC (MeOH/H2O, from 30:70 to 95:5) to give eight fractions (Fr. F1–Fr. F8). Fr. F2 (17.2 g) further afforded six fractions (Fr. F2a–Fr. F2f) after purified by silica gel (CH2Cl2/MeOH, from 100:1 to 0:100). Compounds 13 (MeCN/H2O, 35:65, tR 27.0 min, 2.8 mg), 14 (MeCN/H2O, 35:65, tR 40.0 min, 3.3 mg), and 15 (MeOH/H2O, 48:52, tR 48.0 min, 17.9 mg) were obtained from Fr. F2c, and compounds 16 (MeCN/H2O, 40:60, tR 36.0 min, 17.8 mg), 17 (MeOH/H2O, 48:52, tR 39.0 min, 12.5 mg), 18 (MeCN/H2O, 48:52, tR 26.0 min, 9.8 mg) were yield from Fr.F2d, by semi-preparative HPLC.

The EA layer was purified with silica gel chromatography and afforded eight fractions (Fr. EA-Fr. EH) according to TLC plates. Fr. EB (26.8 g) was further chromatographed on CC (MCI, MeOH/H2O, from 30:70 to 95:5) to give nine fractions (Fr. EB1–EB9). Compounds 2 (MeOH/H2O, 56:44, tR 27.0 min, 12.5 mg), 3 (MeCN/H2O, 45:55, tR 37.0 min, 7.0 mg), 19 (MeOH/H2O, 40:60, tR 48.0 min, 2.5 mg), and 20 (MeOH/H2O, 40:60, tR 57.0 min, 4.2 mg) were purified from Fr. EB5 through semi-preparative HPLC.

Teucrifaride A (1): Yellow oil; UV (MeOH) λmax (log ε): 200 nm: (2.87); \(_}^\) +14.21 (0.02, MeOH); IR (KBr) νmax: 3444, 2935, 1786, 1678, 1118, 1010 cm−1; 1D NMR data: see Tables 1 and 2. HR-ESI-MS [M + Na]+: m/z 367.1149 (cacld for C19H20O6Na+, 367.1152).

Teucrifaride B (2): Yellow oil; UV (MeOH) λmax (log ε): 200 (2.87) nm, 254 (2.00) nm; \(_}^\) +34.62 (0.03, MeOH); IR (KBr) νmax: 2933, 2866, 1734, 1676, 1558, 1508, 1155 cm−1; 1D NMR NMR data: see Tables 1 and 2; HR-ESI-MS [M + Na]+: m/z 353.1346 (cacld for C19H22O5Na+, 353.1359).

Teucrifaride C (3): White and amorphous powder; UV (MeOH) λmax (log ε): 206 nm: (3.28); \(_}^\) −54.17 (0.02, MeOH); IR (KBr) νmax: 3525, 2939, 1689, 1180 cm−1; 1D NMR data: see Tables 1 and 2; HR-ESI-MS [M + Na]+: m/z 411.1408 (cacld for C21H24O7Na+, 411.1414).

Teucrifaride D (4): Colorless acicular crystal; mp 151–152 ℃; UV (MeOH) λmax (log ε): 208 nm: (3.68); \(_}^\)+ 27.03 (0.01, MeOH); IR (KBr) νmax: 3155, 2970, 2939, 1774, 1751 cm−1; 1D NMR data: see Tables 1 and 2; HR-ESI–MS [M + Na]+: m/z 425.1559 (cacld for C22H26O7Na+, 425.1571).

4.4 Single-crystal X-ray diffraction analysis of 4 and 10

A suitable crystal of compounds 4 and 10 were chosen and subjected to X-ray diffraction analysis on a XtaLAB AFC12 single X-ray diffractometer. The crystal was maintained at 100.00 (10) K for 4, and at 99.99 (10) K for 10, during data collection. The structural analysis was initiated using the Olex2 software, where the crystal structures were determined by the SHELXT program with intrinsic phasing. Subsequently, the structures were refined using the SHELXL refinement package, employing least squares minimization techniques [30]. The crystallographic data (CCDC.2383165 for 4 and CCDC.2383166 for 10) can be obtained free of charge via www.ccdc.cam.ac.uk/conts/retrieving.html.

4.5 ECD calculations

ECD calculation methods were the same with previous reports [30].

4.6 Cell viability assay

HT22 cells were grown in a culture medium consisting of DMEM (from HyCyte) supplemented with 1% penicillin/streptomycin (ECOTOP) and 10% fetal bovine serum (HyCyte). The culture was maintained in a 5% CO2 environment at a temperature of 37 ℃ within an incubator. HT22 cells were plated at a density of 3000 cells per well in 96-well plates and allowed to attach for 24 h. Subsequently, RSL3 (1 μM) or erastin (1 μM) along with different concentrations of compounds were introduced. After 24 h, MTT reagent (10 μL, 5 mg/mL) was introduced to each well and incubated for an additional 4 h. Following this, the formazan crystals were solubilized using 150 μL of DMSO. The plates were softly shaken for 15 min at ambient temperature, and then tested the absorbance at 490 nm using a microplate reader.

4.7 Intracellular reactive oxygen species detection

HT22 cells were cultured at 300,000 cells/well in 6-well plates for 24 h. RSL3 (1 μM) and three different concentrations (5, 10, 20 μM) of compound 1 and Fer-1 (1 μM) were added to plates. After a 4 h reaction, the supernatant medium was aspirated off and H2PCFDA (1 mL, 10 μM) was added in an incubator and incubated for 40 min (37 ℃), then washed twice with serum-free medium and photographed with a fluorescence microscope under dark conditions.