Aerial parts of Asterohyptis stellulata Epling, Lamiaceae (Fig. S1) were collected in the locality of Otates, Municipality of Actopan, State of Veracruz, Mexico (Lat, 19.532963 N; Long, − 96.717062 W; Alt, 488 masl) on January 14, 2023, by A.C. Hernández-Rojas and M. Kilian. Samples of the species were identified by Dr. Hernández-Rojas and deposited at the Herbarium XAL with duplicates (accession number 152242) of the Instituto de Ecology A.C. (INECOL, Xalapa, Veracruz). The plant material (1.4 kg) was extracted by maceration 4 × 24 h each at room temperature with petroleum ether. A yellowish-white solid precipitated (250 mg) from the petroleum ether solution (Fig. S3). This solid was washed with MeOH (3 ×) to remove polar impurities and pigments, as the solid was insoluble in this protic solvent. Subsequently, the clean solid was subjected to recrystallization. Initially, it was dissolved in CH2Cl2 (3.5 ml) and, to facilitate a controlled crystallization, a few drops of petroleum ether (0.5 ml) were added. The solution was filtered and allowed to cool at room temperature, resulting in the formation of pale-yellow crystals (125 mg). Further recrystallization (50 mg) with CH2Cl2-petroleum ether (9:1) yielded 40 mg of pure salvigenin (1). This compound was identified by comparison of its physical constants (Moradkhani et al. 2012) and spectroscopic properties, such as 1H and 13C NMR (Fig. S4), as well as HRMS (Fig. S5), with published values (Ayatollahi et al. 2009).

Salvigenin (1): pale yellow crystals, mp 185–187°; 1H NMR (600 MHz, CDCl3) δ 12.77 (s, 1H, C5-OH), 7.82 (d, J = 9.0 Hz, 2H, H-2′ and H-6′), 7.00 (d, J = 9.0 Hz, 2H, H-3′ and H-5′), 6.56 (s, 1H, H-3), 6.53 (s, 1H, H-8), 3.96 (s, 3H, C-7, -OMe), 3.92 (s, 3H, C-6, -OMe), 3.88 (s, 3H, C-4′, -OMe); 13C NMR (CDCl3, 150 MHz) δ 182.8 (C-4), 164.1 (C-2), 162.7 (C-4′), 158.8 (C-7), 153.3 (C-4a), 153.2 (C-8), 132.8 (C-6), 128.1 (C-3′, C-5′), 123.7 (C-1′), 114.6 (C-2′,C- 6′), 106.3 (C-5), 104.2 (C-3), 90.7 (C-8a), 61.0 (C-7, -OMe), 56.4 (C-6, -OMe), 55.7 (C-4′, -OMe). HR APCI-TOF–MS m/z 329.1016 [M + H]+ (calcd. for C18H17O6m/z 329.1019,  δ =  − 0.9 ppm), 314.0778 [M + H – CH3]+, 296.0664 [314 – H2O]+, 268.0723 [296 – CO]+.

Cytotoxicity and drug combination assays were conducted with salvigenin (1) and using human colon carcinoma cells (HCT-116). The cells were cultured in fetal bovine serum medium, 100 U/ml penicillin G, and 100 μg/ml streptomycin at 37 °C under 5% CO2 atmosphere and 100% relative humidity. Cells were used when they reached 60–70% confluence and were maintained in the logarithmic growth phase. A suspension of 104 cells was used. From this suspension, 190 µl was seeded in 96-well plates and 10 µl of different concentrations of test samples in DMSO (10%) was added and the experiments were performed in triplicate. The microplates were incubated for 72 h, and the sulforhodamine B (SRB) method was used. Cell density was determined using an ELISA plate reader at 564 nm. For the combination assays, cells were exposed to test compounds for 72 h. The clinical drugs vinblastine (0.003 µM), podophyllotoxin (0.008 µM), colchicine (0.008 µM), ellipticine (1.6 µM), and doxorubicin (0.7 µM) were individually tested at sublethal concentrations in combination with salvigenin (1), which was evaluated at final concentrations of 50, 30, 20, 10, 3, and 1 µM, followed by SRB method. The growth percentage was plotted along with their respective concentrations using Prisma v. 8.01 to obtain the IC50 (Moreno-Velasco et al. 2024).